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COURS DE VIROLOGIE MEDICALE

1ère Partie : VIROLOGIE GENERALE

SOMMAIRE

Section 1 : Définitions, structures virales et classification des virus


§ 1 Introduction……………………………………………………………………...1
§ 2 Définitions………………………………………………………………………..2
§ 3 Structures virales………………………………………………………………..3
§ 4 Classification des virus………………………………………………………....11
Section 2 : La multiplication virale
§ 1 Aspects originaux de la multiplication virale…………………………………17
§ 2 Les étapes de la multiplication virale………………………………………….17
§ 3 Les interactions entre le virus et la cellule hote………………………………22
§ 4 la variabilité des génomes viraux……………………………………………...23
Section 3 : Pouvoir infectieux viral, mécanismes de défense et épidémiologie des infections
virales
§ 1 Le pouvoir infectieux viral…………………………………………………….24
§ 2 Les mécanismes de défense de l’organisme…………………………………..27
§ 3 Epidémiologie des infections virales…………………………………………..31
Section 4 : Les méthodes d’études des virus
§ 1 Introduction…………………………………………………………………….33
§ 2 la pratique des cultures cellulaires……………………………………………33
§ 3 la détections des antigène viraux et anticorps spécifiques…………………..35
§ 4 La microscopie électronique…………………………………………………..37
§ 5 L’apport de la biologie moléculaire…………………………………………..37
§ 6 le diagnostic biologique d’une infection virale……………………………….38

SECTION 1. DEFINITIONS , STRUCTURES VIRALES ET CLASSIFICATION DES VIRUS

Objectifs :
 comprendre la place des virus dans les maladies infectieuses
 préciser les particularités des virus parmi les microorganismes infectieux
 connaître les critères fondamentaux actuels de définition d’un virus
 étudier les structures virales : génomes, capsides et enveloppes viraux
 connaître et utiliser les critères de classification des virus

§1. INTRODUCTION

On estime généralement que plus de 60% des maladies infectieuses sont dues aux virus. Ex :
les gastro-entérites infantiles, les infections respiratoires aiguës, les hépatites virales et le SIDA. Cela
explique l’intérêt particulier et actuel que soulève l’étude de la Virologie axée sur le diagnostic,
l’épidémiologie et la pathogénie des maladies virales.

La notion de maladie virale a été établie à la fin du 19ème siècle. PASTEUR, IVANOVWSKI
et FROSCH démontrèrent certaines maladies dont les agents pathogènes étaient ultrafiltrables. Ils
passaient à travers les pores des filtres de porcelaine, connus pour retenir toutes les bactéries. Ces
agents étaient invisibles au microscope optique et responsables d’affections transmissibles en série :
Ce sont des virus. A la différence des bactéries le virus donc :
-ne se voit pas en microscopie optique (sauf au microscope
électronique)
-ne se cultive pas sur milieu inerte (tel un bouillon ou une gélose)

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-n’est pas retenu par le filtre de Chamberlain filtre de porcelaine


(qui retient toutes les bactéries )
fig :1 comparaison des tailles

La taille des virus est de l’ordre du nanomètre = 10-9 m (ente 20 et 300 nm) On ne peut
observer les virus que par la microscopie électronique.

Ils affectent pratiquement tous les genres d’animaux de même que les arthropodes, les algues,
les champignons ainsi que les protistes ; en particulier les bactéries par les bactériophages.

Fig 2 : des bactériophages vue au microscope électronique

De cet ensemble nombreux et varié, une centaine d’espèces de virus sont pathogènes pour l’homme et
représente de ce fait une importance médicale.

Le domaine pathologique des virus en virologie médicale est étendu et varié. Beaucoup de
maladies d’origines virales sont en général bénignes, et caractérisées par leur grande fréquence
(certaines conjonctives, le coryza saisonnier ou rhume, les fièvres éruptives de l’enfance).

D’autres affections redoutables sont de plus en plus rares grâce aux vaccinations
(poliomyélite, fièvre jaune). D’autres viroses restent d’actualité, surtout dans les pays en voie de
développement, telles les hépatites virales.

Enfin, certaines sont particulièrement redoutables quand elles atteignent des terrains
particuliers. Chez les femmes enceintes, le virus de la rubéole et le Cytomégalovirus sont
responsables d’embryopathies et ou des malformations.
LE VIH ou virus de l’immunodéfience humaine est l’agent du SIDA, la pandémie de la fin du 20ème
cycle.

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Par ailleurs, les sujets immunodéprimés sont plus susceptibles que les sujets sains, à certains
virus tels, l’herpès virus et le virus de la varicelle et du zona.

D’autres virus, avec des co-facteurs, jouent un rôle dans le développement de tumeurs
malignes. Ces virus sont potentiellement oncogènes (peuvent générer des tumeurs). Ce sont par
exemples, le virus de l’hépatite B dans le carcinome primaire du foie et le virus d’EPSTEIN-BAAR
dans le lymphome de Burkitt.

2. DEFINITIONS

Pendant longtemps, les virus ont été définis de façon incertaine. Les progrès des sciences
biologiques et de la biotechnologie ont permis une définition plus précise des virus.

Ce sont des agents infectieux, des microorganismes invisibles au microscope optique,


dont la structure se résume à un génome entouré d’une coque protéique appelée capside.
L’ensemble de ces deux éléments forme la nucléocapside. Celle-ci s’entoure chez certains virus
d’une enveloppe ou peplos. La taille des virus est de l’ordre de quelques nanomètre, 10-9 m (ente
20 et 300 nm)

Fig 2 un virus nu et un virus enveloppe

A l’inverse des cellules eucaryotes ( cellule humaine, des protozoaires et des micromycetes) le virus
n’a ni cytoplasme ni membrane cytoplasmique, ni organites intra cytoplasmiques tel les ribosomes, le
réticulum endoplasmique, des mitochondries et l’appareil de Golgi. Il n’a pas de membrane nucléaire.
Au contraire des cellules procaryotes (cellule bactérienne) le virus n’a pas de paroi ou
peptidoglycane, ni cils ou flagelles ni capsule.
Ainsi le virus ne possède aucun système enzymatique, aucune réserve énergétique
pouvant assurer sa survie dans un milieu de culture inerte (bouillon de culture bactériologique) ou
dans l’environnement. Le virus n’est pas une cellule ; Ce n’est qu’une information génétique
protégée par des structures périphériques ( capside avec ou sans enveloppe)

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Fig 3 Les différents éléments d’un virus nu ou enveloppe

Fig 4 : la cellule eucaryote

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De plus, les virus ne se multiplient pas par scissiparité comme les bactéries ; ni par mitose
comme les cellules eucaryotes. Ils répliquent leurs génomes. En raison de leur extrême simplicité,
les virus ne peuvent se répliquer qu’en utilisant les macromolécules de la cellule qu’ils ont infecté.
Ils sont donc nécessairement des parasites intracellulaires absolus.

Le virus est une entité biologique dont le génome est formé d’acide nucléique ADN ou
ARN protége par une coque protéique : la capside. Il se réplique dans les cellules infectées en
utilisant l’appareil de synthèse de celles-ci pour coder et diriger la production des structures
spécifiques du virus qui seront assemblés en une entité appelée virion

On appelle virion la particule virale mature, infectieuse et extra cellulaire. C’est la forme de
dissémination, assurant le transfert de l’infection virale, à d’autres cellules ou organismes.

En 1953, LWOFF a énoncé quatre caractères fondamentaux de définition faisant des


virus des entités originaux :
=1/Chaque particule virale ne contient qu’un seul type d’acide nucléique, ARN ou
ADN, qui forme le génome viral. Ceci oppose les virus aux autres êtres vivants ayant les 2 acides
nucléiques ; l’ADN représentant l’information génétique tandis que l’ARN assurant l’expression
génétique
=2/Les virus présentent une structure particulière, les opposent aux autres êtres
vivants à structure procaryote (bactérie) ou eucaryote. Cette structure est le virion.
=3/Les virus se reproduisent uniquement à partir de leur matériel génétique, par
réplication de leur acide nucléique genomique. Ils ne se divisent pas par scissiparité comme les
bactéries, et ne subissent pas de mitose comme les cellules eucaryotes. Les virus se reproduisent
mais ne croissent pas
=4/Les virus présentent un parasitisme intra cellulaire obligatoire. Ce parasitisme
s’explique par le fait que le virus ne possède aucun système enzymatique ou énergétique lui
permettant d’assurer sa propre réplication. En effet, il est limité à une information génétique protégée
par des structures périphériques. Toutefois, certains virus ont des enzymes qui interviennent en partie
dans la réplication du génome virale. Mais la majorité des virus sont amenés à détourner et à utiliser,
pour leur propre biosynthèse, l’ensemble ordonné des macromolécules de la cellule parasitée

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(ribosomes, ARNt , activités enzymatiques diverses, systeme énergétique) ; ainsi est lésée la cellule
parasitée .

En corollaire et pour ces diverses raisons, les virus sont incapables de se multiplier dans
des milieux inertes comme le font les bactéries. Leur isolement passe forcement par l’inoculation à
un système biologique vivant permissif, capable d’assurer leur réplication.
Le détournement, au profit du virus, des principales activités métaboliques de la cellule parasitée,
peut entraîner la mort de celle-ci. Mais ce n’est pas une règle absolue, il peut y avoir tolérance. Il y
aura, donc, diminution ou inhibition complète des synthèses cellulaires pour la cellule parasitée,
bases physiopathologiques de l’infection virale.

En outre, cette étroitesse de relation entre la cellule infectée et le virus explique aussi la
difficulté de la mise au point des traitements efficaces antiviraux. Ceux-ci devraient pouvoir détruire
le virus sans léser la cellule hôte ce qui n’est pas toujours possible.

En conclusion, nous retenons qu’ainsi défini, un virus n’est pas une


cellule, mais l’équivalent d’un fragment de code génétique, qui a pris son indépendance et ne
contient que l’information pour sa propre réplication. Cette information, ne peut s’exprimer,
que grâce aux détournements des systèmes enzymatiques et énergiques de la cellule parasitée,
le virus étant un parasite intracellulaire absolu.

Cette structure et ce mode de réplication conditionnent la plus grande partie des


propriétés biologiques et du pouvoir pathogène des virus.

3. STRUCTURES

Tous les virus sont constitués de deux entités essentielles : le génome, formé d’acide
nucléique et la capside ou coque de nature protéique, entourant ce génome.
L’ensemble génome-capside forme le virus nu. Les virus enveloppés sont constitués de
l’ensemble génome-capside-enveloppe.
Cette enveloppe ou peplos est de composition lipido-glucido protéique.

a) le génome viral

Constitué d’un seul type d’acide nucléique, soit l’ADN ou l’ARN, il contient toute
l’information génétique du virus. Lors de la reproduction virale, il sera répliqué, et transcrit en
éléments structuraux et non structuraux du virus. Cet acide nucléique est caractérisé par un P.M. (en
Daltons) ou leur G + C % ou indice de CHARGAFF, et encore par le nombre de paires de bases
( kilobases) Le Dalton est l’unité de masse atomique = 1/12 de la masse d’un atome nucléide du
carbone ou 12 C.
1Da = 1,66503 x 10-27 kg = 1U de masse

Cet acide nucléique peut être à simple ou double brin, mono ou bicatenaire. Il peut être
fragmenté ou entier, linéaire ou circulaire.
Les génomes a ARN sont monocaténaires a simple brin (a part ceux des Reoviridae avec un
génome a ARN a double brin). Les génomes à ADN sont bicatenaire à double brin.
Les génomes sont constitues de gènes structuraux codant pour les structures virales (capside et /ou
enveloppe) ainsi que des gènes non structuraux qui codent pour des éléments enzymatiques et enfin
des gènes régulateurs de la réplication génomique

1. Génome à ADN ou Acide Désoxyribonucléique


Les génomes viraux à ADN possèdent une grande variété de poids moléculaire, allant de 3
x106Daltons pour certains Papovaviridae à 160 x106 Daltons pour les Poxviridae. Les ADN viraux
sont généralement bi caténaires à double brin et linéaires.
Ex. Les Herpes virus ont un génome à ADN bi caténaire, linéaire, d’un P.M de 80-150 x 106Da

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Fig 5 ADN ou acide desoxyribonucleique

2. Génome à ARN ou Acide Ribonucléique


Le poids moléculaire des ARN varie de 2 x 106 Da pour les Picornaviridae, à 15 x 10 6 Da pour les
Réoviridae.

Ces ARN sont en général mono caténaire à simple brin et linéaire sauf dans le cas de
Réoviridae ou le génome est bi caténaire. Certains génomes à A.R.N sont fragmentés, tels les
génomes des virus grippaux .
Ils sont constitués de plusieurs fragments reliés entre eux par des liaisons covalentes. Cette
disposition favorise les recombinaisons génétiques et les mutations des virus.
Ex : Les Myxoviridae, dont les virus grippaux, ont un génome à A.R.N. monocaténaire, avec 8
segments d’un P.M. de 4-106 Da

Fig
6 :Génome à
ARN de
polarité +,
lineaire,

monocaténaire des Poliovirus formé des gènes VP1, VP2, VP3 de structures

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Fig 7 : Génome du VIH à ARN monocaténaire de polarité positive et ces gène de structures
POL,ENV,GAG de même que des gènes de régulation,vif,vpu ou vpr,nef, tat

b) la capside

C’est une structure polymérisée à base d’unités protéiques, constituées par la répétition
d’une seule ou plusieurs sous unités. Cette structure protège le génome et son information génétique
et facilite le transfert de cette information à la cellule infectée.
Suivant les virus, les unités protéiques de cette capside s’organisent en une capside
hélicoïdale à symétrie tubulaire ou en une capside icosaédrique à symétrie cubique.

L’icosaèdre est un polyèdre régulier constitué de 20 faces triangulaires, 12 sommets et 30


arêtes ; il constitue une boîte creuse contenant l’acide nucléique. L’ensemble forme une
nucléocapside à symétrie cubique ou icosaédrique. Chaque sommet de l’icosaèdre est occupé par un
penton formant un capsomère ou oligomère à 5 unités structurelles ou sous –unités protéiques. Les
faces et les arêtes sont constituées de capsomères à 6 unités de structures ou hexons.

Fig 8 icosaèdre

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Fig 9 Adénovirus avec une capside à symétrie cubique icosaédrique

Les familles de virus à symétrie icosaédrique sont caractérisées par le diamètre de la capside et le
nombre fixe de capsomère. Ainsi les Adénoviridae ont tous une capside de 70 à 90 nm de diamètre,
formée de 252 capsomères, dont 12 pentons et 240 hexons.

Dans les capsides tubulaires à symétrie hélicoïdale, les sous unités ne se regroupent pas en
capsomères, elles s’empilent en colimaçon, maintenant l’acide nucléique enroulé entre 2 tours de
spires. On retient parmi les caractéristiques d’un virus à symétrie hélicoïdale sa longueur et le
diamètre du tube de sa capside.

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Fig 10 virus de la mosaique du tabac avec une capside à symétrie tubulaire hélicoïdale

Rappelons que la structure compacte formée par l’assemblage de la capside autour du


génome s’appelle nucléocapside ou « core ». Les protéines de la capside sont antigéniques ; elles
induisent, chez l’organisme infecté, la formation d’anticorps spécifique.
.
Certains virus ne possèdent pas d’autre structure, ils sont nus. Les autres ont en plus, une
enveloppe ou péplos : ils sont enveloppés.

c. l’enveloppe virale ou peplos

De composition lipido-glucido-protéique, c’est un élément propre aux virus enveloppés. Elle


provient, par bourgeonnement, des systèmes membranaires de la cellule hôte parasitee.
Trois origines sont connues :

• 1. La membrane nucléaire. Ex : les virus de l’herpès


• 2. Les structures intra cytoplasmiques comme le réticulum endoplasmique ou
l’appareil de Golgi. Ex : les Togaviridae
• 3. La membrane cytoplasmique de la cellule que l’on retrouve enveloppant le
Ortho et Paramyxoviridae de même que les Rétroviridae.

La composition macromoléculaire de l’enveloppe reflète celle du composant membranaire dont elle


est issue.

Fig 11 Enveloppe dérivée de la membrane cytoplasmique (VIH ou Virus de la rougeole)

Fig 12 Enveloppe dérivée de la membrane nucléaire ( les Herpesviridae)

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L’enveloppe virale possède 2 types de spécificités antigéniques distinctes :

• Une spécificité cellulaire, le virus emportant avec lui une partie des antigènes des
membranes de la cellule hôte
• Une spécificité virale, on trouve à ce niveau, entre autres,.des protéines à activités
agglutinantes les globules ou hemagglutinine ; une neuraminidase protéine a activité enzymatique,
ainsi que des facteurs toxiques et des facteurs de fusion cellulaire.

L’enveloppe est très sensible aux divers agents physico-chimiques, en particulier aux solvants
lipidiques (détergents, éther, sels biliaires) à la chaleur et la dessiccation
L’enveloppe ne constitue donc pas un élément de protection supplémentaire. Au contraire, c’est un
élément de fragilité rendant le virus enveloppés peu résistant dans le milieu extérieur.

Souvent de formations sont visibles, au microscope électronique, sous forme de spicules


hérissée sur l’enveloppe ou la capside.

Fig 13 Adénovirus hérisse de spicules sur sa capside

VIH 1 dont l’enveloppe est


hérissée de spicules

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§4. CLASSIFICATION

LWOFF, HORNE et TOURNIER ont été les premiers, en 1960, à proposer une
classification fondée sur la structure des virus et non plus sur les caractères des maladies
correspondante.
Le système de classification L.H.T. retient 4 critères objectifs de classification :

1°) La nature de l’acide nucléique (ADN ou ARN) génomique


2°) La symétrie de la nucléocapside, hélicoïdale (H) ou cubique(C)
3°) Le nombre de capsomères chez les virus à symétrie icosaédriques, ou le
diamètre de la nucléocapside si elle est hélicoïdale.

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4°) présence d’une enveloppe distinguant les virus enveloppé (E) des virus
nus (N).

En 1975, un comité international de taxonomie des virus a dressé une classification des virus en
familles (…viridae), en sous- famille (…virinae) en genres (…virus) en espèces et en sérotype.
Dans un texte dactylographie, les noms des familles, des genres et des espèces sont écrits en italique
et souligné. Les noms des familles et des genres s’écrivent en majuscule ; les noms des espèces en
minuscule.
Ex : 1) L’agent de la Poliomyélite Antérieur Aiguë ou P.A.A est un virus
- de la famille des Picornaviridae
- de la sous-famille du groupe C
- du genre Entérovirus
- de l’espèce poliovirus avec trois sérotypes ou types 1,2 et 3

2)L’agent de la Mononucléocse Infectieuse ou M.N.I est un virus :


- de la famille des Herpesviridae
- de la sous-familles des Gammaherpesvirinae
- du genre des Lymphocryptovirus
- de l’espèce du virus d’Epstein-Barr ou E.B.V ou H.H.V.4. pour Human Herpes Virus 4

Deux serotypes d’une même espèce sont différents antigéniquement, sur un détail de leurs structures
antigéniques ou epitope ou déterminant antigénique
Certains virus, tels que les V.I.H et le virus de l’hépatite C, sont pourvus d’une extraordinaire
variabilité génétique. De telle sorte que chez un même individu coexistent, en même temps, plusieurs
variants d’une même espèce. Ces variants forme une « quasi-espèce ». Ces variants sont classes en
génotypes et sous génotypes ou clades.

Ainsi pour l’heure les virus d’une même espèce peuvent être distingué :

1) soit en fonction de différences antigéniques prouvées ; ex : les poliovirus de sérotypes 1,2 et


3 ; ou les V.I.H 1 et 2.
2) Soit en fonction de différences des séquences génomiques, on parle de génotypes.
Ex : pour les V.I.H. il y a deux serotypes : VIH 1 et 2. Ensuite on dénombre pour le VIH 1,
déjà trois groupes génotypiques : le groupe M (Major), le groupe O (Out-lier) et le
groupe N (Non M- Non O). Ces groupes sont des répartitions géographiques différentes.
Le groupe M est cosmopolite responsable de la pandémie du SIDA.

Dans les deux cas, ces distinctions se font vis-à-vis sur une région donnée du virus.Ils
s’agit de la diversité antigénique d’un épitope (un déterminant antigénique), ou de la différence de
séquence de tout ou partie d’un gène viral. Actuellement pour les V.I.H.1, on admet être en présence
de deux génotypes, si au moins 20% de la séquence des acides aminés codés par le gène ENV de
l’enveloppe, diffèrent d’un variant à l’autre

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Fig 14 Schema du VIH 1 virus de l’immunodefiscience humaine type 1

a)Génome : ARN de 9,7kilo base


b) Capside : dense excentrée de forme de trapèze
c) Enveloppe : présente hérissée de spicules de glycoprotéines, gp 120 et gp 41
d) Virion : d’un diamètre de 80-100nm
e) Famille : Retroviridae
f) Genre : Lentivirus
g) Espèce : VIH Virus de l’Immunodéficience Humaine
h) serotypes : VIH1, VIH 2
i) Génotypes du VIH 1: groupe M, groupe O, et groupe N pour Non M et non O
j) -Sous génotypes ou clades du groupe M : 10 clades de A à J de répartition mondiale différente
-Sous génotypes ou clades du groupe O : 4 clades

Ex 2 : La famille des Herpesviridae infectant l’homme

Fig 15 un Herpesviridae

Sous famille Alphaherpesvirinae Betaherpesvirinae Gammaherpesvirinae


Genre 1/ Simplex virus 1/Cytomégalovirus 1/Lymphocryptovirus
2/ Varicellovirus 2/Roseolovirus 2/Rhadinovirus
Genre Simplex virus Cytomégalovirus Lymphocryptovirus
Espèces Herpès simplex virus Cytomégalovirus humain Virus d’Epstein-Baar
HHV 1 et HHV 2 HCMV ou HHV5 EBV ou HHV4

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Genre Varicellovirus Roseolovirus Rhadinovirus


Espèces Virus du Zona et de la Herpes virus humain 6 Herpès virus humain 8
varicelle HHV6 HHV8/KSHV
VZV ou HHV3 Herpes virus humain 7
HHV7

HHV pour Human Herpes Virus

Fig 16 virus à ADN

Virus à AND
POXVIRUS VIRUS DE L'HEPATITE B

HERPESVIRUS

PAPOVAVIRUS

ADENOVIRUS

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PARVOVIRUS

Fig 17 : virus a ARN

Virus à ARN
ORTHOMYXOVIRUS (grippe)

PARAMYXOVIRUS

(rougeole, oreillons, VRS)

FILOVIRUS

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REOVIRUS

RHABDOVIRUS (rage)

ARENAVIRUS

RETROVIRUS
BUNYAVIRUS

CORONAVIRUS
CALICIVIRUS

TOGAVIRUS
PICORNAVIRUS

Tableau 1: Classification de virus d’intérêt médical

FAMILLE TAIL GÉ CAPSI ENV GROUPE VIRUS TYP


LE EN NO DE E
ME OU GENRE
Nm
Adenoviridae 70-90 AD cubique Sans Adenovirus 47
N
Papovaviridae 45-55 AD cubique Sans Polyomavirus Virus BK et 2
N JC

V. des
papillomes

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17

Papovavirus 5a7
Herpesviridae 150- AD cubique Avec Alphaherpesvi HHV1,2,3 3
200 N rinae
HHV5,6,7 3
Betaherpesviri
nae HHV4,8 2

Gammaherpes
virinae
Poxviridae 200- AD comple Avec Poxvirus V. de la 1
300 N xe variole

V. de la
vaccine 1

V. du 1
molluscum
contagiosum

Picornaviridae 20-30 AR cubique Sans Enterovirus Poliovirus 3


N
Coxsackievi- 29
rus A et B
34
Echovirus
3
Enterovirus
Heparnavirus 1
V.hepatite A
Rhinovirus 113
V. du rhume
Retroviridae 80-100 AR Helicoidal Avec Oncornavirus HTLV 1,2 2
N e
Lentivirus VIH 1,2 2

Spumavirus
Togaviridae 40 AR cubique Avec Flavivirus V. de la 1
N fievre jaune

V.de la
dengue 4

V. de 1
l’hepatite C
Rubivirus
V. de la
rubeole 1

Orthomyxovirida 80-120 AR helicoidale Avec Influenzavirus V.de la No


e N grippe A,B mbr

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frag. et C eux
Paramyxoviridae 150- AR helicoidale Avec Pneumovirus VRS 1
300 N
Metapneumo- Metapneum 1
ovirus
virus
V.
parainfluenz 2
a 1 et 3
Respirovirus
V. de la
Morbillivirus rougeole 1

V. des 1
oreillons
Rubulavirus
V. para
influenza 2 3
et 4a , 4b
Rhabdoviridae 60-180 AR helicoidale avec Lyssavirus V. de la rage 6
N

SECTION 2. REPRODUCTION DES VIRUS

Objectifs :
> comprendre les aspects originaux de la reproduction virale
> décrire les principales étapes de la reproduction virale : attachement, pénétration, décapsidation,
réplication génomique et formation des protéines virales, assemblage et libération des virions
 étudier les interactions du virus et de la cellule hote
 expliquer la variabilité des génomes viraux

§1. ASPECTS ORIGINAUX DE LA REPRODUCTION VIRALE


Les virus se divisent très différemment des autres microorganismes. Ces
caractères originaux permettent de comprendre les modalités de l’infection virale et d’envisager des
moyens de s’y opposer. Etant des parasites intracellulaire absolue, ils ne peuvent se reproduire qu’à
l’intérieure de la cellule hôte parasitée. Toutefois, les virus, à la différence d’autres parasites
intracellulaires tels les chlamydia (bactéries) ou les plasmodium (parasites hématozoaires), ils
utilisent le système producteur d’énergie les macromolécule les enzymes, les ARN et les ribosomes
de la cellule hôte.
Ces détournement des voies métaboliques s’accompagnent le plus souvent d’une
diminution, d’une inhibition complète des synthèses cellulaires par la cellule hôte.
Un virus est incapable de synthétiser un autre virus, alors qu’une bactérie est
capable de faire une autre bactérie. La simplicité extrême du virus empêche celui-ci de se multiplier,

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du moins par lui-même. En effet il n’a que son information génétique sous forme d’ADN ou d’ARN.
Mais, il n’a ni réserve de petites molécules, ni réserve énergétique, ni chaînes enzymatiques des
grandes voies métaboliques.
En outre, les virus ne croissent pas, ne se divisent pas. Après pénétration de la
cellule, un seul constituant viral, l’acide nucléique intervient dans la reproduction virale. Il se
réplique et induit la fabrication de protéines virales. Les composants structuraux viraux, synthétisés
séparément, sont alors assemblés pour former le virion et toute une série de virus identique à celui qui
a infecté la cellule hôte permissive.
Donc, la reproduction d’un virus consiste en l’introduction du génome viral
dans une cellule et c’est elle qui va fabriquer de nouveaux virus selon un processus de
biosynthèse que l’on appelle réplication

§2. LES PRINCIPALES ÉTAPES DE LA REPRODUCTION VIRALE


Le cycle complet peut être subdivisé en plusieurs étapes :
- L’attachement ou fixation du virus sur la cellule hôte parasitée
- La pénétration du génome virale
- La désagrégation de la capsides ou décapsidation avec libération du génome viral
- La réplication génomique et formation des protéines virales
- L’assemblage et la formation des virions
- La libération des virions néoformés

Fig 18 : Multiplication virale d’un virus a ARN de polarite +: le poliovirus

a)L’attachement
Cet attachement du virus à la membrane cytoplasmique cellulaire encore appelé fixation et/ ou
adsorption est la 1ère des étapes de la x virale. Elle est due à des interactions électrostatiques entre le virus et la
cellule dite permissive. Cet attachement se fait donc par les protéines de la capside pour les virus nus, par les
glycoprotéines du peplos pour les virus enveloppes. Ces protéines ou glycoprotéines s’attachent aux récepteurs
situes sur la membrane cytoplasmique de la cellule hote Si ce type de récepteurs cellulaires spécifiques aux
structures d’attachement des virus est absent, la cellule sera naturellement résistante à l’infection car ce virus ne
pouvant pas se fixer sur elle.

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Le processus d’attachement est un processus indépendant de la température et qui ne nécessite pas d’énergie.
Ex ; de structure d’attachement sur le VIH 1 ; la Gp 120 ou glycoprotéine de PM 120 est une structure
membranaire de VIH1.
Son récepteur spécifique est la molécule CD4 des lymphocytes T auxiliaires ou helper.
Cette molécule CD4 protéique est présente sur la surface des lymphocytes T auxiliaires. La glycoprotéine 120
est la structure d’attachement viral. Les lymphocytes T auxiliaires sont donc des cellules cibles de VIH.

Les récepteurs utilisés par les virus sont souvent des molécules d'adhésion cellulaires, les CAM
(Cell Adhesion Molecule) qui interagissent avec des molécules portées par la membrane
plasmique d'autres cellules.
Récepteurs cellulaires utilisés par des virus
Virus Récepteur Fonction
Physiologique
Poliovirus 1 PVR 1 Inconnue
virus du sida 2 CD4 reconnaît les molécules
(HIV) (lymphocytes Th, CMH de classe II
macrophages)
Rhinovirus 3 ICAM I 2 molécule d'adhésion
virus d'Epstein-Barr 4 CR2 3 récepteur de C3d
(EBV) (cellules du pharynx,
lymphocytes B)
virus de la grippe 5 NANA 4 constituant des gangliosides
membranaires
1 = PVR Polio Virus Receptor (la fonction normale n'étant pas connue)
2 = ICAM I Inter Cellular Adhesion Molecule
3 = CR2 Complement Receptor 2
4 = NANA N-acetyl neuraminic acid (greffé sur un ganglioside membranaire)

b) La pénétration

C’est un processus actif dépendant de la température. En général, le virus pénètre dans la


cellule par phagocytose ou viropexie. Le virus nu pénètre par pinocytose, c-a-d, sont d’abord contenu
dans une vacuole intra cytoplasmique avant d’arriver à leur site de replication
Pour certains virus enveloppés, on a pu démontrer que l’enveloppe virale fusionne avec la
membrane cellulaire, la nucléocapside est dès lors libérée directement dans le cytoplasme. Ainsi chez
les VIH la gp 41 est une glycoprotéine transmembranaire responsable de fusion entre le peplos du
VIH 1 et de la membrane cytoplasmique des lymphocytes CD 4+
Ce mécanisme de pénétration virale est inhibé par des inhibiteurs usuels de la phagocytose.

c) La décapsidation

C’est la désagrégation de la capside et la libération de l’acide nucléique génomique.


Ce sont des enzymes protéolytique de la cellule parasitée qui lysent les capsides. La décapsidation
peut se faire en même temps que la pénétration virale ou plus tardivement en une ou plusieurs sous
étapes. Elle prend place soit au niveau de la membrane cellulaire, soit dans le cytoplasme ; elle est
totale ou partielle.

d) La réplication du génome et formation des composants viraux.

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Elle commence après la libération de l’acide nucléique virale dans la cellule hôte. A ce stade
le virus a cessé d’exister en tant que particule organisée et naturellement infectant. C’est la phase
d’ECLIPSE.

Pour tous les virus, la réplication repose sur 2 processus fondamentaux :


- La réplication du génome
- La transcription d’ARNm viraux qui seront traduits en protéines virales par des
ribosomes de la cellule hôte.
1/ Pour les virus à ADN, une première transcription conduit à la synthèse d’un lot de protéines
précoce, intervenant en particulier dans la réplication de l’ADN virale.
Ces protéines précoces ne sont généralement pas incorporées dans la particule virale formée ou
virion. On parle alors de protéines dites non structurales principalement des enzymes.
Ensuite l’ADN se réplique souvent au niveau du noyau ; et enfin une transcription tardive aboutit à la
constitution de protéines dites structurales dite encore protéine virioniques qui sont incorporées dans
l’édifice virale définitive. Ce schéma de reproduction virale est celui des Herpesviridae et des
Adenoviridae

2/ Pour les virus à ARN, le processus diffère selon la nature de l’ARN génomique.
S’il a une structure d’ARNm, il est immédiatement traduit en protéine virale structurale. Il est dit de
polarité positive ; comme les Poliovirus
Dans le cas contraire, il ne peut être lu directement par les ribosomes cellulaires et doit être transcrit
au préalable en ARN complémentaire qui jouera le rôle d’ARNm : il est alors de polarité négative,
tels les Rhabdovirus dont le virus de la rage

3/ Pour les Retroviridae , des virus a ARN ( dont les VIH) ainsi que pour les Hepadnaviridae des
virus a ADN ( dont le virus de l’hépatite B) la réplication commence par une retrotranscription grâce
a une enzyme la transcriptase inverse ou retrotranscriptase qui est une polymerase. Ainsi l’ARN
génomique des VIH est transcrit en ADN proviral qui va s’intégrer au génome des cellules parasitées,
grâce cette fois a une endonuclease intégrase. Ces différents enzymes de la réplication des VIH sont
codes par le virus lui-même (gène POL)

Fig 19: Retrotranscriptase d’un VIH

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e) L’assemblage

C’est un auto assemblage à partir de composants fabriqué séparément, successivement ou


simultanément. L’assemblage de la capside se fait à partir des protéines capsidales ; son association
avec l’acide nucléique forme la nucléocapside. Les virus à peplos tire leur enveloppe des membranes
cellulaires cytoplasmiques ou nucléaires qui sont modifiés par l’adjonction de protéines virales, tel les
glycoprotéines sur la face extérieure ou interne de cette membrane.
Ex Chez les VIH1 ; la gp 120 est présente à la face externe de l’enveloppe virale et la gp 41 est en
position transmembranaire.

Fig.20 : virus a ARN enveloppe

Tableau 2 : Résume des réplication des ADN et ARN

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Fig 21 : formation des antigènes de structures et non structuraux des poliovirus

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f) La libération

Elle peut revêtir 2 aspects :

i. Celle des virus nu : se fait par éclatement de cellule hôte


ii. Celle de virus à peplos : se fait par bourgeonnement à travers les
membranes cellulaires.

La nucléocapside détermine alors au sein de ces membranes une invagination qui s’isole ensuite
dans le milieu extérieur permettant ainsi l’acquisition d’une enveloppe.

Remarque :
- La localisation nucléaire ou cytoplasmique du site de la reproduction virale est un facteur de
diversité supplémentaire. C’est ainsi que les Herpèsviridae se répliquent au niveau du noyau de la
cellule alors que les rétrovirus dont les VIH le font au niveau du cytoplasme cellulaire.
- L’agencement des protéines virales synthétisées dans le temps et dans l’espace suppose un contrôle
précis de l’expression des gènes viraux grâce à des protéines de régulation codées par le virus lui-
même. Ainsi le génome des VIH ont 5 gènes de régulation : Tat, Rev, Vif, Nef et Vpr (vih 2) ou
Vpu (vih 1). De plus les régions non codantes LTR joueraient des rôles d’initiation de la réplication

Fig 22 : Génome viral d’un VIH

§3. INTERACTION ENTRE LE VIRUS ET LA CELLULE HÔTE.

L’expression des gènes viraux peut occasionner diverses modifications qui définissent les
interactions entre le virus et la cellule hôte.
Ces interactions sont ainsi résumées :

-a) Le cycle abortif : celui-ci n’aboutit pas à la production de virus. C’est le type d’interaction entre
un virus et une cellule non permissive ne présentant aucun récepteur a ce virus

-b) Le cycle lytique : il aboutit à l’apparition des effets cytopathiques ou E.C.P et à la mort
cellulaire. L’ECP est défini par des modifications morphologiques des cellules infectées directement
visibles au microscope optique sur des cultures in vitro des virus. Ainsi, comme exemple d’E.C.P :
la nappe cellulaire est détruite (aboutissant à une masse discontinue),les cellules sont ballonisées
ou rétractées et réfringente (aspect entre le brillant et le mat est augmentée)

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An illustrated tutorial.

Laboratory diagnosis of viral infections frequently requires the isolation of the virus in cell cultures.
Cell monolayers are inoculated with a suitable clinical specimen, and then observed for cytological
changes that indicate virus growth.

Cytopathic changes

The term "cytopathic effect" (CPE) is frequently applied to virus-induced cellular changes that are
visible by light microscopy. These changes include swelling or shrinkage of cells, the formation of
multinucleated giant cells (syncytia), and the production of "inclusions" (made visible by staining) in
the nucleus or cytoplasm of the infected cell.

The most efficient way to demonstrate cellular changes is by staining with chromatic dyes.
Cell monolayers are fixed and then exposed to basic and acidic dyes that accentuate the nature and
location of the changes.

The use of haemotoxylin (basic dye) and eosin (acidic dye) is often referred to as
H&E staining.

The gross appearance of the cellular changes, and the location and nature of the "inclusions" - i.e.
basophilic or eosinophilic - can in many instances be used as a diagnostic criterion to identify the
causative virus. These will be illustrated for some of the viruses commonly isolated in cell culture:-

• Adenovirus - example of basophilic intranuclear inclusions


• Reovirus - example of eosinophilic intra-cytoplasmic inclusions

Adenovirus

Cells infected with adenovirus have an affinity for


haematoxylin (a purplish-blue dye).
Infected cells become rounded and the cell sheet

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disintegrates. Dark basophilic inclusions within the nuclei represent


accumulated viral proteins at the site of virus assembly.

Reovirus
Replication of reovirus particles occurs in the cytoplasm of the cell, and in the final stages of
assembly the virus particles bud through the endoplasmic reticulum membrane.
Cytoplasmic sites of accumulated viral protein are stained with eosin (deep pink).

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Par ailleurs chez certains virus, il y a fusion des cellules avoisinantes et formation de nappe
cytoplasmique contenant de nombreux noyaux ou syncytium.

Syncitia de cellules infectees par le VIH

Syncytia

Many enveloped viruses possess a fusion


protein in their envelopes. This confers the
ability of the virion to fuse with the host cell
membrane and thus allow entry of the
infectious genomic material into the cell
cytoplasm. During replication of the virus,
expression of the fusion protein at the cell
membrane can result in the fusion of
neighbouring cells, and the formation of multi-
nucleate cells or syncytia.

Measles virus

Very large syncytia can be formed during replication of measles virus in cell culture.
An additional distinguishing feature of measles is the presence of distinct eosinophilic inclusions in
the nuclei of infected cells.

In the syncytium shown on the right, multiple nuclei are clustered around an eosinophilic cytoplasmic
mass that probably represents the Golgi compartments of the fused cells.

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Intra-nuclear inclusions are clearly visible.

Respiratory syncytial virus


As its name implies, respiratory syncytial virus (RSV)
causes large syncytia.
Nuclei do not contain inclusions, but pale eosonophilic inclusions can often be seen in the cytoplasm
(see below).

Mumps virus
Mumps virus CPE is indistinguishable from that of RSV (shown above).
However, mumps virus encodes a haemagglutin protein which is incorporated in the virus envelope,
and appears at the cell surface from which progeny virions will bud. If erythrocytes (red blood cells)
are added to infected cell sheets, they will adhere to the cell surface.
This process is termed haemadsorption
and allows differentiation between mumps and RSV infections.

Haemadsorption of erythrocytes on the surface of cells infected with mumps virus

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Fig 23 ECP sur fibrobasters infectes par du Poliovirus

-c) Une transformation cellulaire : elle se fait par intégration du génome virale dans le génome
cellulaire. Dans ce cas, l’expression de certains gènes viraux ne provoque pas la mort cellulaire,
mais au contraire donne à celle-ci de nouveaux caractères en particuliers des caractères propres aux
cellules malignes. Tels :
- le virus d’Epstein Barr (VEB) qui induit la transformation des lymphocytes B infectés en
lymphomes : c’est une transformation cancéreuse,
- L’HTLV 1, pour Human T Lymphotropic Virus type 1, est un rétrovirus responsable de
leucémies et sarcomes a lymphocytes T de l’adulte dans des zones géographiques particulières
(Caraïbes, Japon, Afrique tropicale)
- Le HPV 16, 18 et 31, des serotypes de Papillomavirus humains sont associes au cancer
du col utérin dont un vaccin est disponible depuis peu.
- Le HHV 8 ou 8eme herpes virus humain est associe a la maladie de Kaposi, un
lymphome diffus retrouve chez les sideens
- Les VIH qui induisent un lymphome cerebral

-d) L’apparition d’infection virale latente : on a mis ainsi en évidence de nombreux virus vivant à
l’état latent dans les cellules hôtes. Ces virus sont susceptibles de se réactiver soit lors de la mise en
culture in vitro ou in vivo sous forme d’infection chronique suraiguës.
- Ex VHB virus de l’hépatite B : Il existe des porteurs sains ou chroniques de l’Ag de
surface du VHB.
Ces porteurs peuvent être considérés comme étant atteint d’une hépatite chronique persistante ou
aggréssive ; cette dernière évoluant vers la cirrhose et le cancer primitif du foie (CPF).
- la varicelle est la primo infection du VZV ou virus du zona et de la varicelle. Le zona est
une réactivation de la forme latente du VZV présent dans les ganglions sensitifs après la primo-
infection patente ou asymptomatique.

§4. La variabilité des génomes viraux

Certains virus en particuliers des virus à ARN sont doués d’une variabilité génétique. Une
des causes principales de cette variabilité génétique est le taux d’erreurs dans la réplication du
génome virale. En effet, la réplication de l’ARN génomique des Rétrovirus par les polymérases
transcriptase inverse ou RT ne présente pas de mécanismes de correction d’erreur ; a l’inverse des
polymerase ADN dépendante de réplication des ADN génomiques
Ce taux d’erreurs est considérablement plus élevé chez les virus à ARN par rapport aux virus
à ADN. Ces erreurs aboutissent à la production de nouveaux virus mutants. Ces mutations ne
concernent pas tout le génome viral ; mais des régions de certains gènes dits « hypervariables » tels la
boucle V3 du gène ENV qui code pour l’enveloppe viral du VIH.

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Ainsi chez les patients atteint autant par les VIH que par le VHC (Virus de l’hépatite C) la
production virale est formée de nombreux variants ou virus mutant différents. Cet ensemble de virus
constitue une « quasi-espèce », un concept nouveau applicable au virus présentant une variabilité
génétique.

SECTION 3 : POUVOIR INFECTIEUX VIRAL, MECANISMES DE DEFENSE,


EPIDEMIOLOGIE DES INFECTIONS VIRALES.

Ojectifs :
> comprendre la notion de pouvoir infectieux viral
> connaître les différents modes de transmission et d’implantation de l’infection virale
> préciser les étiologies des infections virales du fœtus et du nouveau-né
> déterminer les virus responsables des principales infections virales dans l’enfance
> connaître les virus responsables des gastro-entérites et des hépatites
> préciser les étiologies virales des infections du tractus respiratoire
> présenter les virus qui affectent le SNC
> définir les mécanismes de défenses de l’organisme contre l’infection virale
> décrire les moyens de défense non spécifique
> comprendre les mécanismes de défenses spécifiques de type humoral
> comprendre les moyens de défense spécifiques a médiation cellulaire
> comprendre les mécanismes d’échappement aux défenses immunitaires
> étudier l’épidémiologie des infections virales
> préciser les modes epidemiologiques
> comprendre la notion du réservoir des virus
> préciser les voies de pénétration et d’excrétion des virus
> connaître les facteurs favorisant l’infection virale

§1. POUVOIR INFECTIEUX VIRAL

a) contage et pouvoir infectieux viral

L’infection virale se manifeste de façon diverse. Selon les cas, les cellules infectées sont
détruites par le développement des effets cytopathiques caractéristiques de l’infection lytique ;
ou elles supportent une infection tempérée ou latente ; ou encore elles se transforment ou
acquièrent de nouveaux caractères.
En ne considérant que les affections aiguës, on observe déjà une grande variété dans les
expressions cliniques, qui vont de l’infection inapparente aux formes mortelles.
De plus, l’expression clinique peut être modulé par divers facteurs tels que :
- par la souche virale : un nouveau sous type ou nouveau variant provoque une épidémie ;
- par l’exposition antérieure à un virus apparenté : une infection antérieure à Herpès
simplex virus type 1 protège contre une nouvelle infection par l’Herpès simplex virus type 2.
- par l’âge du sujet qui intervient dans divers sens : les manifestations respiratoires sont
plus marquées chez les nourrissons atteints du virus respiratoire syncytial ; les expressions cliniques
du virus de l’hépatite A ou VHA sont accrus chez l’adulte.

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- Par l’état d’immunodépression qui peut majorer les signes cliniques d’une infection
virale, d’une infection à CMV(cytomégalovirus) notamment.

Dans l’organisme, l’infection virale progresse différemment selon les virus et même pour un
virus donné selon les individus. Toutefois, elle commence par investir une porte d’entrée
représentant un site de multiplication primaire du virus.
Comme c’est généralement par inhalation ou ingestion que l’on se contamine, les muqueuses
digestives et pulmonaires sont les sites de multiplication primaire de la grande majorité des virus.
Plus rarement le contage résulte d’une effraction cutanée ; c’est le fait d’une morsure (virus
rabique ou virus de la rage), ou par une piqûre d’arthropodes pour les Arbovirus (Arthropode-
Bore-Virus).
La transmission sexuelle est de règle pour l’herpès simplex virus de type 2(HSV2) ainsi que pour
les VIH. Elle est secondaire pour les virus des hépatites B et C .

On divise les infections virales en viroses localisées et généralisées.


Dans les infections virales localisées, la porte d’entrée et l’organe cible sont confondus ; d’où une
incubation courte (2-3jours) caractérisant les viroses localisées comme la grippe.
Il en est autrement des viroses généralisé ou une longue période d’incubation sépare les
infections du site primaire puis de l’organe cible. Ex. le tube digestif est le site primaire du
poliovirus car c,est un enterovirus ; l’organe cible sera le système nerveux central.

Les infections virales restent extrêmement fréquentes durant l’enfance. La grande majorité
d’entre elles sont particulièrement silencieuses et infra cliniques.
Cela tient au fait que beaucoup de virus sont très peu pathogènes tel les rhinovirus responsable du
rhume. Notre vigilante immunité débarrasse notre organisme de la plupart des virus pathogènes.
Toutefois, certains, tels les poliovirus, ont un redoutable pouvoir neuropathogène.

b) principales infections virales du fœtus et du nouveau-né

- Le virus de la rubéole qui donne une infection in utero


- Le cytomégalovirus avec des infections in utero et périnatales,
- L’herpès simples 2, avec des infections périnatales,
- Le virus de la varicelle et du zona avec des infection in utero et en périnatales,
- Le parvovirus B 19 qui donne des infections in utero,
- Le VIH peut donner des infection in utero et en périnatale,
- Le VHB donne principalement une infection périnatale,
- Les Entérovirus donnent des infections périnatales

c) les principales infections virales de l’enfance

Elles sont dominées par des exanthèmes ou manifestations éruptives sur le revêtement cutané. On
distingue :

- La rougeoles causée par un virus à ARN du genre la Morbillivirus et de la famille des


Paramyxoviridae,
- La rubéole causée par un virus à ARN appartient au genre Rubivirus et à la famille des
Togaviridae,
- Le mégalérythème épidémique ou < 5ème maladie éruptive<, causées par un virus à ADN
du genre Parvovirus B19 de la famille des Parvoviridae.
- L’exanthème subit ou « roséole » du à un virus à ADN, l’Herpès virus humain 6, ou
(HHV6), un virus à ADN du genre Cytomégalovirus de la famille des Herpèsviridae.
- La varicelle qui est une maladie essentiellement bénigne avec de rares complications
chez les enfants et nouveau-nés immunodéprimés. Elle est causée par le virus de la Varicelle et du

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Zona ou VZV ou HHV 3, un virus à ADN, qui appartient au genre Varicellovirus et à la famille des
Herpesviridae Ce virus peut donner une méningite chez les enfants immunodéprimés….
- Le virus des oreillons responsable d’une parotidite, maladie bénigne de l’enfance. Ce
virus, à ARN, appartient à la famille des Paramyxoviridae. Il peut être à l’origine de manifestations
neurologiques type méningites voire méningo encéphalites.
-
d) Les virus responsables d’infections du tractus respiratoire

Ces virus dits respiratoires ont un tropisme presque exclusif pour les cellules ciliées des muqueuses
respiratoires :
-les Virus influenza responsables de la grippe principalement le virus influenza A
-les Virus para-influenza,
-le Virus respiratoire syncytial (V.R.S)
-les Adénovirus respiratoires,
-les Rhinovirus,
-les Coronavirus.
-les Metapneumovirus humain
Suivant les virus, ils provoquent des syndromes respiratoires différenciés en rhumes, laryngites,
trachéites, bronchites, bronchiolites, pneumonies ou grippes.

Chaque syndrome clinique respiratoire viral est en général caractéristique d’un groupe de
virus respiratoire ; par exemple le rhume avec les rhino virus, les laryngites avec les para influenza
virus, les bronchiolites avec le virus respiratoire syncitial et la grippe avec les influenza virus.
Mais il n’existe pas réellement de spécificité étiologique. Plusieurs virus différents peuvent être
responsable du même syndrome clinique.

e) les principales infections virales digestives

Elles sont représentées par les hépatites virales et les gastro-entérites virales. Les hépatites
virales sont dues aux cinq « virus des hépatites » A, B et D, C, E et aux « nouveaux virus des
hépatites »GB-A, GB-B, GB-C/VH-G et TTV. Tous ces virus appartiennent à des familles virales
différentes.

Virus Famille Génome Contamination

VHA Picornaviridae ARN Entérale ou oro-fecal


Heparnavirus
VHB Hepadnavirus ADN Parentérale, mère-
enfant,
sexuelle
VHC Flaviviridae ARN Parentérale

VHE Calciviridae ARN Entérale ou oro-fecal

GB-A Flaviviridae ARN Parentérale


GB-B
GB-C/VHG
TTV Parvovirus ADN Parentérale

Les diarrhées aiguës infectieuses représentent une des maladies les plus fréquentes partout
dans le monde et touchent essentiellement le nourrisson et le jeune enfant. Durant l’année 1977-1978,
une étude recensait 3 à 5 millions de cas de diarrhées en Afrique, Asie et Amérique. Si l’étiologie

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bactérienne était connue de longue date, le rôle important joué par certains virus est de découverte
relativement récente.
Dès les années 1970, l’introduction de la microscopie électronique dans le diagnostic
virologique a permis la visualisation de nombreux agents viraux dans les selles des patients atteints de
gastro-entérites aiguës.
On y trouve ainsi :
-des Rotavirus
-des Astrovirus
-des Adénovirus entériques serotypes 39, 40
-l’agent de Norwack
-des Coronavirus

Tous ces virus associés aux gastro-entérites aiguës, ne se retrouvent pas avec une même
fréquence. Les Rotavirus et les nouveaux Adénovirus entériques sont très fréquemment retrouvés
aux cours des gastro-entérites des nourrissons. Les autres virus intéressent l’enfant et l’adulte et sont
plus rarement isolés. Actuellement un vaccin a été mis au point contre les rotavirus

f) les infections virales du système nerveux central

Elles sont dominés par les syndromes cliniques aiguës du S.N.C : tels les méningites aseptiques
causées par les Poliovirus agents de la PAA (Poliomyélite aiguë antérieure) ou le virus de la rage
humaine et animale.
Quatre maladies chroniques et dégénératives du S.N.C sont liées à des virus lents conventionnels.
Ce sont :
1) la pan-encéphalite subaiguë sclérosante (PESS) due au virus de la rougeole un
Paramyovirus,
2) la leucoencéphalite multiforme progressive (L.M.P) due au virus J.C, un Papovavirus
3) la paralysie spastique tropicale (P.S.T) due au virus humain de la leucémie à lymphocyte T
type 1 (HTLV1) ;
4) la démence associée au syndrome d’immunodéficience acquise ou SIDA due aux virus de
l’immunodéficience humaine de type 1 et 2(VIH 1 et 2).

A ces pathologies chroniques nous pouvons ajouter les maladies de CREUTZFELD-JACOB


(M.C.J) et ces dérivés chez l’homme et l’animal qui sont des encéphalopathies subaiguë
spongiformes transmissibles ou ESST (dont la maladie de la vache folle).
Ces maladies toujours mortelles à évolution très lente sont transmises par des agents transmissibles
non conventionnels (ATNC) ou Prions.

§2. LES MECANISMES DE DEFENSE DE L’ORGANISME


C’est le rôle de l’organisme que d’empêcher au maximum la pénétration virale et la diffusion
des virions. Lors de la pénétration virale, selon le lieu de sa pénétration, sa situation extra ou
intracellulaire dans l’organisme, certains mécanismes de défense vont être privilégiés. L’organisme
va utiliser plusieurs mécanismes de défense, les un spécifiques, les autres non spécifiques.
Les mécanismes que l’organisme mettra en œuvre pour lutter contre l’infection virale devront
non seulement neutraliser les particules virales mais également détruire les cellules infectées.
Les effecteurs de l’immunité spécifique et non spécifique se présentent sous forme de cellules
(essentiellement des leucocytes) et de molécules (anticorps, interférons, complément etc.) ; ils sont
répartis dans tout l’organisme.
Les lymphocytes sont capables de détruire directement les agents infectieux par exemple
grâce aux lymphocytes T cytotoxiques CD8 + ; ou indirectement par des molécules qu’ils
synthétisent tels les anticorps produits par des lymphocytes B activés et différenciés en plasmocytes
sécréteurs d’immunoglobulines ou anticorps.
On oppose en général les moyens de défense spécifique, principalement dû aux lymphocytes,
aux moyens de défense non spécifiques. On peut aussi parler de moyens de défense de « première
ligne », prenant en charge les virus lors de la pénétration dans l’organisme par des cellules

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phagocytaires. Et des moyens de défense de « deuxième ligne », à l’intérieur de l’organisme, qui


font appel plus particulièrement à l’immunité spécifique humorale et/ou cellulaire. Cette immunité
spécifique établira une mémoire immunitaire qui reconnaîtra une réinfection par le même virus

a) les moyens de défense non spécifiques

Les moyens de défenses non spécifiques interviennent en premier.


L’immunité innée ou naturelle préexiste à l’infection, et ne nécessite pas d’immunisation
préalable. Elle intervient dans les heures voire les minutes après l’infection. Elle est non spécifique,
large, distinguant le soi du non soi ( self from non self) étant dirige contre le non soi. Les virus, étant
constitues d’une mosaique d’antigènes, sont perçus comme étrangers à l’organisme. L’immunité
innée met en jeu de nombreux acteurs, (cytokines , cellules sentinelles, cellules NK) aux actions
diverses et enchevêtrées :
- actions proprement antivirales
- potentialisation mutuelle de ces éléments de la défense naturelle
- préparation de la ligne de défense suivante constituée de l’immunité acquise,
spécifique .

L’infection se heurte d’emblée à des défenses mécaniques comme la peau intacte, le tapis
mucociliaire de l’arbre respiratoire, ou l’acidité gastrique.
Des inhibiteurs non spécifiques préexistent à l’infection dans les secrétions muqueuses et le
sang. Ce sont des macrolécules présentant des analogies structurales avec les récepteurs cellulaires
membranaires du virus. Grâce à cette analogie, ces macromolécules bloquent sur la surface virale les
structures permettent l’attachement.

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Dans la défense non spécifique de l’organisme :

1/ les macrophages et les cellules dendritiques jouent un rôle essentiel ; qu’ils s’agissent de ceux
fixés, présents dans les organes (rate et foie), ou ceux libres présents dans les alvéoles pulmonaires et
le sang. (monocytes-macrophages) Ces cellules sentinelles produisent des IFN et autres cytokines.
Elles participent à la mise en place de l’immunité acquise. Ces macrophages interviennent en
premières lignes. Ils phagocytent et digèrent les particules virales grâces aux enzymes de leurs
lysozymes. Ils jouent aussi le rôle, combien important, de cellules présentatrices d’antigènes au
système de défense spécifique. En effet, ils internalisent ( phagocytent) et apprêtent ( processing) les
antigènes viraux avec lesquels ils migrent dans les ganglions lymphatiques, pour les présenter aux
lymphocytes T et B, cellules effectrices de l’immunité spécifique acquise. La grande sensibilité des
nouveau-nés aux infections virales s’explique en partie par l’immaturité de leurs macrophages.

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2/ Les interférons (INF) participent lors de l’infection virale, aux mécanismes de défense non
spécifique. Les IFN-alpha et bêta sont produits essentiellement (mais pas exclusivement) par des
leucocytes et des fibroblastes, sous l’effet de l’infection virale ou de l’administration d’inducteurs
artificiels. Ces interférons inhibent les multiplications virales dans les cellules et protègent les cellules
voisines, sans en altérer leur fonctionnement. Ils interviennent dans les premières heures de
l’infection.
Ils ont une spécificité d’espèce ; seuls les interférons humaines protègent l’homme. En revanche ils
n’ont pas de spécificité de virus et leur spectre apparaît très large. Tous les virus sont inducteurs et
sensibles aux interférons. A titre indicatif, le traitement par interféron alpha a une activité
partiellement bien démontrée dans les hépatites B et C.

3/ les NKC( Natural Killer Cells) Elles ont une activité antivirale directe. Elles reconnaissent les
cellules infectées comme étant anormales et les lysent. ; tout comme elles lysent les cellules
cancéreuses.

4/ le Complément. Constitué de protéines plasmatiques qui agissent en cascades, le système du


complément joue un rôle dans l’immunité anti- virale. Il est activé entre autre par la formation de
complexes antigènes anticorps ou virus anticorps. Une fois activités, les molécules de système
induisent la formation de complexes protéiques doués d’une activités lytique vis à vis des virus
enveloppes ou de cellules infectées.

Pour aboutir :
a) à la lyse des cellules infectées qui présentent des antigènes viraux à leur surface. C’est la
cytolyse
b) à l’inactivation des virus enveloppés ou virolyse
Un des composant intermédiaire du complément appelé C3b, peut également s’incorporer à
certains complexes virus anticorps. Il favorise alors la phagocytose des complexes virus anticorps.

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Fig 24 le complement

5/ la fièvre est un autre moyen de défense de première ligne. Elle est induite par des cytokines de
l’inflammation. Au fur et a mesure que la t augmente la multiplication virale diminue
b) mécanismes de défense spécifiques
Les cellules effectrices sont pour l’essentiel les lymphocytes B (aboutissant a la
formation d’anticorps) et les lymphocytes T cytotoxiques CD8 + (aboutissant a la lyse des cellules
infectées et appelées des lors CTL pour cytotoxic T cells des anglo-saxons) . Chaque lymphocyte
cible un antigène particulier ; d’ou la spécificité. Des récepteurs pour chaque antigène sont codes
par notre organisme et sont formes d’anticorps membranairesou BCR, B Cells Receptor pour les
lymphocytes B et de TCR , T Cells Receptor , pour les lymphocytes T

Les lymphocytes T CD4+ sont en position centrale, les chefs d’orchestre de l’immunité
acquise spécifique. Une fois informes, par les CPA ou cellules présentatrices d’antigènes
(macrophages et/ou cellules dendritiques), les lymphocytes T CD4 + ou auxiliaires , helper (Th)
vont favoriser, par secrétions de cytokines, d’une part l’évolution des lymphocytes B en plasmocytes
producteurs d’anticorps circulants, et d’autre part l’évolution des lymphocytes CD8+ en CTL.
Les cytokines sont des protéines produites par les cellules de l’immunité qui permettent la
communication intercellulaire. Les cytokines régulent la réponse immunitaire. Elles interviennent
dans le métabolisme, la différenciation et la mobilité cellulaires

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1/ réactions immunitaires de types humorales avec productions d’anticorps


Elles sont caractérises, dans l’infection virale, par l’élaboration d’anticorps ou
immunoglobulines spécifiques au virus infectants, dirigés contre les antigènes capsidiques et/ou
membranaires de ces virus.
Ces anticorps anti-viraux sont produits par des lymphocytes B activés en plasmocytes sécréteurs
d’immunoglobulines ou anticorps, qui sont des globulines, des protéines apparaissant dans le sérum,
7 à 15 jours après l’introduction du virus dans l’organisme. On dit alors qu’il y a séroconversion.
Le titre des anticorps va atteindre, en 2 ou 3 semaines, un taux maximal. Ces anticorps
persistent, dans le sérum, plus ou moins longtemps à un taux variable. De façon séquentielle, à la
séroconversion, c’est les IgM qui apparaissent en premier lieu. Cet iso type ou classe
d’immunoglobulines est prédominant à la primo-infection. Les IgM vont rapidement disparaître et
laisser la place aux IgG et /ou IgA (lors de l’infections des muqueuses). Ces derniers signent la
convalescence avec acquisition d’une immunité ; ou la réussite d’une vaccination et l’acquisition
d’une immunité artificielle mais aussi efficace que la naturelle
Dès lors une stimulation antigénique primaire entraîne une réponse immunitaire primaire avec
production d’ IgM : c’est la primo infection. Lors d’une réinfection, lors d’une stimulation
antigénique secondaire, la réponse secondaire immunitaire se caractérise par la production
d’anticorps qui :
a. augmentent plus rapidement en concentration et persistent plus longtemps
b. atteignent des taux plus élevés
c. sont constitués essentiellement d’IgG (immunoglobuline de type gamma

DECLIN
LAG LOG PLAT
E
EAU

Total IgM IgG


AbAg Ab Ab
A

R
T

T
E
b

Days After Immunization


1 o
2o
TE

Ag Ag
Ab

R
T
I

38

Days After Immunization


39

Anticorps spécifique a l’antigène

Certains de ces anticorps suppriment ou réduisent le pouvoir infectieux des préparations


virales in vitro ; de même que le pouvoir infectieux viral chez l’animal d‘expérience. Ces anticorps
sont neutralisants ou protecteurs. Comme des anticorps anti V.H.A (Virus de l’hépatite A), ou des
anticorps anti-virus de la rougeole. Leur présence démontre l’installation d’une immunité naturelle
avec guérison ou l’acquisition d’une immunité humorale artificielle par la vaccination. Ces anticorps
protègent contre une infection ultérieure par les virus V.H.A ou de la rougeole.
L’administration par voie passive d’anticorps spécifiques ou immunothérapie protège les
individus contre de nombreuses infections virales (hépatite A et B, rougeole et la varicelle). Ce rôle
est dévolu aux anticorps neutralisants. Ce rôle préventif des anticorps anti-viraux apparaît comme
primordial.
Cependant, tous les anticorps ne sont pas toujours neutralisants ou protecteurs. Leur détection
témoigne alors, uniquement, de la séroconversion, de l’apparition d’anticorps spécifiques dans
le sérum des patients infectés.
C’est le cas de la séropositivité aux V.I.H. En effet, suite à l’infection aux V.I.H, et lors de la
séroconversion, des anticorps anti V.I.H sont détectés dans le sérum des patients infectés. Ceux-ci
sont donc séropositifs aux V.I.H c-a-d ont été infecte et produisent des anticorps. Mais l’infection
aux VIH continue à se développer malgré la présence des ces anticorps Ces anticorps anti-V.I.H
sont inefficaces, non neutralisants.
Par ailleurs certains virus, pourvu d’une variété génétique (virus grippal, les V.I.H, le
VHC ou virus de l’hépatite C), peuvent échapper à une immunité humorale établie, en changeant
d’épitopes ou déterminants antigéniques (Ce sont des détails structuraux immunodominants des
antigènes viraux). Ces mutations, caractéristiques des virus influenza, agents de la grippe, expliquent
la répétition des épidémies grippales et la nécessité de mettre au point de nouveaux vaccins

2/ mécanismes de défenses spécifiques à médiation cellulaire ou immunité cellulaire

Classiquement, l’immunité cellulaire joue un rôle important dans l’immunité spécifique


contre les virus. En particulier par l’action des lymphocytes T CD8, cytotoxiques qui
reconnaissent les cellules infectées par des virus (car ells expriment en surface des antigènes viraux)
et les détruisent

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Les lymphocytes auxilliaires ou helper CD4 jouent un rôle central dans l’immunité
en activant tous les effecteurs de l’immunité innée ( macrophages), de l’immunité adaptative
spécifique( lymphocytes B et T cytotoxiques CD8).Ce sont les régulateurs de l’immunité.

3) conclusions

En général, ces mécanismes de défense,s non spécifiques et spécifiques, s’avèrent efficaces et


complémentaires pour prévenir l’infection ou éliminer les virus de l’organisme infecté. Les cibles des
défenses antivirales sont :
= pour les anticorps : les virions ou virus extracellulaires
= pour les cellule NK et CTL : les antigènes viraux en surface des cellules infectées
= pour les IFN ou interférons : les ARN messagers viraux des cellules a protéger.

Fig 27 action des lymphocytes cytotoxique CD 8+

D’où l’intérêt de vacciner contre l’infection virale.


Les vaccinations contre les maladies virales sont des moyens privilégiés de lutte
préventive contre les virus. Le PEV (Programme Elargie de Vaccination) cible la vaccination contre
les affections suivantes :
la Diphtérie, la Rougeole, le Poliovirus, le Tétanos et la Tuberculose
La vaccination contre l’hépatite B et l’Heamophylus influenzae type b viennent d’être introduites dans
les PVD ( Pays en Voie de Developpement)
Le PEV couvre dès lors trois maladies virales (polio, hépatite B, rougeole). Rappelons que la variole
fut la première maladie infectieuse a été éradiquée, grâce à la vaccination antivariolique, première
vaccination (découverte par JENNER) contre un microorganisme. Cette éradication a été proclamée
par l’OMS en 1980.

4) mécanismes d’échappement aux défenses immunitaires

Les virus ont évolué en développant des mécanismes d'échappement aux défenses immunitaires.
Entre autre le camouflage et le sabotage.

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Il doit en effet y avoir un modus vivendi entre virus et hôte car la « destruction mutuelle assurée »
n'est pas une option viable en matière d'évolution biologique. L'hôte a des mécanismes de défense
contre les virus mais, en revanche, les virus ont des mécanismes d'échappement aux défenses de
l'hôte.

= le camouflage

Le camouflage des virus consiste en deux mécanismes.

• La modification des épitopes de neutralisation ou de cytolyse par les lymphocytes T (CTL),


cela par mutations. S'y prêtent particulièrement les virus à ARN, comme les virus de la
grippe et le virus de l'hépatite C, car l'ARN polymérase ARN-dépendante qui réplique le
génome n'a pas de mécanisme de lecture et de correction des erreurs, d'où la facilité des
mutations. S'y prête également le VIH, dont la rétrotranscriptase (RT, ADN polymérase
ARN-dépendante) manque également d'un mécanisme de correction d'erreur.
• L'infection latente. Bien des virus en sont capables. C'est le cas, notamment, des
Herpesviridae, des Polyomavirus, des Papillomavirus, du Virus de l'hépatite B, des
Rétrovirus. Après la primo-infection, le génome viral persiste dans la cellule avec, dans
certains cas, intégration dans le génome cellulaire, mais il ne s'exprime pas, ou n'exprime
qu'une partie de son information génétique. Ainsi, il ne produit pas d'antigène et échappe
donc aux défenses immunitaires ; de même, il ne se multiplie pas et échappe donc aux
antiviraux qui sont essentiellement des inhibiteurs de la multiplication virale. Donc, le virus
en phase de latence « survit en faisant le mort », et il est difficile ou impossible de le
déloger.

= le sabotage

Le sabotage des mécanismes de défense de l'hôte peut être brutal comme dans le cas de l'HIV
détruisant les lymphocytes T CD4+, dont le rôle de chef d’orchestre de l’immunité est établi. Ailleurs,
plus subtil, il repose sur la production de protéines virales altérant ou bloquant les différents
mécanismes de défense. C'est le fait des plus gros virus donc des gros virus à ADN (Poxvirus,
Adénovirus, Herpèsvirus) qui sont suffisamment riches en gènes pour, outre se faire reproduire par la
cellule, en consacre à la production de protéines virales capables de remanier la cellule : il s'agit en
particulier de protéines capables d'antagoniser les interférons et autres cytokines antivirales ou le
complément, de perturber la présentation des antigènes viraux, de détruire ou bloquer l'expression du
CMH-I, d'inhiber l'apoptose, etc. Les protéines virales exécutant ces remaniements sont, pour une
part, des homologues de protéines cellulaires de notre système de défenses antivirales, jouant ainsi le
rôle de leurres. Elles viennent sans doute du « piratage de gènes cellulaires ».
Ainsi on parle de virokines, analogues de cytokines cellulaires, de virorécepteurs, analogues des
récepteurs de virokines cellulaires.
Il y a donc adaptation réciproque et co-évolution des virus et des systèmes de défense de l'hôte.

§3. EPIDEMIOLOGIE DES MALADIES VIRALES

a) modes épidémiques

Le développement d’une infection virale au sein d’une population donnée peut s’accomplir
suivant plusieurs modes épidémiques.

a1. Mode sporadique

La maladie survient à intervalles irréguliers par cas isolés sans lien apparent entre eux.

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a2. Mode endémique

La maladie est constamment présente dans une population donnée affectant un petit nombre
de sujet qui n’ont entre eux aucun lien apparent de contagiosité. Comme pour le virus de l’hépatite B
avec 9,75% de porteur de l’Ag HBs (antigènes de surface) chez les donneurs de sang au CHUK.
(Centre Hospitalo-Universitaire de Kamenge).

a3. Mode épidémique

Pendant une période limitée, en même temps ou successivement, la virose frappe plusieurs
personnes. Quand le nombre de personnes est petit, on parle de foyer épidémiques scolaires ou
familiaux d’épidémie de grippe.

a4. Mode pandémique

Le pouvoir extensif de l’épidémie lui permet de franchir les frontières et de se répandre dans
divers pays et continents ; ex. la pandémie du VIH, apparue au courant de la décennie 80-90.

b) réservoir de virus

C’est un organisme dans lequel s’accomplit le cycle complet du développement viral. Le


réservoir de virus peut être humain ou animal mais un grand nombre de virus pathogènes sont
spécifique de l’espèce humaine.
Les enfants se révèlent être le plus fréquemment des réservoirs de virus. Leur contamination
s’opère généralement au moment de leur incorporation dans une communauté, crèche ou maternelle.
Ils sont souvent contaminés par leurs frères et sœurs.

Il existe des maladies virales communes à l’homme et à l’animal : ce sont les


anthropozoonoses, telles les encéphalites, la rage et la fièvre jaune. Les animaux en cause sont des
animaux domestiques (chevaux dans les cas des encéphalites, chien dans la rage) ou des animaux
sauvages (singe dans la fièvre jaune et le renard dans la rage sylvestre). Ces animaux contaminés
constituent « les réservoirs de virus » des anthropozoonoses.

Les arthropodes après un repas de sang virulent, peuvent soit immédiatement ou après une
période d’incubation, devenir capable de contaminer par une nouvelle piqûre un sujet sain. Cette
contamination est propre aux Arbovirus (Arthropode-Bore-Virus) dont les arthropodes vecteurs
forment leur réservoir des virus.

c) voies de pénétration des virus

Les voies principales de pénétration des virus sont par inhalation et ingestion. On peut encore
se contaminer par effraction cutanée, relation sexuelle, transfusion sanguine non contrôlée,
scarification, piqûre par aiguille non stérile contaminée, piqûre d’arthropodes vecteurs, contact de
peau et des muqueuses infectées, par voie transplacentaire, ainsi que dans la filière urogénitale lors de
la naissance.

d) voies d’excrétions des virus

Les agents infectieux d’origine virale, quittent l’organisme par plusieurs voies :
1° avec les produits d’excrétions muqueuses des épithéliums. Ce sont les excrétions
conjonctivales pour la rougeole, rhino-pharyngées pour la grippe et bronchitiques lors d’une
pneumopathie à virus.
2° avec des produits de secrétions glandulaires : le virus des oreillons chez l’homme, le virus
de la rage chez le chien.
3° avec les excrétions intestinales telles les selles contenant des Entérovirus.

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4° avec des produits d’excrétions et d’exfoliations cutanées : sérosités des vésicules et des
pustules dans la varicelle et la variole.

e) facteurs favorisant l’infection virale

Ils sont intrinsèques comme l’âge, le sexe, la race, l’état immunitaire, le terrain réceptif
collectif. Ils seront extrinsèques lorsqu’il s’agit du mode de vie, de l’habitat, des habitudes
alimentaires et vestimentaires, de la profession, des déplacements de population et des facteurs
climatiques.
La somme des actions de ces nombreux facteurs, joint à la virulence et à la contagiosité de la
maladie, détermineront le mode épidémique de la virose.

SECTION 4.LES METHODES D’ETUDE DES VIRUS

Objectifs :
> présenter l’historique du diagnostic virologique
> décrire les pratiques de cultures cellulaires
> décrire la culture sur œuf de poulet embryonne
> expliquer l’intérêt de l’inoculation sur animal de laboratoire
> décrire l’isolement d’un virus a partir de prélèvement biologiques
> connaître les méthodes de détection des Ag et Ac spécifiques des virus : l’ELISA,
l’immunofluorescence direct et indirecte, l’hemagglutination et son inhibition, la seroneutralisation
des ECP
> comprendre l’apport de la microscopie électronique
> connaître les techniques de biologie moléculaire employées pour le diagnostic virologique : la PCR,
l’hybridation in situ, le clonage et le séquençage
> effectuer un diagnostic direct et indirect d’une infection virale

§1. INTRODUCTION

Les virus se trouvent en quantité variable dans l’échantillon biologique issu de l’organe
infecté. Leur étude sera facilitée si on peut les multiplier en culture cellulaire in vitro.
Pendant longtemps, l’étude des virus a reposée sur 2 types d’approches.
1/L’isolement des virus en culture cellulaire in vitro suivie de la détermination du sérotype.
2/Le titrage des anticorps dirigés contres les antigènes viraux pour :
- Détecter la séroconversion ou apparition des anticorps (Ac) circulants dans le sérum du
patient infecté
- Suivre la cinétique des Ac ou rechercher une élévation significative du titre de
Ac entre 2 sérums, l’un précoce, l’autre tardif distants d’une quinzaine de jours.

Depuis 1970, la microscopie électronique et les techniques d’immunofluorescence ou


d’immunoenzymologie ont progressé au point de permettre dans bien de cas, un diagnostic
virologique rapide aboutissant à l’identification du virus le jour du prélèvement ; au préalable, ces
prélèvements sont pratiqués de façon précoce car la virémie dure en règle générale peu de temps lors
de l’infection aiguë.
Ces prélèvements porteront chaque fois que cela est possible sur les lésions.

§2. PRATIQUE DES CULTURES CELLULAIRES

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Les virus ne peuvent se répliquer que dans les cellules vivantes et permissives. Dans l’étude
des virus, les cellules utilisées en culture in vitro peuvent provenir de tissus normaux ou être issu
d’une lignée continue : c à d de cellules cancéreuses.
On peut aussi cultiver le virus dans l’œuf de poulet embryonné ou encore par inoculation à un animal
de labo. sensible.

a) Culture Cellulaire in vitro

La culture cellulaire in vitro est au virus ce que la gélose ou bouillon sont à la bactérie.
Pratiquement toutes les cellules des vertébrés peuvent être cultivées in vitro. Les cultures cellulaires
s’effectuent dans des récipients de verre ou de plastique contenant des milieux de culture
standardisés défini par EAGLE. Ces milieux contiennent en quantité connue de l’eau, de sels
minéraux, glucose, AA, et des vitamines.
Des facteurs de croissance sont apportés par des sérums d’animaux (du sérum de veau) ainsi que les
antibiotiques y sont ajoutés pour prévenir des contamination bactériennes et fungiques. Tous ces
éléments permettront à la cellule de se développer normalement
Avant l’emploi, ces milieux seront stérilisés. Les cellules cultivées dans ces milieux de
cultures conservent globalement leur aspect primitif. Ainsi, les cellules d’origine fibroblastique
resteront fusiformes, celles d’origine épithéliale resteront polygonales. Ces cellules se fixent sur les
parois du récipient contenant les milieux de culture. Elles se divisent jusqu’à former un tapis
continue d’une seule couche cellulaire.
Arrivées à confluence, ces cellules cessent de se diviser par inhibition de contact.

b) Culture sur œuf de poulet embryonnée

Cette technique a été proposée par GOODPASTEUR en 1930. La culture dans l’œuf de
poulet embryonné a permis jusqu’en 1950 de nouveaux progrès en virologie.
Le virus est inoculé avec les prélèvements biologiques dans les divers liquides des membranes
annexes càd la membrane chorioallantoïdienne, l’amnios et le sac vitellin.
Après une incubation de quelques jours, la présence virale est décelée soit par la mort de
l’embryon, soit par des lésions macroscopiques sur la membrane chorioallantoïdienne, soit par la
production d’antigènes viraux dans les liquides allantoïdien ou amniotique.
Bien que largement supplanté par les cultures cellulaires, la culture sur œuf de poulet
embryonné reste le procédé utilisé pour l’isolement des virus grippaux, l’entretien des souches et la
production des antigènes et des vaccins grippaux.

c) L’inoculation sur l’animal de labo

L’inoculation du virus à l’animal de laboratoire sensible était autrefois la seule méthode de


culture de virus. Cette technique est encore utilisée actuellement tant pour la détection des virus non
cultivables que pour le suivi expérimentale de la maladie virale.
Ainsi, les VIH seront inoculés au singe vert d’Afrique, les VHB inoculé sur des canards.

d) Isolement du virus

Le diagnostic étiologique d’une infection virale repose sur l’isolement du virus à partir de
prélèvement biologique. Mais en pratique on se contente de la détection de constituants du virus ou
antigènes viraux comme celle de l’antigène de surface du VHB au Ag VHBs dans le diagnostic d’un
hépatite aïgue à VHB ; ou encore la détection des Ac anti VIH dans le dépistage de l’infection à VIH.
De fait, l’isolement de virus à partir de sécrétions et liquides biologique est une démarche
contraignante nécessitant des technologies avancées.

Schématiquement, on procède par plusieurs étapes :


- D’abord il faut prélever le plus possible au niveau des lésions …

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- Ensuite il faut garder ces prélèvements exploitables dans un milieu de transport adéquat
ou dans un sérum physiologique stérile. avant de les acheminer dans les meilleurs délais vers le labo
de virologie.

Ces labos devront disposer en permanence d’un panel de culture de cellule de différentes
origines prêtes à être ensemencées.
Ces cultures cellulaires sont réalisées à partir de cellules normales ou de cellules cancéreuses. Elles
sont contenues dans des récipients contenant du milieu de culture standardisée tel le milieu de
EAGLE ou un de des dérivés MEM(= Minimal Essential Medium).
Ces cultures cellulaires doivent être quotidiennement entretenues pour être prêtes à l’emploi. A
chaque prélèvement correspond une culture cellulaire de cellules permissives au virus recherché. Ce
sont des fibroblastes pour déceler les poliovirus, et une co-culture de lymphocytes pour la culture de
VIH.
Les prélèvements ensemencés sur les cultures cellulaires sont mis en incubation à des
températures variables à 37°C dans la recherche des poliovirus et à 22°C pour la recherche des
rhinovirus agent du rhume.
Quotidiennement, on surveille l’apparition au niveau du feuillet cellulaire des effets cytopathiques
ou ECP attendus

ECP sur des fibroblastes


Les ECP sont des modifications morphologiques des cellules, en rapport avec la multiplication virale
dans la culture cellulaire.

Au-delà de ces modifications morphologiques induites par la multiplication virale, certains


virus confèrent aux cellules qu’ils infectent des caractéristiques biochimiques et génétiques
nouvelles, telle une transformation maligne ( la cellule devient cancéreuse) ou une acquisition d’un
pouvoir hémagglutinant, pouvoir d’adsorber des hématies ou GR Par diverses techniques, on peut
détecter ces nouveaux comportements de la cellule infectée attestant de la présence d’un virus au
sein de la culture cellulaire.

Toutefois, certains virus tels les Coxsackievirus A et les virus des hépatites ne sont pas cultivables.

§3 LA DETECTION DES ANTIGENES VIRAUX ET DES ANTICORPS SPECIFIQUES

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Parmi les techniques de détection des Ac et des Ag viraux les plus utilisées sont les techniques.
ELISA et les techniques d’immunofluorescence directe ou indirect, l’hemmaglutination et son
inhibition, la séroneutalisation des ECP
ELISA : Enzyme Linked Immuno-Sorbent Assay ou technique immunoenzymatique sur phase solide

Principe de la technique ELISA, dans la détection des Ac :

a) Des Ag viraux natifs ou synthétiques sont adsorbés sur une phase solide faite de cupules de
microplaques, de tubes ou billes en verre ou polystyrènes ,ou sur surface de membrane.
b) Incuber les sérums à tester sur cette phase solide afin de fixer les Ac. présents dans le sérum
sur les Ag viraux.
c) Mise en évidence de cette réaction immunologique entre les Ac du sérum à tester et les Ag
viraux adsorbés sur la phase solide par une antimmunoglobuline marqué par une enzyme ( = c’es le
CONJUGUE).
d) Révélation par un substrat chromogène de l’enzyme qui se colore
e) Mesure de l’intensité colorimétrique par spectrophotomètre. Cette mesure rendue en densité
optique (D.O) est proportionnelle a la quantité des Ac présent dans le sérum

Microplaque d, ELISA

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Distributeur d,echantillon d,un automate d, ELISA

I.F.D de cellules infectees par Herpes


virus

§4 LA MICROSCOPIE ELECTRONIQUE

La microscopie électronique a permis de visualiser les virus car son pouvoir de résolution est de
l’ordre du nanomètre soit 10x-9 mètre. La plupart des virus ont un diamètre de quelques centaines
de nanomètres. Toutefois pour une utilisation optimale du microscope électronique :

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-il faut d’abord « colorer » les préparations d’échantillons avec des métaux lourds pour créer
un contraste
-les prélèvements doivent être riche en particule virale, clarifiés et liquides tels les selles
diarrhéiques ou des sérums
Enfin la microscopie électronique détecte les formes des virus ; dès lors elle ne peut donner
qu’un diagnostic direct des genres différents, elle ne peut fournir un diagnostic différentiel entre deux
espèces du même genre.

Calcivirus en microscopie electronique

§ 5 L’APPORT DE LA BIOLOGIE MOLECULAIRE

La biologie moléculaire est le diagnostic le plus spécifique et le plus sensible des


biotechnologies actuelles. Plusieurs techniques son utilisées dans ce domaine.
1/ l’amplification génique ou PCR pour polymerase chain reaction qui est une des techniques les plus
utilisée dans la détection des génome viraux
2/ l’hybridation in situ
3/ le séquençage qui permet de réaliser le génotypage des génomes
4/ le clonage qui a permis de détecter les virus des hépatites C et E

Certaines de ces techniques sont passées dans la routine des laboratoires de virologie

Genotypage sur membrane de VHC après PCR et Thermocycleur pour PCR

§5 LE DIAGOSTIC BIOLOGIQUE D’UNE INFECTION VIRALE

Elle repose sur deux démarches :


- un diagnostic direct
- un diagnostic indirect

Diagnostics directs en virologie

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a) le diagnostic direct

Par le diagnostic direct on recherche à mettre en évidence le virus lui-même ou ces constituants, à
savoir, son génome et ses antigènes de la capside ou de l’enveloppe :
Pour ce faire on emploie les techniques suivantes :
- PCR et hybridation in situ pour le génome viral
- La microscopie électronique
- L’isolement par culture cellulaire ou inoculation sur œuf de poulet embryonné
- Détection des antigènes viraux par immunofluorescence direct, ELISA et immunoblot,
l’hemagglutination et la séroneutralisation des effets cytopathiques ( enterovirus)
- genotypage

b) diagnostic indirect

Le sérodiagnostic est l’analyse du sérum afin de dépister et quantifier la présence des


anticorps spécifiques dirigés contre l’infection virale
Les techniques actuellement utilisées sont l’ELISA et l’immunofluorescence indirecte ainsi
que l’inhibition de l’hemagglutination

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2 ème Partie : VIROLOGIE SYSTEMATIQUE

VIRAL GASTRO-ENTERITIS

INTRODUCTION

Paediatric diarrhoea remains one of the major causes of death in young children. This is especially
so in Asia, Africa and Latin America where it causes millions of deaths in the age group 0-4 years.

The main factors for high incidence and mortality are unsafe water or inadequate sanitation, requiring
social, economic and political solutions. The immediate causes are often of an infectious nature and
include a variety of pathogenic micro-organisms. A range of bacteria and parasites has been
identified = enterotoxigenic Escheritia coli, salmonella, shigella, cholera, other vibrio bacteria, as
well as cryptosporidium, but these account for well below half of investigated cases.

A number of different viruses cause diarrhoea, of which the most important is the family of
ROTAVIRUSES.

Rotaviruses have been estimated to cause 30-50% of all cases of severe diarrhoeal disease in man.

Two strains of adenovirus (40 and 41) have also been associated with diarrhoeal disease.

A group of "small round viruses" (discovered by electron microscopy) have been linked by genetic
techniques as closely related to the previously described "Norwalk" agent, associated with vomiting
and diarrhoea.

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ROTAVIRUS - (REO virus family)

• Group A subtypes 1, 2, 3, 4 (main human pathogens)


(Further 7 subtypes) also infect animals (monkey, calf, mouse)
• Group B Infects pigs and rats
Found to cause extensive outbreaks in China in past decade
• Group C Infects Pigs (Occasionally Man)
• Group D Infects birds
• Group E Infects pigs

VIRUS MORPHOLOGY:
Particles are 70 nm round, double shelled, enclosing a genome of 11 segments of double stranded
RNA.
The virus is hardy and may even survive in sewage, despite stringent treatment.
Human rota virus has proved difficult to culture in vitro, but the serologically related monkey and calf
rotaviruses grow easily in cell culture.

Click here for electron microscope images of the virus.

A double-capsid particle is shown on the left, and the single (inner) capsid on its right.

CLINICAL:
Essentially an ingestion disease (faecal-oral route)

Incubation is short : 1 to 3 days.


Illness: Sudden onset watery diarrhoea, with or without vomiting. May last up to 6 days (or longer if
immunocompromised). The disease is self limiting.
Complications: Dehydration may result, this can be severe and life threatening in young children.
Treatment: No specific treatment of viral infection is available nor is it really required.
Treatment is aimed at prevention and/or treatment of dehydration by oral and/or intravenous fluids
and electrolytes
Diagnosis: Detection of virus in stools (peaks at day 3 or 4 of diarrhoea):-

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1. Latex agglutination
2. Elisa
3. Electron Microscopy (labour intensive, relatively insensitive)
4. Electrophoresis of RNA segments

(Antibody can be detected but is not clinically useful)


Prevention:
Non specific factors: improved hygiene, education, clean water
Specific - Breast feeding helps to provide passive immunity in the newborn (from maternal
antibodies),
Vaccination are available and in a near r included in imunazation natinal program

EPIDEMIOLOGY

Infection is found world-wide.


All ages can be infected and reinfection can occur (usually asymptomatic).

Age: Infections at < 6 months age and > 5 years of age tend to be asymptomatic and give degrees of
protection against diarrhoeal infection.
Maternity hospitals commonly have resident strains which readily cause asymptomatic infections of
new- borns.
Seasons: In temperate '1st world' populations rota virus is the main cause of winter gastroenteritis .
In tropical and developing countries, rotavirus diarrhoea occurs all the year round ,but with a peak in
summer. However, it is only one of a variety of pathogens causing diarrhoea.
Vaccine: In view of the major role of dehydration from diarrhoea as a cause of childhood death, the
World Health Organisation has waged an intensive campaign for

• (1) oral rehydration solutions to prevent or treat dehydration


• (2) development of a vaccine for rota virus infection.

No vaccine is currently used routinely, but several candidate vaccines are being evaluated in children:
e.g. animal strains, attenuated human strains, animal-human recombinant strains, designed to cover all
4 main human pathogenic strains.
The prevention of severe dehydration is the main aim, rather than totally preventing infection.

ADENOVIRUS

A limited number of strains of ADENOVIRUS have been causally related to childhood diarrhoea.
They do not grow in cell cultures and were discovered by Electron Microscopy. ( Recently there has
been limited success in special cell culture systems).
They are classified in the 40/41 serogroup of adenoviruses.
Viruses can be isolated from stools,as well as throat and respiratory secretions. Adenoviruses in stools
can be detected by latex agglutination, and the 40/41 strains can be detected by specific molecular
techniques.
The exact role or significance of these strains in the global picture of childhood diarrhoea, especially
in developing countries, is not yet fully established.

Apart from this 40/41 group, other adenoviruses may be found in the stools of asymptomatic
children. .

Click here to view electron microscope images of ADENOVIRUSES

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GASTROENTERITIS IN OLDER CHILDREN AND ADULTS

Apart from the severe problem of diarrhoea in young children there have been outbreaks of infectious
gastro-enteritis in adults. Electron microscopy (of the stools) has revealed two main groups of virus
particles which do not grow in cell culture.

• (1) Calici viruses (ssRNA) including Norwalk and related agents ('Hawai'; Ditchling; 'W')
• (2)"small round viruses" about which very little is known

NORWALK agent (33nm in size)

'Common source' type of explosive outbreaks of gastroenteritis, with limited secondary spread to
household contacts, have been described. These often occur in institutions, or follow common source
ingestion episodes e.g. celebratory feasts.
Vomiting with cramps are more common symptoms than the diarrhoea.

At first, the virus was seldom identified as there were no easy diagnostic tests - only expensive
electron microscopy of stools. Serology was limited, as the only antigen available was prepared from
known infected stools - not in plentiful supply!

Recently, molecular techniques have shown that many of these agents from different parts of the
world are essentially similar. Molecular techniques have also enabled the expression of viral antigens
that can be used in serological surveys. For example, a 1993 survey in the UK has shown that
Norwalk infection is apparently a common silent infection in childhood. Antibody prevalence rises
to about 16 years of age and then begins to level off, with 80% of persons above 30 years being
seropositive. It was hitherto believed that Norwalk infections were rare, - it now seems that the
disease is rare, not the infection.

ENTERO-VIRUSES

INTRODUCTION

These are small non-enveloped isometric viruses that multiply in the gut mucosa and are transmitted
from person to person by the faecal-oral route (ingestion disease). They are spread throughout the
body via the blood stream. Most infections occur during childhood, and they are usually transient but
produce lifelong immunity. Clinical syndromes are generally mild, but occasional infections may
cause serious disease e.g. paralytic poliomyelitis, meningitis, or myocarditis. There is a high degree of
serological cross reactivity between the 72 members.

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CLASSIFICATION

Viruses belong to the family Picornaviridae (pico=SMALL - RNA viruses)

Enteroviruses:
Polio 1, 2, 3
Coxsackie A 1-24
Coxsackie B 1-6
ECHO 1-34

Entero 68-71
Entero 72 (Hepatitis A)

Rhino viruses: > 120 serotypes

Other animal viruses: e.g. Foot & Mouth Disease virus

POLIOMYELITIS

Poliovirus has been well studied and is a good example of an enterovirus.

Virus: small (30nm) and stable; an icosahedral capsid enclosing a positive-sense, single-stranded
RNA genome. Relatively resistant to extremes of pH and temperature, and to lipid solvents and
detergents.

Types: 3 types can be distinguished by antigenic properties.

Electron microscopy of Picornaviridae

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Genoma of a Poliovirus

Icosahedral capsid of Poliovirus

CLINICAL

Source: Only known source is infected man

Incubation: After ingestion of the virus, there is local multiplication in the oropharynx and associated
lymph nodes. Local multiplication also takes place in the gut mucosa and regional lymph nodes.
Thereafter a viraemia follows, and the patient may experience a fever about a week after exposure.

Illness: Most infections are asymptomatic, although in some there is a minor transient febrile illness.
Occasionally (between 1/100 and 1/1000 of cases) the viraemia may lead to CNS involvement and
paralysis due to permanent damage to the anterior horn motor neurones of the spinal cord. The patient
may experience degrees of headache, fever, meningism, aseptic meningitis and muscle pains, and
finally muscle paralysis, usually asymmetrical. Paralysis develops more frequently in adults, and may
be precipitated by muscle trauma (injections, exercise), tonsillectomy, pregnancy and steroid drug
administration. The spinal cord may be damaged in a progressive manner from distal to more central -
some cases may progress to involve the medulla and brainstem (bulbar paralysis) with consequent
respiratory paralysis and death, or life on a respirator.

Virus is produced and released into the gut (and throat initially) and can be isolated from the throat or
stools for some weeks following the incubation period. No true long term carrier status occurs.

The host's antibody response begins soon after the viraemia. Good solid lifelong immunity results to
the specific strain of poliovirus, but subsequent infection with other strains may still occur.

DIAGNOSIS

(1) Demonstration of the virus


Virus may be recovered from faeces (also throat swabs), by inoculation of cell cultures and
recognition of cytopathic effects with confirmation by neutralisation of infectivity with specific
antisera. Vaccine strains may be recovered and need to be differentiated from wild strains by
molecular nucleic acid techniques (PCR). Multiple specimens over several days improves chances of
recovery of the virus.

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(2) Serology:
Most cases of poliomyelitis that come to medical attention present with paralysis, i.e. quite late in the
pathogenesis, and antibodies have already been formed. Antibodies are not usually helpful in
providing a positive diagnosis of poliomyelitis, but do give the immune status of an individual
(does/does not need further vaccination). Detection of specific IgM has not been applied to polio
diagnosis. Antibodies are traditionally tested by micro-neutralisation of infectivity in vitro using
antisera to known virus strains

CSF: Polio virus is never found in the CSF but antibodies here mean either CNS infection or a leak
from blood antibodies.

EPIDEMIOLOGY

Before the introduction of a vaccine, (< 1960), polio was endemic in the tropics, with rapid
circulation in young children (poor hygiene facilitates faecal-oral spread) with minimal paralysis (?
protective effect of residual maternal antibody). This ensured high "herd immunity" without
epidemics.

In temperate regions, polio showed peaks in summer/autumn. As conditions of hygiene improved,


viral spread was hindered and the age of primary exposure rose. Primary infection in adulthood
resulted in a much higher incidence of paralytic disease. These tendencies rose to a climax in
Scandinavian countries just before the vaccine era ,when they experienced devastating epidemics of
paralytic poliomyelitis.

Post Vaccine. Universal vaccination commencing in the early 1960s, and has eliminated polio from
the Western world, including North, Central and South America. The disease has also largely been
controlled it in Africa and Asia. In South Africa, polio has been effectively eliminated but
reintroductions into regions with deficient vaccination programs has resulted in localised outbreaks
(e.g. 1982 and 1988).
The World Health Organisation is forging ahead with a total elimination plan (c.f. smallpox) "by the
year 2000".

POLIO VACCINES

(1) Live attenuated virus (SABIN)(1963)

Strains of poliovirus 1, 2 and 3 which have been attenuated by passage in unnatural conditions to lose
neurovirulence.
3 live strains mixed, given as oral drops (easy administration).
Given on 3 occasions plus boosters (one strain may interfere with uptake of another, hence must be
given repeatedly to ensure immunity to all 3 types).
Wild enteroviruses coincidentally present in the gut may also interfere, especially in the tropics.

Live vaccination mimics natural infection with good immunity including IgA in the gut. This vaccine
is used in RSA, USA and most other countries. It is very important to maintain 'cold chain' when
storing and distributing vaccine as it may lose potency.

(2) Killed whole virus (SALK)(1957)

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Polio 1, 2 and 3 grown in cell cultures, mixed, killed with formalin.


3 injections at 3 to 6 months of age; later boosters.
Much antigen is required which makes the vaccine expensive.
Effective when coverage is good (nearly 100% immunity in people regularly vaccinated).
Still used in some countries (Netherlands, Scandinavia) in 1990's.

SABIN POLIO VACCINE POLICY, RSA( 1989 Onwards)

A. Neonatal monovalent P1 at birth

B. Standard Trivalent (P1 + P2 + P3) at 3 months, 4½ months and 6 months of age

C. Boosters: at 18 months, and again at time of School entry.

Polio is controlled (?eliminated) by:

(1) Education
(2) Vaccination
(3) Surveillance

ENTEROVIRUSES - OTHER THAN POLIO

i.e. Coxsackie, Echo, Entero 68-72

Virus structure, Epidemiology, Pathogenesis of all the enteroviruses are remarkably similar and
follow the pattern described for polio.
Most infections are silent. Viraemia may lead to degrees of involvement of secondary 'target organs'
and clinical symptoms and signs related to those organs. For example, the most common type of
meningitis seen in the Cape is aseptic meningitis caused by coxsackie or echo viruses (which can
often easily be isolated from the CSF, in contrast to polio). Viral meningitis resolves spontaneously
without treatment but bacterial meningitis is a medical emergency requiring treatment.

Enteroviruses may be found in the gut of healthy as well as sick children; the association with any
illness may be purely co-incidental.

CLINICAL SYNDROMES

Information given in the following format:-

CLINICAL SYNDROME
Common virus associated with the clinical syndrome
Less commonly associated virus

ASYMPTOMATIC
All enteroviruses

PARALYSIS - permanent

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Polio 1, 2, 3
Coxsackie A7

PARALYSIS - temporary
Coxsackie B1-6

MENINGITIS (aseptic)
Echo, Coxsackie A and B
Polio, Entero 71

ENCEPHALITIS
Entero 71
Polio, Echo

RASH
- macular
Many enteroviruses
- vesicular - (e.g. 'Hand Foot Mouth')
Coxsackie A

SUMMER FEBRILE ILLNESS


Many enteroviruses

VESICULAR PHARYNGITIS ('Herpangina')


Coxsackie A

MYOCARDITIS
Coxsackie B

EPIDEMIC MYALGIA ('Bornholm')


Coxsackie B

UPPER RESPIRATORY INFECTION (cold)


Echo, Coxsackie A

PANCREATITIS
Coxsackie B

GASTRO-ENTERITIS
Many enteroviusess

CONJUNCTIVITIS (Haemorrhagic)
Entero 70

HEPATITIS
Entero 72 (hepatitis A virus)

VIRAL HEPATITIS

The termVIRAL HEPATITIS is usually used to describe infections caused by agents whose primary
tissue tropism is the liver.
To date, at least five hepatitis viruses have been recognised, and these have been named:-

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Hepatitis A, B, C, D and E.
Acute hepatitis may also occur as part of the clinical course of a number of viral infections, including
human cytomegalovirus, Epstein-Barr virus, herpes simplex virus, yellow fever virus and
rubella.

Clinical Features

Hepatitis due to all these viruses presents clinically in a very similar fashion, especially during the
acute phase of the illness. Thus a specific diagnosis can only be made in the laboratory.
The majority of infections are totally asymptomatic, but common clinical features include: anorexia,
nausea, vomiting, right upper quadrant pain and raised liver enzymes AST and ALT.
Jaundice is the hall mark of infection, but tends to develop late.
Anicteric cases are also very common.

ENTERICALLY TRANSMITTED HEPATITIS: A and E

PARENTERALLY TRANSMITTED HEPATITIS B , C , D and G

Hepatitis A - "Infectious Hepatitis"

Caused by a picornavirus, Enterovirus 72


This is a small, non-enveloped icosahedral particle, 27 nm in diameter, containing a ssRNA genome

Clinical Features
Incubation period 3-5 weeks (mean 28 days)
Milder disease than Hepatitis B; asymptomatic infections are very common, especially in children.
Adults, especially pregnant women, may develop more severe disease.
Although convalescence may be prolonged, there is no chronic form of the disease.

Complications:
Fulminant hepatitis is rare: 0.1% of cases

Pathogenesis
Virus enters via the gut; replicates in the alimentary tract and spreads to infect the liver, where it
multiplies in hepatocytes.
Viraemia is transient. Virus is excreted in the stools for two weeks preceding the onset of
symptoms.

Epidemiology
World-wide distribution; endemic in most countries. The incidence in first world countries is
declining. There is an especially high incidence in developing countries and rural areas.
In rural areas of South Africa , the seroprevalence is 100%.

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Transmission - Enteric
Large numbers of virus particles are excreted in
stools, before the onset of symptoms.

1) Case-to-case, via faecal-oral route.


Outbreaks in creches are very common.
2) Contamination of food or water with sewage
Infected food handlers
Shell fish grown in sewage-polluted water

Diagnosis
Virus cannot be cultured in vitro from clinical material, and diagnosis is made on the presence of
HAV-specific IgM in the patient's blood.

Prevention
1) Passive immunisation -

Normal immunoglobulin given to:


Travellers to third world countries
Household contacts of acute cases

2) Active Immunization
Inactivated cell culture-derived vaccine has recently become available; not in general use

Hepatitis E

Recently identified cause of enterically transmitted non-A, non-B (NANB) hepatitis

Calicivirus
spherical, non enveloped, 27-34 nm particles containing a ssRNA genome.

Clinical Features
Incubation period 30-40 days
Acute, self limiting hepatitis, no chronic carrier state
Age: predominantly young adults, 15-40 years

Complications
Fulminant hepatitis in pregnant women. Mortality rate is high (up to 40%).

Pathogenesis
Similar to hepatitis A; virus replicates in the gut initially, before invading the liver, and virus is shed
in the stool prior to the onset of symptoms.
Viraemia is transient. A large inoculum of virus is needed to establish infection.

Epidemiology
Little is known yet. The incidence of infection appears to be low in first world countries.

1) Large outbreaks have been described in India, Mexico and North Africa where the source of
infection is usually gross faecal contamination of drinking water supplies.
2) Case-to-case transmission to household contacts appears to be uncommon. This suggests that a
large inoculum is needed to establish infection.

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The incidence of infection in South Africa is unknown.

Diagnosis
No routine laboratory tests are available as yet. Virus cannot be cultured in vitro.

1) Calicivirus-like particles in the stool, by electron microscopy


2) Specific IgM in serum
3) PCR HEV-specific sequences in stool

PARENTERALLY TRANSMITTED HEPATITIS


B, C, D and G

Hepatitis B

Hepadna virus
42nm Virions (also known as "Dane particles") contain a circular dsDNA genome

HBV Antigens
HBsAg = surface (coat) protein
produced in excess as small spheres and tubules
HBcAg = inner core protein
HBeAg = secreted protein; function unknown

You can view a diagram and electron micrographs of the virus


structure.

A diagrammatic representation of the hepatitis B virion and


the surface antigen components
.

Virions are 42nm in diameter and possess an isometric nucleocapsid or "core" of 27nm in diameter,
surrounded by an outer coat approximately 4nm thick. The protein of the virion coat is termed
"surface antigen" or HBsAg. It is sometimes extended as a tubular tail on one side of the virus
particle. The surface antigen is generally produced in vast excess, and is found in the blood of
infected individuals in the form of filamentous and spherical particles. Filamentous particles are
identical to the virion "tails" - they vary in length and have a mean diameter of about 22nm. They
sometimes display regular, non-helical transverse striations.

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Core particles: The actual number and arrangement of the capsomers has not been established with
certainty, but certain images suggest a lattice formation, as shown below.
A group of hepatitis B virions (right) and enlargements of the two exposed cores (indicated by
arrows).

Clinical Features
Incubation period 2 - 5 months
Insidious onset of symptoms. Tends to cause a more severe disease than Hepatitis A.
Asymptomatic infections occur frequently.

Pathogenesis
Infection is parenterally transmitted. The virus replicates in the liver and virus particles, as well as
excess viral surface protein, are shed in large amounts into the blood. Viraemia is prolonged and the
blood of infected individuals is highly infectious.

Complications
1) Persistant infection:-
Following acute infection, approximately 5% of infected individuals fail to eliminate the virus
completely and become persistantly infected.

Those who are at particular risk include:


babies, young children
immunocompromised patients
males > females

The virus persists in the hepatocytes and on-going liver damage occurs because of the host immune
response against the infected liver cells.

Chronic infection may take one of two forms:


Chronic persistent Hepatitis - the virus persists, but there is minimal liver damage
Chronic Active Hepatitis - There is aggressive destruction of liver tissue and rapid progression to
cirrhosis or liver failure.

2) Patients who become persistently infected are at risk of developing hepatocellular carcinoma
(HCC)..
HBV is thought to play a role in the development of this malignancy because:

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a) 80% of patients with HCC are


carriers of hepatitis B.
b) Virus DNA can be identified in
hepatocellular carcinoma cells.
c) Virus DNA can integrate into the
host chromosome.

3) Fulminant Hepatitis
Rare; accounts for 1% of infections.

Epidemiology

Prevalence of disease in Africa


World-wide there are 450 million persistant carriers of hepatitis B, 50 million of which are in Africa.
Carriage rates vary markedly in different areas. In South Africa, infection is much more common in
rural communities than in the cities. Hepatitis B is parenterally transmitted

1) Blood:

• Blood transfusions, serum products,


• sharing of needles, razors
• Tattooing, acupuncture
• Renal dialysis
• Organ donation

2) Sexual intercourse

3) Horizontal transmission in children, families, 'close personal contact'.


This is the major mode of transmission in South Africa where the majority of individuals become
infected at between three and nine years of age.
Horizontal transmission also occurs in children's institutions and mental homes.

4) Vertical transmission - perinatal transmission from a carrier mother to her baby

• transplacental (rare)
• during delivery
• post natal , ??breast feeding , ??close contact

( This is the major mode of transmission in South East Asia)

Diagnosis: Serology

A. Acute infection with resolution

Viral antigens:
1) Surface antigen (HBsAg) is secreted in excess into the blood as 22 nm spheres and tubules. Its
presence in serum indicates that virus replication is occurring in the liver
2) 'e' antigen (HBeAg) secreted protein is shed in small amounts into the blood. Its presence in serum
indicates that a high level of viral replication is occurring in the liver
3) core antigen (HBcAg) core protein is not found in blood

Antibody response:
1) Surface antibody (anti-HBs) becomes detectable late in convalescence, and indicates immunity

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following infection. It remains detectable for life and is not found in chronic carriers (see below).
2) e antibody (anti-HBe) becomes detectable as viral replication falls. It indicates low infectivity in a
carrier.
3) Core IgM rises early in infection and indicates recent infection
4) Core IgG rises soon after IgM, and remains present for life in both chronic carriers as well as those
who clear the infection. Its presence indicates exposure to HBV.

B. Serology of the chronic carrier

Ag agHBe Ac IgM Ac Ac Diagnostic


HBs HBc HBe HBs
+ - - - - Porteur chronique
(si persistance au delà de six mois)

+ + - - - Porteur avec forte


infectivité (multiplication virale)

+ - + - - hépatite aigue (symptomatique ou


asymptomatique )

- - - + + Immunisation naturelle : guerison

- - - - + Immunisation artificielle par


vaccination

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Prevention

1) Active Immunization

Two types of vaccine are available:


Serum derived - prepared from HBsAg purified from the serum of HBV carriers
Recombinant HBsAg - made by genetic engineering in yeasts

Both vaccines are equally safe and effective. The administration of three doses induces protective
levels of antibodies in 95% of vaccine recipients.
Universal immunization of infants was introduced in April 1995. Infants receive 3 doses at 6, 10 and
14 weeks of age.

Vaccine should be administered to people at high risk of infection with HBV:


1) Health care workers
2) Sexual partners of chronic carriers
3) Infants of HBV carrier mothers

2) Passive Antibody
Hepatitis B immune globulin should be administered to non immune individuals following single
episode exposure to HBV-infected blood.
For example: needlestick injuries

Hepatitis C

The major cause of parenterally transmitted non A non B hepatitis. It eluded identification for many
years. In 1989, the genome was cloned from the serum of an infected chimpanzee.

Virology

Putative Togavirus related to the Flavi and Pesti viruses.


Thus probably enveloped.
Has a ssRNA genome
Does not grow in cell culture, but can infect Chimpanzees

Clinical Features

Incubation period 6-8 weeks


Causes a milder form of acute hepatitis than does hepatitis B
But 50% individuals develop chronic infection, following exposure.

Complications
1) Chronic liver disease
2) Hepatocellular carcinoma

Epidemiology

Incidence endemic world-wide; high incidence in Japan, Italy and Spain


In South Africa, 1% blood donors have antibodies

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Transmission

• Blood transfusions, blood products


• organ donation
• Intravenous drug abusers
• community acquired: mechanism unclear. ?Vertical transmission
• ?sexual intercourse

Diagnosis

1) Serology
Reliable serological tests have only recently become available.
HCV-specific IgG indicates exposure, not infectivity

2) PCR detects viral genome in patient's serum

Delta Agent
Defective virus which requires Hepatitis B as a helper virus in order to replicate. Infection therefore
only occurs in patients who are already infected with Hepatitis B.

Clinial Features
Increased severity of liver disease in Hepatitis B carriers.

Virology
virus particle 36 nm in diameter
encapsulated with HBsAg, derived from HBV
delta antigen is associated with virus particles
ssRNA genome

Epidemiology
Identified in intra-venous drug abusers in Italy. Incidence in South Africa is unknown.

Hepatitis G (HGV)

A virus originally cloned from the serum of a surgeon with non-A, non-B, non-C hepatitis, has been
called Hepatitis G virus. It was implicated as a cause of parenterally transmitted hepatitis, but is no
longer believed to be a major agent of liver disease. It has been classified as a Flavivirus and is
distantly related to HCV.

Virus Famille ou Génome Contamination Expressions


Genre epidemiologiques

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VHA Picornavirida a ARN Entérale , Epidemique


e Oro-fecale ( sporadique)
Heparnavirus
VHB a ADN Parenterale, endemique
Hepadnavirus sexuelle,
Mere a enfant

VHC a ARN Parenterale,


Flavivirus (sexuelle) endemique

Parenterale, endemique
VHD a ARN sexuelle,
Viroïde un Mere a enfant,
virus défectif toujours associe
ou satellite a une infection
a VHB

Entérale ,
VHE a ARN Oro-fecale Epidemique
Calciviridae

GB-A, a ARN Parenterale Endemique


GB-B Flavivirus ( sporadque)
GB-
C/VHG

TTV a ADN Parenterale Endemique


Parvovirus (sporadique)

HERPESVIRUSES

Sous famille Alphaherpesvirinae Betaherpesvirinae Gammaherpesvirinae


Genre 1/ Simplex virus 1/Cytomégalovirus 1/Lymphocryptovirus
2/ Varicellovirus 2/Roseolovirus 2/Rhadinovirus
Genre Simplex virus Cytomégalovirus Lymphocryptovirus
Espèces Herpès simplex virus Cytomégalovirus humain Virus d’Epstein-Baar
HHV 1 et HHV 2 HCMV ou HHV5 EBV ou HHV4
Genre Varicellovirus Roseolovirus Rhadinovirus
Espèces Virus du Zona et de la Herpes virus humain 6 Herpès virus humain 8
varicelle HHV6 HHV8/KSHV
VZV ou HHV3 Herpes virus humain 7
HHV7

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The family of herpesviruses is very large, and its members infect most animal species.
There are 8 herpesviruses which are known to infect humans:

• Herpes Simplex Virus type 1 and type 2


• Varicella-Zoster Virus
• Cytomegalovirus
• Epstein-Barr Virus
• Human Herpesvirus 6
• Human Herpesvirus 7
• Human herpes virus 8

DISEASE
It is a characteristic of all herpesviruses that, following primary infection, the virus establishes a
latent infection in the host and may reactivate at any stage. Reactivation is frequently, but not always,
associated with further disease.

Structure of the virion

All herpesviruses are morphologically identical.

They have a large double stranded DNA genome. The VIRION


consists of an icosahedral nucleocapsid of about 100 nm in diameter,
which is surrounded by a lipid bilayer envelope. Between the capsid
and the envelope is an amorphous layer of proteins, termed the
tegument.

Click here to view electron microscope images of the virus.

Members of the family herpesviridae are found in a wide range of host


systems. To date, at least eight different species are known to infect man, including herpes simplex
virus (HSV); cytomegalovirus (CMV), varicella zoster (VZV); and Epstein Barr virus (EBV).

Herpesviruses have an envelope surrounding an icosahedral capsid, approximately 100nm in


diameter, which contains the dsDNA genome.

When the envelope breaks and collapses away from the capsid, negatively stained virions have a
typical "fried-egg" appearance.

The herpes virus CAPSID is an icosahedron of triangulation number T = 16. There are 12 pentavalent
capsomers (one at each apex) and 150 hexavalent capsomers. Each capsomer has a deep central
indentation.

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The ENVELOPE that surrounds the nucleocapsid is derived from the inner nuclear membrane of the
host cell.
Virus-encoded glycoproteins are incorporated into the virion envelope and are visible as "spikes" that
project from its surface.

In the micrograph of HSV, the glycoprotein B (gB) is clearly visualised in clusters of spikes
appoximately 10 nm in length.

Between the capsid and the envelope is an ill-defined layer of proteins, collectively known as the
tegument.

Herpes Simplex Virus

There are two closely related viruses termed :


herpes simplex virus type 1 (HSV1), and
herpes simplex virus type 2 (HSV2).
Both viruses cause painful vesicles on the skin at the site of inoculation.
HSV1 is usually associated with oro-facial lesions.
HSV2 is usually associated with genital lesions.
EPIDEMIOLOGY

Infection with HSV1 is almost universal. This is known because, although many infections are sub-
clinical, virtually 100% of adults have antibodies in their serum. Most individuals become infected in
the first few years of life.
Virus is shed from the infected area and spread occurs as a result of direct contact with lesions. For
example, through kissing (HSV1) or sexual intercourse (HSV2).
Virus may also, however, be shed in saliva and genital secretions and can thus be transmitted in the
absence of clinical lesions.

CLINICAL FEATURES

There are 2 clinical patterns of disease:

a) Primary Infection , and

b) Recurrent disease

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Primary Infection

Most primary infections are silent.


In clinically apparent cases, vesicles usually develop at between 1-3 days post exposure and remain
localized to the site of inoculation. However, in immunocompromised individuals the virus may
disseminate.

The nature of the disease is determined by the site of inoculation:


Gingivo-stomatitis
This is the most common form of primary infection; inoculation is usually through kissing. There is
a wide spectrum of severity, from trivial to extensive disease. Painful vesicles develop inside the
mouth on the bucchal mucosa and gums, on the lips and skin around the mouth. The vesicles inside
the mouth ulcerate and become covered with a greyish slough. Lesions may occur at other sites on
the head and neck as well.

The primary eruption is often associated with fever and cervical lymphadenopathy. The illness is self
limiting and lesions usually heal within 14 days.

Kaposi’s Varicelliform eruption


Super-infection of eczematous skin with HSV.

Herpetic Whitlow
Inoculation of virus into the fingers - an occupational hazard of doctors, nurses and dentists.
May be mistaken for a paronychia and incised .

Conjunctivitis, Keratitis

Herpetic lesion on the cornea - is called a dendritic ulcer because of its branching appearance. Pain
and photophobia are prominant features. Conjunctivitis and oedema of the lids commonly accompany
primary infection. Lesions usually heal within 3 weeks.

Genital Herpes
Sexually transmitted herpetic lesions. Usually due to HSV2 but 20-30% of cases are due to type
HSV1. Primary eruption lasts ±10-14 days.
Acute necrotizing encephalitis
Infection of the brain by HSV. Neurons of the temporal lobe are most commonly involved. Infection
is severe and necrotising. Clinical features include: sudden onset of fever, headache, confusion and
alteration in personality. Mortality is high and neurological impairment in the survivors is invariable.
Encephalitis may be due to primary infection or reactivation.
Neonatal Infection

This is a very rare condition. Neonates have poor cell mediated immunity and are therefore at
increased risk of disseminated infection if they are exposd to HSV in the perinatal period.

Exposure may occur:


1) at birth, if the mother has genital herpes at the time of delivery. (This is only a significant risk if the
mother is experiencing a primary infection).

2) In the post natal period, if the infant is handled by people with herpetic lesions.

The disease may take one of three forms:-

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Cutaneous lesions:-
These are confined to the skin, and the prognosis is good.
Generalized infection:-
This is a serious condition, with a high fatality rate. Virus disseminates throughout the organs.
Cinical features include jaundice,
hepatosplenomegaly, thrombocytopenia,
pneumonia and encephalitis. Lesions on the skin
may be trivial.
Encephalitis:-
Direct infection of brain tissue.

Latency and Recurrent disease

HSV1 and HSV2 can establish a latent infection in


the ganglia of the nerves that supply the site of the
primary infection
Genital area - sacral ganglia
Oro-facial - trigeminal ganglion

Following primary oro-facial infection, the virus enters sensory nerve endings and travels up the axon
and establishes a latent infection in the trigeminal ganglion.

The viral genome persists in an episomal form (plasmid) in the nucleus of the neurone. No viral genes
are expressed. This state of latency may persist for many years.

In a percentage of people,
the virus will reactivate: - A cycle of viral replication occurs in the neurone and virus particles travel
down the axon to reinfect the skin or mucous membrane in the area supplied by the nerve.

Reactivation may be provoked by a number of stimuli:


including sunlight, stress, febrile illnesses, menstruation or immunosuppression.
Clinical manifestations of reactivation:

1. Cold sores (follows gingivo-stomatitis):


Following one of a variety of stimuli, vesicles erupt on the muco-cutaneous junctions of the nose or
mouth. These are more localized than the primary infection and heal more rapidly (7-10 days). The
eruption is often preceeded by paraesthesia of the involved area.

2. Recurrent genital herpes:


Recurrence with HSV 2 infections is more common than with HSV 1. Lesions are less extensive and
heal more rapidly than the primary infection.

3. Keratitis:
The virus reaches the cornea via the ophthalmic branch of the trigeminal nerve; the clinical lesion is
termed a dendritic ulcer. It heals more rapidly than the primary infection.

LABORATORY DIAGNOSIS

Direct detection by electron microscopy of herpesvirus particles in vesicle fluid

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Culture: Isolation from clinical material from skin lesions may be inoculated onto cell monolayers
which are monitored for the development of characteristic cytopathic effect. This is usually detected
within three days

Direct immunfluorescence on material from lesions

Serology is not very useful because there is a high prevalence of antibody in the normal population.

Varicella Zoster Virus

CLINICAL FEATURES

There are two clinical entities:

(1) Varicella or chicken pox, and


(2) herpes zoster or shingles

Varicella (chicken pox)

(Primary infection)

This is a common childhood infection that presents as a mild febrile illness associated with a
generalized vesicular rash. The incubation period is long, about 21 days. Vesicles appear as
successive "crops", so that lesions of different ages are present at the same time.
The lesions progress from macule to papule to vesicle to pustule to scab.

In children the disease is usually trivial and complications are rare. If infection is delayed until
adulthood the disease may be more severe and complications more frequent. Infection is transmitted
either by respiratory droplets or by direct contact with skin lesions.

Varicella is followed by long lasting immunity.

Complications

Post Infectious Encephalomyelitis - self limiting condition, with a good prognosis.


Haemorrhagic varicella - fulminating infection in immunocompromised patients: high
fatality rate
Pneumonia - common complication of varicella in adults.

Congenital varicella syndrome

Infants born to mothers who have varicella in early pregnancy may develope a syndrome associated
with; limb hypoplasia, muscular atrophy, mental retardation and skin scarring. This condition is
extremely rare.

Perinatal varicella

If the mother acquires varicella more than 5 days before delivery, the disease in the infant is mild.
This is because the severity of the disease is ameliorated by the presence of passively acquired

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maternal antibody. If the mother acquires infection less


than five days before delivery the neonate is likely to
develope severe disease. An injection of special
hyperimmune globulin should be administered to the
baby.

Zoster (shingles)

Reactivation lesion of VZV

Like HSV, the virus (VZV) establishes a latent infection in sensory ganglia. Reactivation usually
occurs many years after primary infection and is often associated with immunosuppresion of the host.

The virus travels down the axon and re-infects the dematome supplied by the sensory ganglian to
produce painful vesicles on the skin.
Common sites include the thoracic dermatomes ( more than 50% of cases) and those supplied by the
trigeminal nerve.
Post herpetic neuralgia is a common complication.

LABORATORY DIAGNOSIS

Direct detection : electron microscopy can be used to detect herpes virus particles in vesicle fluid.
Isolation: -by inoculation of cell monolayers. The virus grows slowly with the production of typical
cytopathic effect usually evident in seven to ten days.

All herpesviruses replicate in the nucleus of the cell. An accumulation of viral proteins results in the
formation of characteristic eosinophilic intranuclear inclusions.
Minor differences in CPE allow distinction between the different types of herpesvirus.

Herpes simplex virus

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1. Prominent foci, or plaques, of rounded cells typifies


infection with HSV-1.
2. The nuclei become distorted and contain eosinophilic inclusions.
3. Other manifestations of CPE include the formation of "giant cells", and also multi-nucleate
syncytia (most common with HSV type 2).

Varicella zoster virus will replicate in either epithelial cells


(as shown here) or fibroblasts.
CPE takes much longer to become apparent than with HSV.

Nuclei of infected cells become enlarged and display prominent eosinophilic intra-nuclear inclusions.
Rounding of cells and strong affinity for eosin, is characteristic at late stages of infection.
Direct immunofluorescenc on material from lesions
Serology can detect specific IgM; present in both primary varicella as well as zoster

Therapy

ACYCLOVIR

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Nucleoside anologue of Guanosine.


Converted to acyclovir-tri-phosphate in herpesvirus-infected cells:

Acyclovir* - Acyclovir-MP* - Acyclovir-DP* - Acyclovir-TP


viral kinase* cellular kinases ACTIVE FORM

During viral DNA replication, the viral DNA polymerase incorporates Acyclovir-TP into the growing
DNA chain. This interferes with viral DNA synthesis by: Binding irreversibly and blocking the viral
DNA polymerase, and causes chain termination of the viral DNA

NB! Only HSV and VZV are sensitive to acyclovir.

See Antiviral Therapy

Cytomegalovirus

Human cytomegalovirus (HCMV) rarely causes disease in healthy people. This is particularly true
when infection occurs in childhood. Most individuals are infected in the first few years of life and by
adulthood 70-90% of people have IgG antibodies. Virus is secreted in the saliva of healthy carriers
and transmission usually occurs through close contact. When infection occurs in adulthood it may
cause an infectious mononucleosis- like illness associated with hepatitis, fever and lymphacytosis.

Like other herpes viruses, following primary infection, the virus becomes latent and may reactivate at
any stage.

Transmission:

(1) Close contact


(2) Blood transfusion

There are two clinical situations where infection with CMV may cause serious disease.

• (1) Congenital infection


• (2) Immunosuppressed individuals

Congenital infection

If a mother becomes infected with HCMV during pregnancy, the infant is at risk of congenital
infection.

Congenital HCMV infection is a difficult clinical problem to manage because


(1) maternal infection is usually asymptomatic
(2) the foetus may be damaged at any stage during pregnancy
(3) either re-activation or primary infection in the mother can result in foetal infection
(4) the vast majority of HCMV infected babies do not develop congenital abnormalities.

Clinical features

Most infected babies appear normal at birth. Affected infants may, however, later develop deafness
or mental retardation.
Rarely a severe generalized CMV infection of the neonate may occur (Cytomegalic inclusion
disease). This condition is associated with jaundice, hepato-splenomegaly, thrombocytopenia,

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haemolytic anaemia and microcephaly.


The histology of affected organs shows enlarged cells (‘cytomegalo’) with owls eye inclusions.
The prognosis for this condition is poor.

Infection in immunosuppressed individuals

Transplant patients and patients with AIDS, may develop life-threatening disease following either
primary infection with HCMV or reactivation.
Common syndromes include:

Interstitial pneumonia, retinitis, enteritis, or disseminated infection


Laboratory Diagnosis

Direct detection :-HCMV antigen in patient’s WBC’s by immunofluorescence


Culture: -monolayers of human fibroblasts.
Virus grows slowly and produces a characteristic cytopathic effect - (see colour photographs of
CPE)

Cytomegalovirus

Human cytomegalovirus replicates only in fibroblast cells (not in epithelial cells).


The virus is slow-growing and thus foci are small and discrete. Characteristic eosinophilic "owl-eye"
inclusions are evident in the nuclei of infected cells.

Serology :- IgG and IgM antibodies

Treatment

Gancyclovir or Foscarnet
These drugs are highly toxic and should only be used to treat life threatening infections

Epstein Barr Virus

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Infection is widespread. Most people have antibodies by the time they reach adulthood. Following
primary infection, the virus persists in a latent form in the B lymphocytes of the host. Periodic
reactivation of the virus is associated with shedding of virus in saliva.

Transmission is by close contact, especially kissing.

Clinical Syndromes associated with EBV infection

Primary Syndromes:-

1) Infectious Mononucleosis
2) ‘Chronic EBV infection’
3) X-linked lymphoproliferative syndrome
Re-activation Syndromes
4) Lympho-proliferative disorders in immunocompromised patients
5) Burkitts Lymphoma
6) Naso-pharyngeal Carcinoma

Primary Syndromes

Infectious Mononucleosis
Illness results from primary infection with EBV. A clinically apparent illness only develops when
primary infection occurs in adolescence or adulthood.

Clinical Features

The incubation period is 4 to 7 weeks

The route of infection is usually close contact. Virus is secreted intermittantly in the saliva of
asymptomatic carriers. Two main periods when the virus is transmitted are:
amongst small children particularly in day care settings where mouthing of toys is common, and
in adolescence where kissing is common.

Signs and symptoms

Fever, generalised lymphadenopathy, sore throat, malaise, tiredness, splenomegaly, hepatomegaly,


abnormal liver function tests and atypical lymphocytosis in the peripheral blood. The disease is self
limiting, but convalescence may be prolonged in some cases

Laboratory Diagnosis

Paul-Bunnell test (Monospot):

70-80% of patients with acute IM make heterophile antibodies that agglutinate sheep red blood cells.
These antibodies can be adsorbed (removed) with ox RBC’s, but not guinea pig kidney.

Specific serological tests:

• Antibody to the viral capsid and nuclear antigens are useful for confirming the diagnosis of
acute IM:
• IgG and IgM to Viral capsid antigen (VCA) is detectable early during the acute phase

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• VCA IgM is only present during acute phase


• IgG to EBV nuclear antigens (EBNA)becomes detectable late in convalescence

X linked Lymphoproliferative syndrome

Rare X-linked recessive disorder in which affected individuals develop a fatal primary infection when
exposed to EBV.

B cell Latency

EBV infects B lymphocytes and establishes a latent infection. The viral genome enters the nucleus
and persists in an episomal form. Six viral genes, termed EBNA 1-6 are expressed during this stage.
They transform the B cell into an immortal, continuously dividing cell. A small number of these
"EBV-transformed" B cells circulate in the blood of healthy carriers. Their numbers are kept in check
by the host's immune response.

Lymphoproliferative disorders

Immunosuppressed patients may develop a polyclonal proliferation of EBV- transformed B cells.

Burkitt’s Lymphoma (BL)

This is a high grade B cell lymphoma which occurs endemically in equatorial Africa and New
Guinea and, sporadically, world-wide. Almost 100% of equatorial BL tumours are known to be
associated with EBV.
EBV is believed to be an important co-factor in causing this malignancy, based on the following
evidence:

• 1) BL is a B cell lymphoma and EBV is known to transform B cells


• 2) All patients have high levels of antibodies to to EBV antigens
• 3) EBV genome can be detected in tumour cells.
• 4) Virus particles have been detected in cultured BL cells.

The areas which are endemic for BL are also hyper-endemic for Malaria and it is thought that the
malignant change in EBV-transformed B cells may follow infection with Malaria.

Nasopharyngeal Carcinoma (NPC)

This is a malignant tumour of the squamous epithelium of the nasopharynx.


Undifferentiated forms of this tumour are associated with EBV:

• 1) Patients have high levels of antibodies to EBV antigens.


• 2) EBV genome is present in NPC cells.
• 3) Virus can be recovered from cultured NPC cells.

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Other Malignancies:

EBV has been implicated as a cofactor in a number of other malignancies, including leiomyosarcomas
in AIDS patients and Hodgkins disease. But the role of EBV in the aetiology of these conditions has
yet to be resolved.

Human herpesviruses 6 and 7

Human Herpesvirus 6 (HHV6)

In 1986 a new herpesvirus was isolated from the peripheral blood mononuclear cells of six patients
suffering from a variety of lymphoreticular disorders. It has since become apparent that infection is
widespread in the population. Primary infection is usually acquired early in life and, like other herpes
viruses, the virus establishes a latent infection.

Clinical Features

Most infections are probably asymptomatic. Two types of disease have been found to be associated
with HHV6 infection:

1) Roseola Infantum
a febrile illness with a rash that usually occurs in the first few months of life.

2) Mononucleosis
with cervical lymphadenopathy in adults experiencing a primary infection.

HHV6 has been isolated from many patients with lymphoproliferative disorders, but its significance
is unknown.

Human Herpesvirus 7 (HHV7)

This virus is closely related to HHV6 and has been isolated from peripheral blood mononuclear
cells.
It is not known whether it causes any disease.

HHV 8 which is associate to different epidemiological manifestations of the Kaposis’s carcinoma


encountered in SIDA

ARBOVIRUSES
and viral
HAEMORRHAGIC FEVERS

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GROUP COMMON FEATURES


There is a large heterogenous group of RNA viruses (>400), which cause essentially zoonotic
infections of wild animals and are spread by insects (hence the name arboviruses) and occasionally
cause problems in man. They have the unusual ability to multiply in the vertebrate host (usually
mammals) as well as in the very different metabolism of the insect vector.

DISTRIBUTION
They are mostly found in the warmer parts of the world (tropics) but some range into temperate
regions. Rapid transport today means every country is at some risk.

NATURE
The majority are zoonoses maintained silently in nature by their animal (vertebrate) hosts which form
their natural reservoir. In their normal hosts they have usually adapted well and do not produce overt
disease. They produce high blood titres of virus which favours their chances of being picked up by a
blood-sucking insect vector. Within that vector they can multiply again, which favours their chances
of being transmitted from the insect vector to a new member of their definitive animal host. Crossing
into non-normal hosts may produce disease.

HUMAN INFECTION
Man usually forms an accidental, non-normal host, with sporadic - end point’ infections which have
no further transmission. However, there are some notable exceptions to this generalisation, where
human infection and disease can be spread to other humans via human-feeding insects or even
directly person to person. Human infections thus tend to be isolated or sporadic, unless climatic
conditions have favoured large-scale vector proliferation when human (and animal) infections may
become seasonal and epidemic. Some vaccines but no specific treatments are available.

TRANSMISSION
Climate has a major role in facilitating transmission, eq. mosquito proliferation after heavy (warm)
rains. Viral replication only occurs in the insect vector in warm conditions (>25’C). Human activity
can bring man into contact with an otherwise silent cycle in nature, eg. clearing of forests, agriculture.
Creating vector promoting activities, such as dams and irrigation, may enhance human arbovirus
disease potential. Within the vector the virus multiplies without making the insect sick and in some
viruses there is evidence there may be trans-ovarial (vertical) spread, ie. the virus can overwinter from
one season to the next inside the insect eggs. The virus is present in the insect saliva and the infection
is lifelong in the insect.

LABORATORY
Arboviruses are single-stranded enveloped RNA viruses with haemagglutinating properties. They
grow in suckling mice and/or in cell cultures. Mouse inoculation is hazardous to human workers and
must only be done in special containment facilities (National Institute for Virology in RSA).
Diagnosis is made by isolation of the virus (usually done in a specialist laboratory) and by detection
of IgM and IgG antibodies by haemagglutination-inhibition, or ELISA.

CONTROL
Human disease control is limited to prevention by

1. control of vector, eg. mosquito control which con be very effective; and,
2. use of vaccines in a few types of infection, eg. Yellow Fever,
3. surveillance.

HUMAN DISEASE

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Infection of man may produce a wide spectrum of disease - from subclinical infections to fulminant
haemorrhagic disease with death. By and large, there are 4 major clinical patterns of disease. Any one
virus characteristically tends to produce mostly one pattern, but overlapping may occur.

Major Clinical Categories of Arboviral Disease

• No clinical illness.
• Febrile systemic illness.
• Encephalitis
• Haemorrhagic Fever.

1) No clinical illness

Severe clinical disease in man attracts attention, and many arboviruses were first discovered (and still
are today) by investigation of unexplained, possibly infectious, disease cases. As the investigation
progressed, it was commonly found that there were milder disease cases in the vicinity of a severe
disease case, aa well as subclinical cases of infection. We now know that subclinical cases are
probably more common than frank disease in any one outbreak, eg. even in a yellow fever epidemic.
New Arboviruses have also been discovered by intensive cultural and serological investigation of
possible insect vectors (mosquitos, ticks) and possible definitive hosts (rodents, birds, small
mammals, etc.).

2. Febrile systemic illness

After inoculation there is a short incubation period of a few days, and then an abrupt onset of disease
at a time of high viraemia. There is a sudden onset of fever with chills; headache; muscle, bone and
joint pains; nausea and vomiting, and often a rash and lymphodenopothy. These clinical features may
be mild or very severe and prostrating.
The disease is typically self-limiting and short lived (few days to a week). Occasional cases may go
on to a more fulminant form (- see haemorrhagic fever section).

Examples

In South Africa.

West Nile Virus.


Asia, Central and South Africa (Transvaal).
Birds and mosquitoes. Human outbreaks after rains.
Rift Valley Fever.
East Africa, Egypt, Israel, South Africa.
Disease (with abortions) in sheep, cows, goats, spread by mosquitoes.
? occasional direct droplet spread.
Definitive host not identified.
Human outbreak follows animal outbreak after rains.
Sindbis
Africa & SE Asia (Transvaal, OFS)
Birds/mosquitoes.
Mild "Summer popular fever" epidemics in man.

Wesselsbron, Chikungunya

Other than South Africa:

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Dengue.
SE Asia,, Carribean and Central America.
Mosquitoes
Definitive host is unknown (? is none).There are seasonal epidemics in man.
4 Serological strains without cross-protection.
Previous exposure may predispose to haemorrhagic form.
(Has been imported into RSA.

3. Encephalitis

Almost all arboviruses may infect the CNS but some characteristically produce an encephalitis in
man.
Symptoms start with fever, progressively severe headache, nausea and vomifing, neck stiffness,
paralysis, decreased consciousness, convulsions and death in severe cases.

Examples (none in South Africa)

Japanese B Encephalitis

SE Asia - seasonal.
Birds/mosquitoes - transmitted to man.
2 Human vaccines available..
(Closely related Saint Louis Encephalitis - USA, Murray Valley Encephalitis - Australia)
Eastern Equine Encephalitis. (EEE).
Eastern USA, Central & South America.
Birds/mosquitoes.
Severe CNS disease in horses and man.
Western Equine Encephalitis. (WEE)
North & South America.
Birds/mosquitoes.
Severe disease in horses. Milder disease in man.
Tick Borne Encephalitis (TBE)
Central Europe, East Russia.
Goats/ticks.
Goats milk seems to be infectious.

4. Haemorrhagic Fever

A number of arboviruses (Toga viruses and Bunyaviruses) and some similar viruses (Arenaviruses
and Filoviruses) may cause an illness similar to the rather non-specific flu-like illness described under
(2) above, but which rapidly progresses to a severe disseminated illness with a marked bleeding
tendency and multi-organ failure.

There is usually a considerable mortality in these haemorrhagic fever infections.


Note:- Overwhelming parasitaemia/septicaemia may produce a similar clinical picture and
require to be excluded,
eg. meningococcus, rickettsia (Tick Bite Fever), leptospirosis, hepatitis, malaria, snake bite,
tryponosomiasis.

Mild and subclinical cases also do occur, but are less easily recognised.

Examples:

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In South Africa:

Crimean Congo Haemorrhagic Fever


(Eastern Europe, Central and Southern Africa)

Hyalomma ticks are known to transmit the disease to animals and man. There is a brief but high-titre
viraemic phase in mammals, at which stage their blood is highly infectious.
Note
Antibodies to CCHF have been detected in cattle, sheep and small mammals over a wide area in
Southern Africa without evidence of disease, and antibodies have been found in farm workers with no
confirmation of Congo Fever disease.

Several human outbreaks and individual cases have been reported previously in South Africa.
In November, 1996, a number of cases of Congo Crimean Haemorrhagic Fever (CCHF) were
identified in individuals working in an ostrich abbatoir in the town of Oudtshoorn, Western Cape
Province, South Africa.
It remained to be determined whether ostriches can be infected (and themselves serve as an infectious
source), or whether they may merely carry infected ticks (they certainly do carry ticks) that can be
passed to humans.
See: "Some Fascinating Facts about the 1996 CCHF Outbreak," by Bob Swanepoel

What is Congo-Crimean Haemorrhagic Fever?

The following is an extract from an article in the SAMJ, Vol. 62, p576-580, October,1982
by James H. S. Gear et al.

ONLY FOR INFORMATION

Congo-Crimean haemorrhagic fever

Congo-Crimean haemorrhagic fever was first observed in the Crimea by Russian scientists in 1944
and 1945. At that time it was established by studies in human volunteers that the aetiological agent
was filtrable and that the disease in man was associated with the bite of the tick Hyalomma
marginatum. The agent was detected in the larvae and in adult ticks, as well as in the blood of patients
during the fever. This agent, presumably a virus, was not maintained in the laboratory and was lost.
Congo virus was first isolated in Africa from the blood of a febrile patient in Zaire in 1956. In 1967
Simpson et al. described 12 cases of a feverish illness of which 5 were laboratory infections; the virus
was isolated by the inoculation of blood into newborn mice. Simpson showed that these viruses were
similar to the one isolated in 1956. Casals then showed that the viruses isolated in cases of Crimean
haemorrhagic fever and the Congo virus were serologically indistinguishable and demonstrated that
other virus strains from Central Asia, the USSR and Bulgaria were similar.
The virus has been classified as a Nairovirus in the genus Bunyavirus in the family Bunyaviridae. It
contains RNA and is inactivated by lipid solvents and detergents.
Laboratory studies have shown that Congo virus is related to Hazara virus isolated from ticks in
Pakistan, and to Nairobi sheep disease virus; together they form the Nairovirus group.
In Africa the virus has been isolated from a variety of animals, including cattle, sheep, goats, hares
and hedgehogs, and from a number of ticks which parasitize them, including Hyalomma sp.,
Amblyomma variegatum, Boophilus decoloratus and Rhipicephalus sp.

The most important transmitters of the infection to man are species of the genus Hyalomma, the life
history of which is shown in the figure below.

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The larval and nymphal stages of some species parasitize birds, including migratory birds, some of
which fly from south-eastern Europe to South Africa and thus may carry the infection over long
distances. To verify whether this actually happens will require further study of the ticks and their
hosts in South Africa and on their way from Europe.

Clinical picture

The infection is usually transmitted to man by the bite of a tick, but an increasing number of cases
have occurred among the medical and nursing staff caring for patients in hospital and in laboratory
personnel carrying out investigations of these patients. In these cases the infection has apparently
been acquired by contagion, particularly by contact with the patient's blood or blood-contaminated
specimens. Exposure to the blood of infected animals, especially cattle and sheep, has led to severe
and often fatal infections.

The incubation period is 2 - 7 days. The onset of the illness is sudden, with fever, chills, severe
muscular pains, headache, vomiting and pain in the epigastric and lumbar regions. A haemorrhagic
state develops from the 3rd to the 5th day and manifests as petechial haemorrhages or purpura in the
skin, and bleeding from the mucous membranes manifests as epistaxis, haemoptysis, haematemesis,
melaena and haematuria. At this stage the conjunctivae are injected, the face is flushed and the tongue
is dry, often coated with dry blood. The pulse is slow in the beginning, but with continuing loss of
blood becomes fast and feeble; the blood pressure drops and the heart sounds become weak - clear
signs of impending shock and vascular collapse. The liver is enlarged and tender and there is
tenderness over the epigastrium and splenic region. In patients who recover, the temperature falls
between the 10th and the 20th day and bleeding stops, but convalescence is prolonged up to 4 weeks
or longer. In fatal cases, death from massive haemorrhage and cardiac arrest occurs, usually 7 - 9 days
after the onset of the illness. Massive haemorrhage into the gastro-intestinal tract, with scattered
haemorrhages into the viscera, is found at autopsy.

The diagnosis is suggested on clinical grounds when the patient has a history of a tick bite or of
exposure to ticks in the environment, and after an incubation period of 2 - 7 days develops an illness
of sudden onset of muscle pains, headache fever and a rapidly evolving severe illness with the
development of a haemorrhagic state with bleeding from the mucous membranes and petechiae in the
skin, associated with thrombocytopenia and leucopenia.
The diagnosis may be confirmed in the laboratory by intracerebral inoculation of baby mice with
blood of a patient; the mice sicken about 1 week after inoculation. The virus is identified by using
known specific Congo virus antiserum in an immunofluorescent test. The development of antibodies

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in patients' serum as the illness progresses may be demonstrated in immunofluorescent tests using
chamber slides with tissue culture cells infected with Congo virus.

The authors are grateful to Professor 0. W. Prozesky, Director of the National Institute for Virology,
and to Dr R. Swanepoel, Dr K. Struthers, Mrs E. Rossouw and Miss G. McGillivray, staff members of
the high-security laboratory, and to Dr P. Jupp of the Arbovirus Unit of the National Institute for
Virology for undertaking, as a matter of urgency, the investigations which led to the incrimination of
the Congo virus as the cause of this patient's fatal illness. The authors are grateful to Mrs M.
Anderson, who prepared the chart of its life cycle.

REFERENCES

1. Gear JH S. Haemorrhagic fevers of Africa, an account of two recent outbreaks. J. S Afr Vet
Assoc 1977; 48: 5-8.
2. Pattyn SR, cd. Ebola Virus Haemorrhagic Fever. Amsterdam: Elsevier North Holland
Bioniedical Press, 1978: 301.
3. Simpson DIH, Knight EM, Courtois G et al. Congo virus: a hitherto undescribed virus
occurring in Africa. 1. Human isolations - clinical notes. East A fr Med J 1967; 44: 86-92.
4. Hoogstraal H. The epidemiology of tick-bome Crimean-Congo hemorrhagic fever in Asia,
Europe and Africa., J Med Entomol 1979; 15: 307-417.
5. Burney MI, Ghafoor A, Saleen M, Webb PA, Casals J. Nosocomial outbreak of viral
hemorrhagic fever caused by Crimean hemorrhagic fever - Congo virus in Pakistan, January
1976. Am J Trop Med 1980; 29: 941-947.
6. .Tantawi HH, AI-Moslih MI, AI-Janabi NY et al. Crimean-Congo haemorrhagic fever virus
in Iraq: isolation, identification and electron microscopy. Acta Virol (Praha) 1980; 24: 464-
467.
7. Suleiman MN, Muscat-Baron JM, Harries JR et al. Congo/Crimean haemorrhagic fever in
Dubai: an outbreak at the Rashid Hospital. Lancet 1980;ii: 939-941.

See "What is CCHF?"


A rare disease, but the most common viral haemorrhagic fever in South Africa.
Most frequent in Kimberley area, but antibodies are widespread in cattle herds all over South Africa.
Transmitted by ticks with striped legs (bont-poot bosluis, Hyalomma species).

Host:
cattle, ticks, ? other small mammals.
History:
visit to country in post week;
contact with animals;
tick bite;
contact with known human case (blood is highly infectious, can spread direcfly).
Illness:
short incubation (sudden onset - headache, fever, chills, body pains; diarrhoea & vomiting;
dizzyness, confusion, abnormal behaviour,.
pharyngitis, conjunctivitis, red face and neck,
haemorrhage starts day 3 - 6 (not all cases),
multi-organ failure, that leads to death.
Treatment:
isolation and barrier nursing;
transfusions,.

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ribavirin.

Outside South Africa:

YellowFever (YF)
Africa:
Central, West East.
Central South America (Amazon basin).
Hosts: Monkey - mosquito (=silent reservoir).
Jungle (sylvan) YF
sporadic cases in forest worker/visitor.
Urban )YF
YF introduced into towns;
now get cycle: man - mosquito - man.
NB. Mosquito control is important.
Illness: as above, but jaundice is a prominent feature (liver necrosis).
Vaccine:
i) "17 D strain" live attenuated virus
Good safe long lasting protection but labile and expensive to administer.
Used in the RSA.
ii) "French Dakar Vaccine" live attenuated brain tissue-derived vaccine
Inoculated by skin scratch.
More stable and cheap to administer.
Occasionally causes CNS complications.
Widely used in Froncophone Africa.
Vaccination is required for travel to endemic zones (or rather, for return to non-endemic
zones!)
Lassa Fever
West Africa.
Multimammate mouse, ? vector.
Human blood is highly infectious.
Ebola
Zaire & Sudan.
No known reservoir/vector hosts.
Human blood is highly infectious - gives rise to outbreaks in hospitals and clinics, if great
care is not taken.
Marburg
(Green Monkey Disease) ? Central Africa.
Has occurred in RSA. - ? imported.
Hanta viruses
Asia, Europe, North America ? more widespread.
Rodent borne, not true arboviruses.
Droplet and rodent excreta spread.
"Hoemorrhagic Fever with Renal Syndrome" (Korean HF)
Recently: Hanta virus respiratory illness in USA.

ARBOVIRUS
Togaviridae Alphavirus =Chikungunya
Flaviviridae Flavivirus =Fièvre jaune
=Dengue
=Maladie de la forêt de
Kyasanur ( en Inde)
=Fièvre hémorragique d’Omsk (

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en Russie)
Bunyaviridae Nairovirus =Fièvre hémorragique de
Crimée Congo
Phlébovirus =Fièvre de la vallée du Rift
Virus de Haantan =fièvre hémorragique avec
syndrome rénal
AUTRES VIRUS
Arenaviridae Virus Lassa =Fièvre de Lassa
Virus Junin =Fièvre hémorragique
d’Argentine
Virus Machupo =Fièvre hémorragique de
Bolivie

Filoviridae Virus Ebola =Fièvre hémorragique à virus


Ebola
Virus Marburg =Fièvre hémorragique à virus
Marburg

INFLUENZA

Influenza is a disease caused by a member of the Orthomyxoviridae. Many features are


common with those of the paramyxovirus infections of the respiratory tract.
CLINICAL FEATURES

Influenza is characterised by fever, myalgia, headache and pharyngitis. In addition there may be
cough and in severe cases, prostration. There is usually not coryza (runny nose) which characterises
common cold infections.

Infection may be very mild, even asymptomatic, moderate or very severe (see letter from 1918, -
attached at end of page).

Source
The reservoir is acute infection in other human beings.
Spread
Is rapid via aerial droplets and fomites with inhalation into the pharynx or lower respiratory
tract.
Incubation
Is short: 1-3 days. Rapid spread leads to epidemics
Complications
Tend to occur in the young, elderly, and persons with chronic cardio-pulmonary diseases

Consist of:
1. Pneumonia caused by influenza itself; and

2. Pneumonia caused by bacteria


- Haemophilus influenzae
- Staphylococcus aureus
- Streptococcus pneuminiae

3. Other viral superinfection, eg. Adenovirus.

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Overall death rates increase in times of influenza epidemics.

LABORATORY DIAGNOSIS

A. Viral Isolation:

Respiratory secretions:
- direct aspirate
- gargle
- nasal washings

a) Rapid examination of cells by immunofluorescence.


b) Inoculation of cell cultures (or eggs).

B. Serology
serum antibodies by haemagglutination inhibition

INFLUENZA VIRUSES

Diagramatic representation of the morphology of an influenza virion.

The virion is generally rounded but may be long and filamentous.


A single-stranded RNA genome is closely associated with a helical nucleoprotein (NP), and is
present in eight separate segments of ribonucleoprotein (RNP), each of which has to be present for
successful replication. The segmented genome is enclosed within an outer
lipoprotein envelope. An antigenic protein called the matrix protein (MP 1)
lines the inside of the envelope and and is chemically bound to the RNP. The
envelope carries two types of protruding spikes. One is a box-shaped protein,
called the neuraminidase (NA), of which there are nine major antigenic
types, and which has enzymic properties as the name implies.

The other type of envelope spike is


a trimeric protein called the haemagglutinin (HA)
(illustrated on the right)
of which there are 13 major antigenic types. The haemagglutinin functions
during attachment of the virus particle to the cell membrane, and can combine
with specific receptors on a variety of cells including red blood cells.
The lipoprotein envelope makes the virion rather labile - susceptible to heat,
drying, detergents and solvents.

CLICK here to see Electron micrographs of the virion

Influenza virus (an Orthomyxovirus) is responsible for acute upper respiratory disease, usually
accompanied by fever and myalgia.

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Virions are usually roughly spherical and about 200nm in diameter.


The envelope contains rigid "spikes" of haemagglutinin and neuraminidase which form a
characteristic halo of projections around negatively stained virus particles. .

The viral genome is composed of eight segments of ssRNA.


The helical ribonucleo-protein is not often seen,
but occasional particles show evidence of internal helical components.

REPLICATION

The Life Cycle of Influenza Virus

Receptor-bound viruses are taken into the cell by endocytosis. In the low pH environment of the
endosome, RNP is released from MP1, and the viral lipoprotein envelope fuses with the lipid-bilayer
of the vesicle, releasing viral RNP into the cell cytoplasm, from where it is transported into the
nucleus. New viral proteins are translated from transcribed messenger RNA (mRNA). New viral RNA
is encased in the capsid protein, and together with new matrix protein is then transported to sites at the
cell surface where envelope haemagglutinin and neuraminadase components have been incorporated
into the cell membrane. Progeny virions are formed and released by budding.

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The cell does not die (at least not initially).

Flu is one of a rare few viruses that has its genome in separate segments (eight). - This increases the
potential for recombinants to form (by interchange of gene segments if two different viruses infect
the same cell), and may contribute to the rapid development of new flu strains in nature - can also be
duplicated in the laboratory (used for making vaccine strains). Avian and human strains recombining
in pigs in the Far East may permit virulent human strains to evolve.

CLASSIFICATION of virus STRAINS


Is done on the basis of antigenicity of NP and MP into three main groups:

Influenza A -HA undergoes minor and occasional major changes - very important.
- NA some variation.
Influenza B) Undergoes relatively slow change in HA with time. Known only in man.
Influenza C) Uncommon strain, known only in man.

NOMENCLATURE
Influenza strains are named in the following way:
A SINGAPORE 6 86 (H1N1)
TYPE TOWN NUMBER YEAR MAJOR
of influenza where first isolated of isolates of isolation TYPE of HA and NA

EPIDEMIOLOGY

Influenza A virus is essentially an avian virus that has "recently" crossed into mammals. Birds have
the greatest number and range of influenza strains. Avian haemagglutinins sometimes appear in pig
human and horse influenza strains.
Every now and then (10 - 15 years) a major new pandemic strain appears in man, with a totally new
HA and sometimes a new NA as well (antigenic shift). This variant causes a major epidemic around
the world (pandemic).
Over the subsequent years this strain undergoes minor changes (antigenic drift) every two to three
years, probably driven by selective antibody pressure in the populations of humans infected.
See chart below indicating main pandemic strains in previous years.

Influenza A Evolution
1874 --- (H3N8)
1890 --- (H2N2) .........................Pandemic
1902 --- (H3N2)
1918 --- (H1N1)..........................Pandemic
1933 --- (H1N1)..........................First strains isolated
1947 --- (H1N1)..........................Variation detected
1957 --- (H2N2).........................."Asian" Flu pandemic
1968 --- (H3N2).........................."Hong Kong" Flu pandemic
1976 --- (H1N1).........................."Swine" Flu, non-epidemic
1977 --- (H1N1) + (H3N2)........."Russian" Flu epidemic

This constant antigenic change down the years means that new vaccines have to be made on a
regular basis.

New influenza strains spread rapidly in children in schools and créches and in places where people
crowd together. Influenza epidemics may cause economically significant absenteeism.

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TREATMENT

Antibiotics are often prescribed - have no effect on virus but may prevent or cure bacterial
superinfection. The drug Amantadine may prevent influenza if taken continuously by high-risk
persons at the time of an epidemic, but is not used widely.

PREVENTION

Vaccines at best give about 70% protection.


They may sometimes not be effective against the most recently evolved strains because the rate of
evolution outpaces the rate at which new vaccines can be manufactured.

Types of Vaccine

Killed Whole Virus


Rather pyrogenic, not used today.

Live Virus
Attenuated strains were widely used in Russia but not elsewhere.

Virus Subunit
HA extracted from recombinant virus forms the basis of today's vaccines.
For example, the WHO Recommendation for Influenza Vaccine, 1995-1996, contains two A
strains and one B strain:-

A / Singapore / 6 / 86 (H1N1)
A / Johannesburg / 33 / 94 (H3N2)
B / Beijing / 84 / 93

Synthetic
Much research is being done to try and find a neutralising epitope that is more stable, and can
therefore be used for a universal vaccine.

PROSPECTS

Because another devastating pandemic strain (cf 1918 pandemic) may appear at any time, the World
Health Organisation (WHO) maintains worldwide surveillance of flu strains and makes predictions of
suitable strains for vaccine production.

ONLY FOR INFORMATION

Pandemic influenza 1918

An illustration of what influenza can be like: a copy of a letter by Professor N R Grist (Glasgow)
published in the British Medical Journal of 22-29 December 1979:-

Epidemic influenza remains the biggest and unconquered acute threat to human health, inflicting
damage and death far beyond familiar notification data. The impact of influenza A is particularly
severe during periodic pandemics owing to novel antigenic variants which override immunity from
experience of earlier subtypes. It is salutary to remember that we do not really understand why the
devastating pandemic of 1918-19 was so severe, and that we cannot therefore be confident that our

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modern medical measures would succeed against a similar future challenge.


As a reminder of the grim reality of that pandemic, the following letter may be of interest. It was
found in a trunk in Detroit among other medical papers handed to the department of epidemiology of
the University of Michigan. A copy was given to me in 1959 by the late Dr T Francis, jun, and it is
now published with the agreement of Dr V Hawthorne, his successor as head of department.
N R GRIST

Copy of original letter found in Detroit in 1959

Camp Devens, Mass.


Surgical Ward No 16
29 September 1918
(Base Hospital)

My dear Burt-
It is more than likely that you would be interested in the news of this place, for there is a possibility
that you will be assigned here for duty, so having a minute between rounds I will try to tell you a little
about the situation here as I have seen it in the last week.
As you know I have not seen much Pneumonia in the last few years in Detroit, so when I came here I
was somewhat behind in the niceties of the Army way of intricate Diagnosis. Also to make it good, I
have had for the last week an exacerbation of my old "Ear Rot" as Artie Ogle calls it, and could not
use a Stethoscope at all, but had to get by on my ability to "spot" ' em thru my general knowledge of
Pneumonias. I did well enough, and finally found an old Phonendoscope that I pieced together, and
from then on was all right. You know the Army regulations require very close locations etc.

Camp Devens is near Boston, and has about 50,000 men, or did have before this epidemic broke
loose. It also has the Base Hospital for the Div. of the N. East. This epidemic started about four weeks
ago, and has developed so rapidly that the camp is demoralized and all ordinary work is held up till it
has passed. All assembleges of soldiers taboo.
These men start with what appears to be an ordinary attack of LaGrippe or Influenza, and when
brought to the Hosp. they very rapidly develop the most viscous type of Pneumonia that has ever been
seen. Two hours after admission they have the Mahogany spots over the cheek bones, and a few hours
later you can begin to see the Cyanosis extending from their ears and spreading all over the face, until
it is hard to distinguish the coloured men from the white. It is only a matter of a few hours then until
death comes, and it is simply a struggle for air until they suffocate. It is horrible. One can stand it to
see one, two or twenty men die, but to see these poor devils dropping like flies sort of gets on your
nerves. We have been averaging about 100 deaths per day, and still keeping it up. There is no doubt in
my mind that there is a new mixed infection here, but what I dont know. My total time is taken up
hunting Rales, rales dry or moist, sibilant or crepitant or any other of the hundred things that one may
find in the chest, they all mean but one thing here -Pneumonia-and that means in about all cases death.

The normal number of resident Drs. here is about 25 and that has been increased to over 250, all of
whom (of course excepting me) have temporary orders-"Return to your proper Station on completion
of work". Mine says "Permanent Duty", but I have been in the Army just long enough to learn that it
doesnt always mean what it says. So I dont know what will happen to me at the end of this.
We have lost an outrageous number of Nurses and Drs., and the little town of Ayer is a sight. It takes
Special trains to carry away the dead. For several days there were no coffins and the bodies piled up
something fierce, we used to go down to the morgue (which is just back of my ward) and look at the
boys laid out in long rows. It beats any sight they ever had in France after a battle. An extra long
barracks has been vacated for the use of the Morgue, and it would make any man sit up and take
notice to walk down the long lines of dead soldiers all dressed and laid out in double rows. We have
no relief here, you get up in the morning at 5 .30 and work steady till about 9.30 P.M., sleep, then go
at it again. Some of the men of course have been here all the time, and they are TIRED.

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If this letter seems somewhat disconnected overlook it, for I have been called away
from it a dozen times the last time just now by the Officer of the Day, who came in to
tell me that they have not as yet found at any of the autopsies any case beyond the
Red. Hepatitis. stage. It kills them before they get that far.

I dont wish you any hard luck Old Man but I do wish you were here for a while at
least. Its more comfortable when one has a friend about. The men here are all good
fellows, but I get so damned sick o Pneumonia that when I go to eat I want to find some fellow who
will not "Talk Shop" but there aint none nohow. We eat it live it, sleep it, and dream it, to say nothing
of breathing it 16 hours a day. I would be very grateful indeed if you would drop me a line or two
once in a while, and I will promise you that if you ever get into a fix like this, I will do the same for
you.

Each man here gets a ward with about 150 beds, (Mine has 168) and has an Asst. Chief to boss him,
and you can imagine what the paper work alone is - fierce,-- and the Govt. demands all paper work be
kept up in good shape. I have only four day nurses and five night nurses (female) a ward-master, and
four orderlies. So you can see that we are busy. I write this in piecemeal fashion. It may be a long
time before I can get another letter to you, but will try.

This letter will give you an idea of the monthly report which has to be in Monday. I have mine most
ready now. My Boss was in just now and gave me a lot more work to do so I will have to close this.

Good By old Pal,


"God be with you till we meet again"
Keep the Bouells open.
(Sgd) Roy.

VIRAL RESPIRATORY INFECTIONS

Respiratory infections are common, eg. colds in both adults and children. Most are fairly mild, self-
limiting and confined to the upper respiratory tract (URT). Most are probably viral induced - at
least initially. However, in infants and children, URT infections may spread downwards and cause
more severe infections and even death.

CLINICAL-ANATOMICAL DEFINITIONS

Upper Respiratory Tract


1. Colds
Main feature: watery to mucoid, sometimes purulent nasal discharge "coryza".
Often preceded by a sore throat, sometimes accompanied by fever and often
followed by transient opportunist bacterial infection.

2. Pharyngitis ("sore throat")


Generalised erythema of pharynx, not localised to the tonsils and not associated
with coryza. Some fever present.

3. Tonsilitis
Local infection of tonsils = red, swollen with exudate on the surface. (Bacterial tonsilitis is quite
common.)

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4. Sinusitis & Otitis Media


Painful inflammatory conditions of sinuses and middle ear. Drainage of these spaces may be
impaired and lead to bacterial infection. (Bacterial infections are usually secondary to viral
infection of the nose and pharynx.)

5. Influenza
Fever, myalgia, sore throat, headache, prostration - usually NOT much nasal discharge compared to a
cold. Maybe some cough.
See also separate lecture notes on INFLUENZA

Lower Respiratory Tract

a) Laryngo-Tracheo Bronchitis (Croup)


An acute viral inflammation of larynx and trachea in small children. Often preceded by a "cold".
Accompanied by pyrexia, hoarseness, croaking cough, stridor, restlessness (respiratory insufficiency).
Can be fatal - ie. life-threatening disease.

b) Acute Bronchitis
Inflammation of bronchi, accompanied by fever, cough, wheezing and "noisy chest".

c) Acute Bronchiolitis
Inflammation of terminal bronchioles in small children. Bronchiole diameter is larger during
inspiration than during expiration and this leads to hyperinflation of air sacs distal to bronchiole.
Complete plugging of bronchiole with air resorption leads to collapse. These features can be seen on
x-ray. These changes cause respiratory embarrassment and can be life-threatening. Bronchiolitis
appears in seasonal epidemics in Britain but it can be seen all the year round in the Red Cross
Childrens' Hospital, in the poorer communities of the Cape Peninsula. Usually preceeded by coryzal
symptoms which later develops into the major pulmonary illness. Clinically there is fever, rapid
respiration, exhausting cough and wheezing.

d) Pneumonia & Bronchopneumonia


Acute respiratory disease accompanied by fever, restlessness and cyanosis. Often not much clinical
"consolidation". Again, can be life-threatening.

WHAT CAUSES THESE INFECTIONS?

Studies in Britain have shown, for example, that of fifty cases of children with lower respiratory tract
infection, only one showed evidence of a bacterial pneumonia. By far the majority appeared to be
viral in origin. Bacterial infections are important because they can be life-threatening and are
treatable with antibiotics, whereas there are as yet no widely-used antiviral agents for respiratory
viruses.

One of the problems is that any respiratory infection requiring medical professional attention is almost
invariably treated with antibiotics. Only the "problem cases" can get into the hospitals where viral
diagnostic facilities exist. Even so, a high proportion of respiratory problems in small children do not
seem to respond to antibiotics and are presumed to be viral.

An important concept is that a wide variety of respiratory pathogens may cause one clinical syndrome
and, vice versa, any one pathogen may cause a wide range of clinical diseases - see diagram attached,
followed by a list of the main respiratory viruses.

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Viral Respiratory Pathogens

• ADENOVIRUS
• PARAINFLUENZA VIRUS
• RESPIRATORY SYNCYTIAL VIRUS
• RHINOVIRUS

In addition, all the following viruses may cause "UNKNOWN -


presumed viral" respiratory disease

• COXSACKIE
• ECHO
• HERPES I
• CORONA
• EBV
• CMV

- infection may be present without disease, ie. "healthy children" -

PARAMYXOVIRUSES

General Properties of the Paramyxoviruses


The whole important group of Paramyxoviruses is fairly homogeneous.

Morphology
Generally fairly similar to influenza: roughly spherical sometimes filamentous, easily distorted. A bit
larger than influenza virus - 100-300nm. Composed of inner helical nucleocapsid containing
protein/RNA, contained within a membranous envelope studded with "spikes" of:
Haemagglutinin (Haemadsorption) and Neuraminidase (Haemolysis)

See electronmicrographs.

The family of Paramyxoviridae contains viruses that induce a wide range of distinct clinical illnesses
in humans:-
These include measles virus, which in rare instances is followed by subacute sclerosing
panencephalitis (SSPE); mumps virus, which has symptoms of parotitis, orchitis and encephalitis,
and the parainfluenza viruses which are respiratory pathogens .

.
Virions are enveloped and enclose a helical nucleocapsid containing non-segmented single-stranded
RNA.

Most virions are roughly spherical (about 200nm in diameter) but they can be much larger and more
pleomorphic.

The virus envelope is a lipid bilayer, studded with virus encoded glycoproteins which have properties
of haemagglutination
and fusion (the F protein).

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By transmission electron microscopy, and negative staining, the helical ribonucleo-protein capsid has
a "herring-bone" appearance.

Antigens
Antigenically distinct from influenza virus. Within the paramyxovirus group there is considerable
overlapping, eg:
Parainfluenza 1, 2, 3 and mumps. Exposure of an adult to any one of these viruses often induces a
rise in serum antibodies to the others. Thus, interpretation of serum antibody levels in this group of
viral infections may be difficult.

As in influenza vrus, there are two major antigens:


1. The haemagglutinin, present in the envelope of the intact virion (this is sometimes called the "V"
antigen);
2. On breaking open the virion, or rupturing of infected cells before virion assembly, a "soluble"
virus antigen is released, which is thought to be the protein component of the nucleocapsid. This is
sometimes called the "S" antigen.

Infection induces antibodies to these two major antigens, amongst others.


The antigens are fairly stable. In contrast to influenza virus, there is no antigenic drift or epidemics of
infection

Primary infections
usually occur in (early) childhood, with some resultant degree of protection against developing
clinical disease later on in life. However, re-infections do occur in adulthood, but disease is
subclinical or very minor.

Spread
The human paramyxoviruses are essentially diseases of man only, and are spread by droplets from the
nose and mouth to fairly close contacts. Many of them are fairly highly infectious and go around the
community in epidemics - often seasonal, eg. Winter coughs and colds. Fomites might also assist
spread.

Laboratory Diagnosis

1. Viral Detection
Isolation of a paramyxovirus from a patient is strong evidence for a cause of a respiratory infection.
All the Paramyxoviruses are to be found in the respiratory tract secretions - and this is the best
material to send to the laboratory. The paramyxoviruses are unstable and do not survive well outside
cells.

a) Direct Examination
Viral antigens or viral infected cells in the secretions may be directly and rapidly detected by
immunofluorescence or ELISA tests.

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b) Culture
Primary Monkey kidney cells will support replication of all the Paramyxoviruses and many of the
other respiratory viruses.

An intriguing property of most Paramyxo's is their ability to induce cell fusion. Neighbouring cells
join up to form large multinucleate syncytia or giant cells. Viral fusion to cell for penetration follows
proteolytic cleavage of fusion protein on virion surface.
One of the first discovered members of the Paramyxo group - probably a mouse virus - called
Sendai , is used as a cell fusing agent in experimental work on cells. The Sendai virus is first
inactivated by UV (which damages the nucleic acid) so that the virus will not grow and interfere with
the experiment. The inactivated virus still induces cell fusion - even across species, eg. heterokaryons
of murine and human cells.

Multinucleated giant cells can sometimes be seen in lung sections from children dying with
Paramyxovirus infection in the lung. Not all Paramyxos do this readily, and haemadsorption is usually
used to detect Paramyxovirus infection in culture cells.

Once isolated, a haemadsorbing virus can be further typed using standard specific antisera in various
laboratory procedures:

• haemadsorption inhibition;
• neutralisation; and
• fluorescent antibody.

2. Serological Diagnosis
Antibody determinations in acute and convalescent blood specimens by complement fixation, Elisa or
haemagglutination inhibition may sometimes be helpful in arriving at a specific diagnosis.
Respiratory syncytial virus and measles virus infections are fairly clearcut.
Mumps and Para influenza viruses seem to share a group common antigen, and serological
interpretation may be difficult.

Clinical Picture

PARA-INFLUENZA Types 1, 2, 3 and 4

All can cause minor infections in children and adults.


Types 1, 2 and 3 may be associated with more severe lower respiratory tract disease in children. For
instance, in an American series of cases, 30% of acute laryngo-tracheo-bronchitis (LTB) cases
yielded para-influenza viruses.
Type 1 is especially associated with LTB, sometimes also type 2.
Paraflu's have also been isolated from patients with pneumonia.

The virus grows locally in the respiratory tract lining of the URT and it may then spread down into
the lungs.
IgA type antibodies are induced. These are present in the respiratory secretions and seem to be more
important than the IgG antibodies in the serum with regard to protection.
However, IgA antibodies do not cross the placenta and babies thus have no maternal protection
against this type of infection. Primary infections with para-influenza viruses usually occur in the first
year or years of life. Re-infection usually causes only minor infection of the URT - one of the causes
of a common cold in children and adults.

Diagnosis
In cell cultures, the para-influenza viruses produce a recognisable cytopathic effect, and
haemadsorption is also used to detect their presence.

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Each virus type has specific antigens by which they can be "typed", and there are also a few minor
subtypes.

Treatment
No specific treatment is available. Killed virus vaccines have been tried but are of limited value in an
infection which is so widespread and usually of trivial significance.

Respiratory Syncytial virus - (RSV)

This is an unusual but very important member the Paramyxovirus group:


while it resembles the other members morphologically, no haemagglutinin or neuraminidase or
haemolytic properties have been detected, and there is no antigenic similarity to other members. In
cell cultures it readily induces many large syncytia with cytoplasmic inclusions - hence its name.

Clinical
RSV was first isolated from chimpanzees with colds, and it was soon found to cause colds in man as
well. (Chimps were probably infected by their human handlers.) However, it was also found to be
associated with severe pulmonary infections in infants - especially Bronchiolitis. RSV is the prime
cause of Bronchiolitis.

In Britain, RSV is the single major pathogen in respiratory infections of childhood. The figures
from Newcastle by Gardner are startling:

under 1 year of age:


78% of Bronchiolitis
38% of LTB
36% of Pneumonia
35% of Bronchitis
12% of minor respiratory illness,

were all caused by RSV.

RSV causes a fairly localised infection of the respiratory tract, and infants have no maternal passive
protection.
Re-infections do occur but seem only to produce fairly minor URT illness.

Vaccine
Because of its apparent importance, an attempt was made at vaccinating infants. However, when the
RS epidemic arrived, the vaccinated children suffered more severe reactions than unvaccinated
children. Somehow the killed virus vaccine had unfortunately sensitised these children and exposure
to the live virus then induced a type of allergic reaction. This drew attention to the possibility that RS
viruses may in fact cause much of its lung damage by immune mechanisms. Because of the
importance of RSV in childhood infections, intensive efforts to make a vaccine have continued,
especially along the lines of sub-unit vaccines (parts of virions) avoiding those antigens which seem
responsible for hypersensitisation. However, no vaccine is as yet in general use.

RHINOVIRUSES

(Common Cold virus)

Over 100 serotypes of this Picorna virus family are responsible for about 50% of common colds.

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Clinical
An inhalational infection of the URT. Incubation period is short: 1 to 3 days followed by headache,
sore throat, fullness in the nose. Then there is a profuse watery discharge from the nose which
gradually thickens and becomes mucopurulent and decreases in volume. The infection resolves in
about a week. Following a rhinovirus cold, there is a short period of immunity to all colds but
prolonged immunity to the specific serotype causing the recent infection.

Treatment
No specific treatment but numerous symptomatic treatments are available.

Complications
A cold may temporarily upset the mucosal cilia and predisposes to secondary invaders especially
bacterial infections, eg. sinusitis (pneumococcus, haemophilus, etc) and bronchitis and possibly
pneumonia. These may require antibiotic treatment.

Epidemiology
An infected person is infectious in the first two days of coryza. Colds are readily acquired from
breathing room air from a room crowded with coldy people. Wet cold weather per sé does not cause
colds, but may predispose to infection from other persons. Colds are ubiquitous around the world
except in very isolated communities.

Prevention
The enormous diversity of cold-causing viruses essentially rules out a vaccine. Vitamin C and
bacterial vaccines are unproven.

ADENOVIRUSES

Adenoviruses are non-enveloped icosahedral particles.


The capsid is built up from 252 capsomers (T=25), of which
240 are hexavalent and 12 (situated at the apices) are
pentavalent. A "penton fibre"projects from each apex.

The penton fibres consist of a slender shaft with a globular head.


They are involved in the process of attachment of the virus particle to the host cell.

Fibres easily become detached during preparation for electron microscopy.

Adenoviruses that infect humans are usually mild pathogens, and can cause respiratory illness or
conjunctivitis (so-called "pink eye"), but under laboratory conditions some human strains can
transform cells in culture.
Most adenoviruses grow easily in cell culture, and give rise to large intranuclear inclusions.

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1. Group Common Antigen


Shared by all adenoviruses, eg.a single complement fixation test can detect a rise in antibody to any
adeno infection also, a single immunofluorescent reagent can be made which will react with all
adenoviruses.

2. Type Specific Antigen


Can identify a specific type by neutralisation of infectivity or haemagglutination. 47 Serotypes are
grouped into about 6 larger groups (labelled A ....... to F).

Clinical
Adenoviruses cause infections of man (many animals have their own adenoviruses) and are spread by
droplet, fomites and ingestion. They infect the mucous membranes of the eye, respiratory and gastro
intestinal tract, occasionally urinary tract. Local lymph nodes often involved (enlarged and tender).
Infections are usually self-limiting.

Syndromes

1. Asymptomatic Infection
Adenoviruses may be present in healthy persons, eg. in stools of children, and may also cause
persistent silent infection of the tonsils.

2. Acute pharyngitis with fever


Adenoviruses are a common cause of an acute sore throat.

3. Pharyngoconjunctivical fever
Adenoviruses may cause an acute conjunctivitis together with a shore throat and fever.

4. Acute follicular conjunctivitis


This is a non-purulent contagious inflammation with pearly, translucent lymphoid hyperplasia.

5. Epidemic kerato-conjunctivitis (shipyard eye)


Mild trauma to the eye may facilitate a damaging adeno infection of cornea (spread by multi- shared
towels).

6. Pneumonia (and pneumonitis in children)


In some cases there may be a severe destructive pneumonia (and death) caused by an adenovirus
infection following measles or other predisposing factors, eg. artificial airway.

7. Acute respiratory disease (ARD)


Is an epidemic form of acute pneumonic disease characteristically appearing in military camps. Has
been prevented by enteric capsulation of a live vaccine strain which bypasses the respiratory tract and
sets up a silent infection in the gut, giving protection against acute respiratory infection.

8. Diarrhoea
In young children many adenoviruses may cause a generalised infection - upper and lower respiratory
tract infection with fever and diarrhoea. Quite separately, some adenos (40/41) have been
specifically associated with causing acute gastroenteritis in children, which may lead to dehydration
and death.

9. Mesenteric Adenitis
Children may present with abdominal pain due to enlarged tender adenovirus infected mesenteric
lymph nodes. Occasionally an enlarged node may invaginate the bowel wall and be gripped by
peristaltic waves, leading to intussusception of the bowel.

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10. Immunocompromised host


In transplant patients, AIDS or other immunocompromised patients, adenoviruses may cause a variety
of infections - renal, disseminated, or a haemorrhagic cystitis.

HUMAN PAPILLOMAVIRUSES

(WART VIRUSES)

FAMILY: PAPOVAVIRIDAE
GENUS: PAPILLOMAVIRUS

CLINICAL FEATURES AND VIROLOGY

Papillomaviruses occur in many animals as well as man but have narrow host specificity and tissue
specificity. Human papillomaviruses do not infect any animal hosts.

In humans, HPV is one of the most common viral infections. Spread is by contact, eg. hand to hand;
sexual transmission in the case of genital infections; infection during birth in the case of juvenile
laryngeal papillomas.

There are more than 80 different types of HPV. Typing is based on the DNA sequence of the virus.
Specific lesions are associated with specific HPV types. There are three major groups of HPVs.

1. Cutaneous warts
2. Epidermodysplasia verruciformis
3. Mucosal HPV infections

VIRUS STRUCTURE

The papillomaviruses are small non-enveloped icosahedral viruses (55nm) and have a circular double
stranded DNA genome of about 8kb. This link will allow you to view electron micrograph images of
the virus particle.

Papillomavirus

Many types of papillomavirus cause benign skin tumours (warts) in their natural hosts. These warts
often regress spontaneously, but human genital warts (tumours caused by specific types of

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papillomavirus, particularly types 16 and 18) regularly become


malignant if they persist for a sufficiently long time.

Papillomavirus particles are approximately 55nm in diameter.

The capsid is composed of 72 morphological units, or capsomers,


arranged on the surface of a T=7 icosahedron. The capsomers located at each of the 12 vertices, are
pentavalent (i.e. each is surrounded by five adjacent capsomers), and the other 60 capsomers are
hexavalent (each adjacent to six capsomers).

A model of the papillomavirus capsid is shown beside a computer colorized EM image.

All 72 capsomeres are pentamers of the major structural protein, termed L1. This arrangement of
subunits violates the theory of quasi-equivalence put forward by
Caspar and Klug in 1962.

LIFE CYCLE OF HUMAN PAPILLOMAVIRUS

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HPVs infect the germinal epithelial cells through damaged epithelium. The viral DNA replicates in
the lower layers of squamous epithelium. However, expression of the late genes coding for the viral
capsid proteins is linked to the differentiation of the epithelial cells and viral particle are only
detectable in the upper layers of the epithelium. Therefore if the cells fail to differentiate as observed
in malignancy, there is a non-productive infection. This is the reason why it was only when molecular
techniques were developed for the detection of HPV DNA that it became possible to link HPV and
cervical carcinoma.

Cutaneous warts
(HPV types 1-4, 7, 10, 26-28)

These viruses cause warts (benign tumours) of keratinised squamous epithelium, ie. skin. They are
very common and the peak incidence is in children between the ages of 10 and 14 years of age. The
cutaneous group of viruses has traditionally been classified according to clinical appearance and
location. There are four clinical types:

• verruca vulgaris or common wart


• deep hyperkeratotic palmoplantar wart or myrmecia
• superficial mosaic type palmoplantar wart
• verruca plana or flat wart

In most people skin warts regress spontaneously.

Epidermodysplasia verruciformis
(HPV types 5, 8, 9, 12, 14, 15, 17, 19-25)

This is a rare life-long disease associated with specific HPV types. There appears to be a genetic
component to the disease as large proportions of people with the disease are descendants of
consanguineous marriages. It is probably an autosomal recessive based inheritance. The infected
people develop widespread warts with about 30% possibility of the lesions progressing to malignancy.
Malignancies are most likely to develop in sun-exposed and traumatised area.

Mucosal HPV lesions

These HPV viruses infect the genital tract, oral cavity, conjunctiva and respiratory tract. The majority
of infections are latent with no visible lesions.
Table 1 lists the major HPV types associated with mucosal HPVs.

HPVs are also the most common sexually transmitted viruses. It is estimated that there are 450 000
new cases of cervical cancer worldwide, 10 million cases of high grade dysplasia, 30 million cases of
low grade dysplasia and 300 million cases of cervical HPV infection without cytological
abnormalities (IARC, 1999). About 200 000 women will die of cervical cancer each year.

Condylomata acuminata (genital warts): This is the most common benign genital tumour found in
both sexes. These tumours hardly ever progress to malignancy. In the USA and UK, 1-3% of sexually
active people have genital warts.

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Laryngeal papillomas: This is a rare condition found in both adults and children. The juvenile
laryngeal papillomas are a result of transmission from the mother to the infant during birth. The same
HPV types are found in laryngeal papillomas and genital warts. Although this is a benign tumour they
have to be removed on a regular basis to prevent blockage of the respiratory tract.

Table 1. Major clinical association of genital tract and other mucosal HPVs (from Fields et al., 1996)

CLINICAL ASSOCIATION HPV TYPES


GENITAL TRACT
Subclinical infection All types
Exophytic condyloma - any site HPV 6 , 11
Flat condyloma - especially cervix HPV 6, 11,16, 18, 31, others
Bowenoid papulosis HPV 16
Giant condyloma HPV 6, 11
Cervical cancer
• Strong association HPV16, 18, 31, 45
• Moderate association HPV 33, 35, 39, 51, 52, 56, 58, 59, 68
• Weak or no association HPV 6, 11, 26, 42, 43, 44, 53, 54, 55, 62
Vulvar cancer HPV 16
Penile cancer HPV 16

RESPIRATORY PAPILLOMAS HPV 6, 11


CONJUNCTIVAL PAPILLOMAS HPV 6, 11

ORAL CAVITY
Focal epithelial hyperplasia HPV 13, 32
Infection with genital tract HPVs HPV 6, 11, 16
Lesions on Lip HPV 2

Cervical cancer and other cancers:

A significant proportion of cancers is associated with HPV infection: 11% of all cancers in women
and 2% of all cancers in men. South Africa has one the highest incidences of carcinoma of the cervix
in the world. This is particularly true in our disadvantaged communities. The malignant and
premalignant lesions (cervical intraepithelial neoplasia) can be detected by routine cervical screening
(PAP smears). If detected early, treatment is very successful. The high incidence of the disease in
South Africa is mainly due to poor cervical screening programmes.

It is now accepted that specific types of HPV play a causal role in cervical cancer. There is
epidemiological evidence that cervical cancer is associated with a sexually transmitted agent. This
includes strong risk factors such as early age of first intercourse, multiple sexual partners, high parity
etc. The disease is virtually unknown in nuns and virgins and is relatively common in commercial sex
workers. Over the years a number of sexually transmitted agents have been investigated as possible
aetiological agents for cervical cancer. The strongest association has been found between specific
HPV types and cervical cancer. HPV types 16 ,18, 31 and 35 are most commonly found in cervical
cancer biopsies with HPV 16 being found in half of the cervical cancer cases worldwide. They are
also found in penile and anal carcinomas. Recent studies also support the hypothesis that cervical
intraepithelial neoplasia is caused by HPV. HPV DNA can be detected in over 95% of cervical cancer
and intraepitheial neoplasia biopsies.

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MOLECULAR BASIS OF HPV INDUCED GENITAL CANCERS

Experimental evidence supports the epidemiological findings that specific HPV types do play a role in
progression to malignancy.

HPV types associated with malignancy (HPV 16 and 18) have different characteristics compared with
those types associated with benign lesions (HPV 6 and 11). One of the important steps in progression
to cancer is the disruption of the activity of two host tumour suppressor proteins; namely p53 and the
retinoblastoma protein (pRb). Tumour suppressor gene products have the normal task of halting cell
growth. The HPV E6 gene product binds p53 and the HPV E7 gene product binds pRb. However the
E6/E7 proteins of HPV 16 and HPV 18 have a much higher affinity for the tumour suppressor gene
products than the E6/E7 proteins of HPV 6 and 11. This difference helps explain the difference in the
transforming abilities of the virus associated with benign and malignant lesions. If the function of p53
is disrupted then the cell cycle does not pause to allow repair of damaged DNA. This results in an
accumulation of mutations in the cells and it is postulated that eventually one of the proto-oncogenes
will mutate. Proto-oncogenes are genes involved in normal cell growth and division. When mutated
they promote uncontrolled cell growth and are known as oncogenes.

In the "normal" life cycle of the virus the production of E6 and E7 are controlled by the E2 gene
product. However in cancers the E2 gene is disrupted during integration of the viral genome into the
host cell. This results in uncontrolled production of E6 and E7 for interaction with the tumour
suppressor gene products. Once again integration is mainly observed with the HPV types associated
with malignancy ie HPV 16 and 18.

It is important to realise that very few women infected with HPV will progress to cervical carcinoma.
In a study done at UCT, 13% of women with normal cervical cytology were found to be infected with
HPV. However, the incidence of cervical carcinoma in the Black population is 38 per 100 000.
Cancer is a multistep process and HPV is only one of the steps in the progression to cancer. Several
co-factors have been identified including immunosuppression, smoking and the presence of other
sexually transmitted diseases.

DIAGNOSIS OF HPV INFECTION

- No serological diagnosis
- No convenient cell culture
- Cytology
- Histology
- Colposcopy in the case of genital HPV infections.
- Electron microscopy. Only applicable in benign lesions as in malignant lesions the viral DNA is
integrated and viral particles are not produced.
- Immunocytochemistry can detect major capsid protein but are generally group specific not type
specific
- DNA detection techniques. This is the only way to type HPVs. Typing is considered to be important,
as specific HPV types are associated with malignancy. Polymerase chain reaction can be used to
detect HPV DNA. At present the only commercial test available is the "Hybrid CaptureTM" test
which will differentiate between oncogenic and non-oncogenic HPV infections.

PREVENTION

Since a few years there are vaccines to prevent HPV infection (prophylactic vaccination) There is
also research taking place to develop therapeutic vaccines to treat people infected with HPVs.

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MUMPS

Mumps virus causes a febrile illness and has a tropism for glandular tissue. It is also

relatively neuroinvasive. Mumps virus is spread by the respiratory route, and has a relatively long
incubation of about 21 days.

It causes a febrile illness and inflammation of the salivary glands, classically the parotid and
submaxillary glands. The swelling of the glands may be asynchronous, and lasts about 1 week.

Aseptic meningitis is a fairly common complication of mumps, and by corollary, mumps is a fairly
common cause of aseptic meningitis (up to 1/3 of cases in Cape Town). In about half of the mumps
meningitis cases, parotitis will not be apparent.

Mumps meningoencephalitis is rarer but a more serious development.

Orchitis can occur, more often after puberty, unilateral or bilateral, but is rarely followed by
infertility. Other glandular tissue is very occasionally involved viz. pancreatitis, oophoritis or
thyroiditis.

Diagnosis can be confirmed by positive IgM antibodies. In cases of meningitis the virus is readily
isolated from cerebro-spinal fluid.

Prevention: see MMR vaccine under Rubella

Mumps is a paramyxovirus, the virus family with the tubular spaghetti-like nucleocapsid. Measles
is also a paramyxomvirus. The other human paramyxoviruses cause respiratory infections - they are
the parainfluenza viruses and respiratory syncitial virus.

Mumps is considered as a common old garden infection that you may have had or seen someone with.
It causes swelling of the salivary glands, especially the parotids. The swelling is usually bilateral, and
can be uneven, ie. one side starts before the other. The child will already have been a bit feverish and
unwell for a few days. Now mumps virus fancies glandular tissue, and the next most common gland
that it affects, after the salivary glands, is the testis; so a common and unpleasant complication in
boys after puberty is orchitis. Contrary to old wives tales, it is rare to become infertile from mumps
orchitis. The ovaries can also sometimes be affected causing oophoritis; and the pancreas causing
pancreatitis.

Apart from glands, mumps virus crosses the BBB (blood brain barrier) rather easily, and if you did a
lumbar puncture on every child with mumps you would find that 10% of them had meningitis, even if
they didn't have overt symptoms. Mumps meningitis is common - it causes about 1/3 of viral
meningitis in Cape Town, but it has a good outlook. Sometimes the virus goes further and actually
invades the brain, ie meningoencephalitis. This is a bit more serious, and the most common long-term
consequence is unilateral deafness.

For suspected mumps meningitis it is quite easy to grow the virus from cerebro-spinal fluid.

Mumps virus CPE is indistinguishable from that of RSV (shown above).


However, mumps virus encodes a haemagglutin protein which is incorporated in the virus envelope,
and appears at the cell surface from which progeny virions will bud. If erythrocytes (red blood cells)
are added to infected cell sheets, they will adhere to the cell surface.

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This process is termed haemadsorption


and allows differentiation between mumps and RSV infections.

RUBELLA and
PARVOVIRUS B19

RUBELLA and PARVOVIRUS B19 are world-wide childhood illnesses caused by unrelated
viruses:

Rubella virus and parvovirus B19 cause similar, mild rash diseases, and arthritis is a common
complication of both. Both may endanger the foetus if the mother is infected in pregnancy, but in
very different ways.

Parvovirus B19 has other unique complications in children and adults because of its tropism
(predilection or affinity) for immature red cells.

For both of these viral infections, man is the only natural host and reservoir, so that the viruses must
keep circulating in human populations, and tend to occur in small epidemics. However, because of
long-lasting immunity, these infections usually occur only once in one person's lifetime .
Note that the person is infectious for about a week before the onset of symptoms, and then for a few
days from the onset of symptoms.

RUBELLA

Rubella virus is shed in oropharyngeal secretions and is spread by the respiratory route. After a 2 - 3
week incubation, a rash of pink macules appears on the face and behind the ears, and spreads
downwards to the trunk and limbs. There is associated low-grade fever and lymphadenopathy,
specifically of the posterior cervical and occipital nodes. These signs resolve in a few days.
Arthralgia / arthritis, usually of the fingers and knees, can occur, especially in adult women.

It is relatively common for rubella infection to occur without a rash, so called "subclinical" infection.

Post-infectious encephalitis or thrombocytopaenia are very rare complications.

Congenital rubella syndrome

Primary rubella infection of the mother in the first 12 weeks of pregnancy is very likely to infect the
foetus, with a high risk (80%) of congenital abnormalities. As in other congenital infections, the

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baby at birth may be of low weight, and have a thrombocytopaenic rash and hepatomegaly with
jaundice.

Specific features of congenital rubella syndrome are: cataracts, micropthalmia, heart defects,
sensorineural deafness, mental retardation. Infection between about the 13th and the 17th week of
pregnancy may result in deafness alone, but infection beyond the 17th week is no longer a hazard.

A confirmed laboratory diagnosis of rubella in a pregnant woman is thus very important, as


termination should be offered for 1st trimester infections.
Rubella IgM by ELISA becomes detectable a few days after the rash appears. The presence of IgM
antibodies indicates an acute infection, and will be detectable for approximately 8 weeks. (Rubella
IgG will appear almost as early as the IgM, but then persists for life).
Suspected congenital rubella in a neonate is also diagnosed by IgM in the baby's blood, or by culture
of the virus from urine.
Note that maternal IgG class antibodies can cross the placenta, but not IgM, and so the presence of
IgM in the baby indicates production of its own antibodies in response to its own infection.
Congenitally infected babies may shed virus for many months, and staff and family contacts should be
aware that the baby is infectious for this time.

PREVENTION:

Rubella vaccine is not given routinely in childhood in South Africa. The vaccine is a live attenuated
strain called RA27/3. In some countries it is given as the combined MMR (measles, mumps, rubella
- all live, attenuated) vaccine to all children at 15 months of age, with a second dose at school entry.
Some countries have tried a policy of rubella vaccine alone given to girls at high school entry age (+/-
13 years) specifically to try to prevent congenital rubella. In practice, good herd immunity of both
boys and girls is necessary to prevent circulation of the virus and protect vulnerable pregnant women.
Women should be screened for immunity to rubella (indicated by IgG antibodies) prior to pregnancy
so that they can be vaccinated if necessary.
(Note that live virus vaccines should not be given during pregnancy, but inadvertent rubella
vaccination during pregnancy is not grounds for termination as there have been no cases of
congenital abnormalities associated with the vaccine.)
Often women are only screened during their first pregnancy, but they can at least be vaccinated prior
to the next pregnancy if they are non-immune.

PARVOVIRUS B19

This virus was only discovered in the 1970's, although the disease it causes, erythema infectiosum,
has long been described. It is also sometimes called "fifth disease" from an historical enumeration of
the rash diseases of childhood, or "slapped cheek disease" because of the facial rash. Its name "B19"
refers to the batch number of a serum in the bloodbank in which the virus was discovered.

Parvo B19 is also spread by the respiratory route, and the rash appears after about 17 days. It's
distinctive feature is the red cheeks, with circumoral pallor. On the rest of the body it is a lacy, pink
macular rash that fades quickly, but may reappear after a warm bath. Arthralgia or frank arthritis, in
fingers and knees most commonly, is more frequent and troublesome than in rubella. The arthritis can
in some cases persist for months, and can even imitate juvenile rheumatoid arthritis.

Parvo B19's site of replication is red cell precursors in the bone marrow. It utilises as its receptor the P
antigen (a red cell surface glycoprotein) which determines the P blood group phenotype.
The infection will cause a temporary shut-down in red cell production until the virus is eliminated by
the immune system, usually within 10 days. In a normal child this causes an insignificant drop in
haemoglobin of around 1g/dl. However, people with hereditary red cell disorders (eg sickle cell

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anaemia, hereditary spherocytosis, thalassaemia) have either red cell under-production or rapid red
cell destruction by haemolysis. In this context, the brief cessation of red cell supply caused by parvo
B19 infection will precipitate an "aplastic crisis" ie. severe anaemia. These patients present with
extreme pallor, lethargy and sometimes in cardiac failure, and require blood transfusion. A parvo
B19 vaccine will soon be available and will benefit this vulnerable group.

In persons with AIDS, or other immunodeficiency states eg. children with leukaemia on
chemotherapy, inability to clear the virus can cause chronic anaemia.
Normal immunoglobulin preparations for intra-muscular injection contain parvo B19 antibodies and
will usually successfully eliminate the infection in immuno-compromised patients.

Maternal infection with parvo B19 in pregnancy can cause foetal infection, and foetal anaemia. In the
worst cases (rare, usually second trimester infection) this may manifest as foetal hydrops. The
mechanism here is severe foetal anaemia causing cardiac failure with oedema. This may end in intra-
uterine death, but it has been possible to treat severely affected foetuses by intra-uterine transfusion,
given the right technology. The milder cases tend to resolve spontaneously.

Note that parvovirus B19, unlike rubella, is not teratogenic, and outcome of infections in pregnancy is
usually good.

Because the rash and mild illness cause by parvo B19 is very similar to rubella, it is essential in
pregnant women to distinguish between the two virus infections by laboratory diagnosis.
Laboratory diagnosis of acute parvo B19 is also based on the presence of IgM antibodies. The virus
cannot be cultivated in routine cell culture lines, but direct detection of the viral DNA may be
achieved by PCR.

Some extra information which is just for interest

The 2 viruses described above are not related to one another.

Rubella is a togavirus. Toga means coat. All the other togaviruses that can affect humans are
mosquito-borne viruses. Only one of them occurs in South Africa - it is is chikungunya virus, which
occurs in the E. Transvaal and quite often affects baboons, and occasionally man. It causes a fever,
rash and severe arthritis, which is very similar to rubella.

However, since rubella is a lone togavirus that does not have an insect vector, and spreads directly
from person to person, it has been put in a genus on its own within the togavirus family.

Parvovirus B19 is a parvovirus - these are the smallest virus particles known. Parvovirus B19 is
the only parvovirus that causes disease in man. Its name doesn't mean anything, the virus was found
in unit of blood in a bloodbank, and B19 was just the batch no. for that blood.

There are important animal parvoviruses , eg. feline parvovirus and canine parvovirus. These cause
leukopaenia (ie. depletion of white cells) and enteritis (ie. bowel inflammation with diarrhoea) in
kittens and puppies. When your pets have their routine vaccinations, you should ask the vet what they
are being vaccinated for. You'll find that the routine domestic animal vaccinations include these
parvoviruses, because they are lethal for young animals.

Both viruses cause really trivial childhood infections, BUT they all have interesting complications.

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We'll start with rubella, also called German measles because it was described by a German physician
in the 18th Century. This virus causes a rash, some lymphadenopathy (all the lymph nodes at the back
of the head and neck for some reason - post auricular, posterior cervical & occipital ) and quite often
arthritis in adults, especially women.

But generally it is a very mild illness, and seemingly of no consequence…

However back in 1940 there was an astute Australian opthalmologist called Gregg. He saw 78 cases
of cataracts in babies in one year, one after the other, and often these babies had other serious
congenital abnormalities, especially cardiac. He linked these cases to a directly preceding epidemic
of rubella in Australia, and suggested that the babies' defects were a consequence of rubella during
pregnancy. He published his observations in the Transactions of the Opthalmological Society of
Australia, and there was huge scepticism - everyone said, rubella is a common and trivial disease,
how could we have missed this consequence up till now? But because of the awareness he had raised,
others started to confirm his observation in other populations. Gregg was the first to introduce the
concept of viruses as teratogens. A teratogen is an agent that causes MALFORMATION in the
developing foetus.

In 1964 there was a major epidemic of rubella in the USA. An estimated 20 000 babies suffered
permanent damage from in utero rubella infection - CONGENITAL RUBELLA SYNDROME.

The risks of rubella in pregnancy have now been carefully studied and its clear there is only a risk to
the foetus in the early part of pregnancy.

So what are the defects that these babies get ?


The virus specifically damages the HEART, commonly causing a patent ductus arteriosus, sometimes
pulmonary artery stenosis, the EYES (typically causing cataracts, but also other abnormalities) and
the NERVOUS SYSTEM causing mental retardation predominantly. Deafness is the commonest
sequelae of congenital rubella and should actually be included here as it is a nerve type of deafness.
What has also been observed is that the majority of major defects occur in the earliest weeks, up to
week 12.

It is worth mentioning that an interesting late manifestation in children who survive congenital rubella
, is DIABETES which becomes evident only in young adulthood.

After the epidemic in the USA, the general public became well aware of the risk of rubella in
pregnancy, and apparently there was a phenomenon of "rubella parties" - if a child in the
neighbourhood got rubella, all the little girls would be invited round to try to get them infected with
rubella so that they would be immune in adulthood before they ever fell pregnant.

However, in the late 60's a live attenuated vaccine became available commercially and was put into
use for all children in the USA - today in the USA they use the combined measles-mumps-rubella
vaccine, and children must have this vaccine before they start school. Rubella, and especially
congenital rubella syndrome, is now extremely rare in the USA. For the last few years they have had
about 4 cases of congenital rubella per year, and that is in a population of 260 million.

Those countries where they tried to vaccinate JUST HIGH-SCHOOL GIRLS have been less
successful in controlling rubella and congenital rubella. This is because with this strategy you will still
allow the virus to circulate in younger children and males, and at the same time it is impossible to
achieve vaccine immunity in 100% of women - so these 2 factors together will allow cases of
congenital rubella to occur.

Congenital rubella is not rare in South Africa, but given that very few in our population are
vaccinated, it's not that common either. Rubella is not part of the state childhood immunisation

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schedule here, and rubella vaccination is only offered in private medicine. So "wild " rubella virus
circulates freely, and in fact, the majority of women in South Africa will be immune to rubella by the
time they reach adulthood. For instance, new female nursing recruits are screened for rubella
antibodies when they start nursing, so that they can be vaccinated if they are non-immune; and about
80% of them are already immune.

Let's look at the sequence of events in rubella infection, and especially the immune response to
rubella. In fact this is very similar to events in parvovirus B19 infection, and in principle this scheme
applies to almost any type of viral infection.

If you take as the reference point the rash, the things to note are:

1. the virus has been incubating in the person for at least 2 weeks, and

2. they have been shedding virus from the throat (and therefore potentially infecting others) for a
week already;

3. the lymph nodes swell up before the rash and stay swollen after the rash (the rash itself is quite
brief).

4. the joint involvement starts at the end of the rash, towards the end of the illness;

5. the IgM becomes positive a day or 2 into the rash, and the IgG simultaneously or shortly
afterwards;

6. the IgM only lasts for about 8 weeks, but the IgG persists for life.

***********************

Now we'll move on to parvovirus B19.


This virus also causes a rash disease with arthritis - the rash is rather typical. Arthritis, again
especially in adults and especially in adult women, is common and can be quite severe. It usually
affects the finger joints, and also some of the bigger joints such as the knees, wrists, ankles. The
symptoms of arthritis are swelling and pain and sometimes redness.

But the key to understanding this virus's life-threatening complications is knowing that it is
erythrotropic, in other words it really replicates best in red cells, specifically young red cells which
haven't even left the bone marrow. It is directed into these red cells via the P antigen which is its
cellular receptor- Like the ABO blood group, and the Rhesus blood group and the Kell and Duffy
systems, there is a P blood group which is determined by whether you have a gene (big P) for the P
antigen or not. In fact almost everyone in the world is P positive ie they have at least one big P gene
(big P being a dominant gene).

The gene frequency of small p is very low. There are literally a handful of people who have two small
p genes and are P blood group negative, and studies of these people have clearly shown that they are
resistant to parvovirus B19 infection - they cannot get infected because the virus can't get access to
their red cells where it replicates.

The consequence of parvovirus B19 replicating in your bone marrow is that you can't make any red
cells until you get rid of the virus. Now for you and me this doesn't really matter - our red cells last
for 120 days on average, and 10 days without new red cells is not a problem. But there are people
who need to keep up red cell production all the time, and they are people with congenital red cell
abnormalities - whether of the red cell membrane, red cell enzymes or haemoglobin - they either have

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poor production of red cells or the red cells are so abnormal that they are rapidly broken down. These
people cannot afford to stop producing new red cells, and if they get infected by parvovirus they go
into severe anaemia; or what is known as aplastic crisis. For years, no-one knew what caused
aplastic crisis in these patients, but when parvovirus was discovered in the 1970's it was soon
recognised as a cause. Fortunately parvovirus infections are normally self-limiting, but these patients
will need transfusions of red cells to tide them over.

Another type of person who cannot afford to stop producing red cells is the rapidly growing foetus,
who needs a rapidly expanding blood volume. Maternal infection with parvovirus B19 can result in
foetal infection which can, in turn, result in a varying degree of foetal anaemia. Most foetuses seem
to get through the anaemic patch, but some don't and become very anaemic. The consequence of a
severe anaemia is cardiac failure, and a major manifestation of cardiac failure is oedema - these
foetuses become extremely oedemic, a condition which is called foetal hydrops (this translates as
"water on the foetus") and is visible on ultrasound. Parvovirus is just one cause of foetal hydrops.

Now, parvovirus is NOT a teratogen. It doesn't cause irreversible malformations, so if the baby
survives a parvo infection, and most of them do, it won't have congenital abnormalities, and its
outlook is good.

We know therefore that the outlook for rubella infections in utero is very bad in the first trimester, but
the outlook for parvovirus infections at any stage in pregnancy is quite good. With rubella there is a
strong case for termination of pregnancy, NOT for parvovirus.
The infections in the mother look very similar, so you must use a lab test to distinguish the two.

*********************

Diagnostically it is useful sometimes to think in terms of clinical syndromes -


"If I see a case of arthritis, what viruses should I consider? If I see a child with a rash, what viruses
could be responsible?", etc., rather than thinking of each virus in isolation.

DIAGNOSIS of these infections is quite simple today - an acute infection is diagnosed by an IgM
assay. And you should all know by now that this is because IgM is the first class of antibody that is
made in response to an infection, and it is transient; so if it is detectable, it means that the infection is
current or very recent.

Finding IgG alone means that the person has been infected or vaccinated sometime in the past.

Diagnosing infection in newborn BABIES - congenital rubella babies are usually IgM positive at
birth, and IgM stays positive for quite a long time. You can also culture virus from them at birth and
for a long time afterwards; their immune response is deranged and ineffectual because they were
infected at such an early, immature stage.

In congenital parvovirus babies, IgM is not reliably positive at birth, but if you suspect this congenital
infection it can be confirmed by PCR to detect the viral nucleic acid itself. These babies also tend to
be persistently infected for some time after birth because of an immature immune system.

RABIES

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INTRODUCTION
Rabies is an infectious disease of animals caused by a bullet-shaped, enveloped RNA virus, 180 x 75
nm. Man is occasionally infected, and once infection is established in the CNS, the outcome is almost
invariably fatal.

HUMAN RABIES
Is acquired from virus in saliva entering a bite wound caused by an infected animal, usually a rabid
dog. The severity of the bite determines the risk of infection. The disease does not usually spread
from man to man.

Incubation
After inoculation, the virus enters small nerve endings at the site of the bite. The virus slowly travels
up the nerve to reach the CNS where it replicates and then travels down nerves to the salivary glands
where there is further replication. The time it takes to do this depends upon the length of the nerve - a
bite on the foot will have a very much longer incubation period than a bite on the face. The
incubation period may last from two weeks to six months. Very often the primary wound is healed
and forgotten by the time of clinical presentation.

Clinical Presentation

A) Furious Rabies
When the virus reaches the CNS the patient presents with headache, fever, irritability, restlessness
and anxiety. This may progress to muscle pains, salivation and vomiting. After a few days to a week
the patient may experience a stage of excitement and be wracked with painful muscle spasms,
triggered sometimes by swallowing of saliva or water. Hence they drool and learn to fear water (*
Hydrophobia). The patients are also excessively sensitive to air blown on the face. The stage of
excitement lasts only a few days before the patient lapses into coma and death.
Once clinical disease manifests, there is a rapid, relentless progression to invariable death, despite all
treatment.

B) Dumb Rabies
Starts in the same way, but instead of progressing into excitement, the subject retreats steadily and
quietly downhill, with some paralysis, to death. Rabies diagnosis may easily be missed.

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ANIMAL RABIES
Very similar picture to human rabies. In the stage of excitement the animal may bite vigorously and
viciously at anything: sticks, stones, grass, other animals and humans, without provocation.

Wild animals may be abnormally tame or appear sick - beware of approaching or picking up such an
animal ("dumb rabies").

Epidemiology

1. The disease is endemic in wild animals in most parts of the world although some countries (UK,
Australia) are rabies free through vigorous control. The wild animal cycle constitutes the natural
reservoir.

2. Wild animals may bite and infect domestic animals (cattle, horses, pigs, dogs and cats) which in
turn may infect man. Occasionally wild animals may infect man directly.

3. In recent decades, a separate form of dog rabies (spread from dog to dog) has been recognised as
spreading from West Africa eastwards and southwards in Africa. Via Mozambique, it reached Natal
Kwazulu in the late 1970's and early 80's. Semi-wild dogs in Natal have formed the highest endemic
rabies reservoir and source for human cases in the whole of the RSA.

ANIMAL RESERVOIR

• MONGOOSE (main reservoir in RSA in the wild)


• SURICATE
• JACKAL
• BAT (some evidence to suggest carrier status and droplet infection)
• FOX (in Europe)
• SKUNKS, RACCOONS (in USA)
• SEMI-WILD DOGS (in Natal)

Note: As in man, an infected animal becomes sick and dies. There is no substantial evidence of a true
carrier status in apparently well animals, except perhaps in bats.

DIAGNOSIS
By assessment of:
1.Bite
Geographical area, type of animal, severity and site of bite.

2. Animal

Live - observe in cage:


If survives > 8 days, then NOT rabies.
Dead - brain sent to Onderstepoort
- Negri bodies
- IFA
- virus isolation

3. Man

Live - difficult diagnosis


- clinical picture, skin biopsy, corneal impression (antibodies only appear very late)
Dead - brain sent to Onderstepoort

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"Negri bodies" in cytoplasm of brain cells;


immunofluorescence virus isolation
TREATMENT

• 1. Wash wound (soap, detergent and water)


• 2. Anti-rabies serum (human). Passive immunisation.
• 3. Vaccine (intensive course). Active immunisation.

RABIES VACCINE
A good but expensive killed virus vaccine (Human Diploid Cell Vaccine, HDCV) grown in human
fibroblasts is available for safe use in man.
The unusually long incubation period of the virus permits the effective use of active immunisation
with vaccine post-exposure. When used, vaccine has dramatically cut the rabies death rate. Supplied
free by the State through district surgeons in South Africa.
(Older killed virus vaccines, made from infected neural tissues, were poorly immunogenic and had
allergic encephalitic side effects, but are still used in developing countries.)

Prophylaxis
High-risk persons, eg. veterinarians, may be immunised before exposure, and then merely require one
or two booster doses if they should be exposed to rabies.

Animal Vaccines
A range of live and killed virus vaccines are available for domestic animals (farm animals, cats and
dogs).

Experimental vaccination of wildlife by using recombinant vaccinia vaccine (live) in bait for foxes
in Europe and North America has been quite promising.

CONTROL OF RABIES (Government Department of Veterinary Services)


1. Education
2. Vaccination of dogs, cats and farm animals.
3. Notification
- animals (district vet officer, police, magistrate) - human (district surgeon)

NEUROLOGICAL DISEASES CAUSED BY VIRUSES

Neurological disease is one of the most serious complications of virus infection.


Clinically, viral neurological disease can be divided into
acute diseases and chronic syndromes.
The pathology may be due either to viral multiplication in the cells of the brain, or due to the
(misdirected) immune response of the host - post infectious encephalo-myelitis.
Where virus replicates in the brain, virus can usually be isolated from brain tissue or from
cerebrospinal fluid. This is not the case with the post infectious syndromes.

Acute neurological syndromes


Virus may reach the brain either by the blood stream or by spread along peripheral nerves.
Asymptomatic infection of the brain is common.

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There are four main syndromes:

• 1. Encephalitis
• 2. Flaccid paralysis
• 3. Aseptic meningitis
• 4. Post infectious encephalo-myelitis

Acute viral encephalitis

Viral replication occurs in the brain tissue itself, causing destructive lesions in the grey matter. The
main symptoms include: fever, drowsiness, confusion, convulsions and focal neurological signs.
Morbidity and mortality is very high.
Viruses that cause encephalitis include
Herpes Simplex, Rabies and some of the Arboviruses.

The Arboviruses include a miscellaneous group of enveloped, RNA viruses, that are transmitted
from one vertebral host to another via blood sucking arthropods. The main reservoirs are wild birds
and small mammals. Man may be infected if bitten by the insect vector. Arboviruses cause two types
of disease:

• 1. Encephalitis, and
• 2. Fever (often with haemorrhagic manifestations)

Arboviral encephalitis occurs in most parts of the world; but different agents are responsible for
disease in different areas. In South Africa, there are no endemic arboviruses that specifically cause
encephalitis. Rarely, however, encephalitis may occur as part of the clinical course of infection with:
West Nile virus, Rift Valley Fever virus, Sinbis virus, Crimean-Congo haemorrhagic fever or
Chickengunya virus. These viruses are endemic in livestock herds in certain parts of the country and
farm workers or vets may occasionally be infected.

[For more information, see lecture notes on viral haemorrhagic fevers].

Flaccid Paralysis

Due to direct infection of motor neurones. Patients present with fever and flaccid paralysis of a
group of muscles. The lower limbs tend to be involved more commonly than the upper limbs. Signs of
meningitis such as headache and neck stiffness are frequent accompanying features. The most
common aetiological agents include the polioviruses 1-3, but with the reduction in prevalence of wild
type polio, a number of non-polio enteroviruses have also been implicated as rare causitive agents.

Aseptic meningitis

Infection of the meninges. A relatively mild disease with a good prognosis. Patients present with
fever, headache, neck stiffness and photophobia. Common viral agents include: enteroviruses,
mumps virus and lymphocytic choriomeningitis virus.

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Post infectious encephalomyelitis

This uncommon complication may develop in the convalescent phase of a number of common virus
infections, including: measles, mumps, rubella and primary varicella-zoster virus infection, as
well as following the administration of certain vaccines, such as: vaccinia virus and the older
neurotissue rabies vaccines. Wide spread demyelinating lesions develop in the brain and spinal cord,
associated with lymphocytic infiltration and perivascular cuffing of adjacent blood vessels. Virus
cannot be isolated from brain tissue or CSF. The aetiology is somewhat obscure, but it is believed to
be an auto-immune phenomenon, triggered by exposure to foreign antigens which are closely related
to host proteins normally expressed in brain tissue (molecular mimicry).

Gillain Barre syndrome

Poly-neuritis which develops a few days after the acute phase of a viral infection. The disease is due
to demyelination of peripheral nerves. Patients present with an ascending paralysis, associated with
paraesthesia. Like post infectious encephalomyelitis, it is believed to be an immunological
phenomenon. Patients usually recover spontaneously over a few weeks or months. There is no
specific treatment.

Chronic Neurological Conditions:

A number of viruses and virus-like agents can cause chronic neurological disaese. Characteristically,
these conditions have a long incubation period followed by slow development of symptoms and a
progressive, uniformly fatal course.

• Subacute-sclerosing panencephalitis (SSPE)


• Progressive multifocal leuco-encephalopathy (PML)
• Retrovirus disease
• Spongiform encephalopathies

Subacute Sclerosing Pan Encephalitis

This is a rare, slowly progressive degenerative disease of the brain which develops six to eight years
following a primary uncomplicated infection with measles virus. It is due to persistent measles virus
infection in the CNS.

Clinical Features
Patients usually present with personality and behavioural changes followed by progressive intellectual
impairment, convulsions, myoclonic movements, coma and death.

Pathogenesis
It is thought that the virus gains entry into the CNS during the viraemia that occurs at the time of the
primary infection. The pathology is due to infection of the brain with a measles virus mutant that
lacks an essential structural gene and cannot therefore undergo complete cycles of replication. This
has two effects:

1.) It enables the virus to establish a persistent infection in the brain - because there is limited
expression of viral antigens on the surface of infected neurones, an effective immune response cannot
be mounted against the infected cells.

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2. ) The disease proceeds very slowly - because, as no infectious virus particles are released, spread
of infection to new cells only occurs through fusion with adjacent infected cells.

Epidemiology
Occurs in about 1 per million cases of measles. The incidence has declined sharply since the
introduction of vaccination against measles.

Laboratory diagnosis
Affected individuals have high titres of measles specific antibodies in their serum and CSF.
Measles virus antigens can be demonstrated in brain tissue

Progressive multifocal leuco-encephalopathy

There are two members of the Papovavirus family which are known to infect man, namely JC virus,
which replicates in neural tissue and BK virus which has been isolated from the urinary tract. Neither
of them cause any pathology in healthy people.
JC virus - is associated with progressive multifocal leuco-encephalopathy

Virology

The Papovavirus family contains two main groups:-

Papilloma viruses (which cause warts),

Many types of papillomavirus cause benign skin tumours (warts) in their natural hosts. These warts
often regress spontaneously, but human genital warts (tumours caused by specific types of
papillomavirus, particularly types 16 and 18) regularly become malignant if they persist for a
sufficiently long time.

Papillomavirus particles are approximately 55nm in diameter.

The capsid is composed of 72 morphological units, or capsomers, arranged on the surface of a T=7
icosahedron. The capsomers located at each of the 12 vertices, are pentavalent (i.e. each is surrounded
by five adjacent capsomers), and the other 60 capsomers are hexavalent (each adjacent to six
capsomers).

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A model of the papillomavirus capsid is shown beside a computer colorized EM image.

All 72 capsomeres are pentamers of the major structural protein, termed L1. This arrangement of
subunits violates the theory of quasi-equivalence put forward by
Caspar and Klug in 1962.

Polyoma viruses,

these are slightly smaller than papilloma viruses, and in animals are sometimes associated
with tumours.
The two known human polyoma viruses are:
JC - Progressive multifocal leuco-encephalopathy
BK - isolated from urine of immunosuppressed patients

dsDNA genome

small icosahedral particles, 42-45 nm

grows in human foetal glial cell cultures (JC) Progressive multifocal leuco-encephalopathy is a
progressive neurological disorder caused by reactivation of JC virus in the brain. Infection is
common, but neurological disease is rare; it only occurs in immunosuppressed patients.

Clinical Features
Patients may present with a variety of neurological signs, including: hemiparesis, dementia,
dysphagia, muscular inco-ordination or impaired vision. The condition is progressive and invariably
fatal.

Pathology
Multiple foci of demyelination are found throughout the cerebral hemispheres and cerebellum. The
virus infects oligodendrocytes, which have a bizzare histological appearance.

Epidemiology
Infection with JC virus is common, but invariably asymptomatic: Seroprevalence surveys have shown
that about 50-60% of adults have antibodies.

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Spongiform Encephalopathies
(Prion diseases)

A number of transmissable neurological syndromes, caused by unconventional virus-like agents, have


been identified in man and other animals. These diseases are characterized by the following features:

1. Confined to the CNS


2. Long incubation period
3. Progressive, uniformly fatal course
4. Typical brain histology: reactive gliosis, vacuolation of neurones, deposition of amyloid
protein in the brain and the absence of an inflammatory response.
5. Infection is believed to be transmitted by means of a novel infectious protein, termed prion or
PrP.

Diseases falling into this category include:


Scrapie - sheep, goats
Bovine spongiform encephalopathy - cattle
Transmissable Mink encephalopathy - mink
Creutzfeldt-Jakob disease - man, sporadic and familial
Gerstmann-Streussler disease - man, familial
Fatal Familial Insomnia - man, familial
Kuru - man, Fore people, New Guinea

Properties of Prions:

• 1. Infectivity is associated with a novel infectious protein, termed prion (PrP)


• 2. Infectious particles are very small - about 20-30 nm in size.
• 3. No nucleic acid has yet been found to form part of the agent
• 4. Prions are extremely resistent to inactivation by: ultra-violet light, formaldehyde and heat.
• 5. Prions can be transmitted by intra-cerebral or sub-cutaneous inoculation of material from
infected brain.

Pathogenesis of prion diseases


The pathogenesis of prion diseases is still a great mystery. The pathology appears to be both
infectious as well as genetic.

Recent evidence has demonstrated that the prion protein has the same primary amino acid
sequence as a host protein which is normally present at low levels in healthy brain tissue. The
cellular protein has been termed PrPc. Although the amino acid sequence is the same as the cellular
one, the prion protein, has an abnormal conformation, due to different post translational modification.
Exposure to the abnormal form triggers a conversion of the cellular isoform to the infectious form.
This is followed by progressive accumulation of the abnormal form (prion protein) in the brain,
which is deposited as amyloid.

LEARNING ONLY FOR INFORMATIION

Scrapie

Scrapie is a neurological disease occurring in sheep and goats. It was first identified in Britain, but it
is now known to occur world wide. It is the prototype disease of the spongiform encephalopathies; the
scrapie prion is termed PrPsc. The disease is clearly infectious as it can be transmitted to sheep, mice

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and hamsters by intra-cerebral or sub cutaneous inoculation of material from the brains of infected
sheep. Susceptibility to infection appears to be genetically determined.

Clinical features
The disease is transmitted by contact in flocks of sheep and also vertically, from ewes to lambs. In
natural infection, the incubation period is 2 to 5 years. Following intra- cerebral inoculation,
however, the incubation is much shorter, only 3 to 24 months.

Affected sheep develop ataxia, tremor and muscular in co-ordination; the symptoms progress to
paralysis and death.

Bovine Spongiform encephalopathy

Similar disease to scrapie which occurs in cattle. It is believed that scrapie was introduced into cattle
herds fed on feeds containing scrapie-infected sheep offal. The condition is now epidemic in cattle
herds in England and Wales ("Mad Cow Disease"). There is concern that the infection may be
transmitted to humans, and a report of an increased number of cases of CJD in young people linked to
the consumption of beef burgers sparked a world wide furore.

Transmissable Mink encephalopathy

Scrapie has been introduced into minks, bred in captivity through feeds containing sheep brains.

Creutzfeldt-Jakob disease (CJD)

In 1920, Creutzfeldt described a progressive dementing illness in a 22 year old woman. The following
year, Jakob described four older patients with a clinically similar presentation and course. Since then,
numerous cases of CJD have been described. CJD occurs world wide. It is very rare, with an
incidence of about 1 case per million population. While most cases are sporadic, 5-10% are familial.
In the familial form, CJD is inherited as an autosomal dominant condition.

Clinical features
The onset of the disease typically occurs between the ages of 50 and 65 years. There are two main
modes of presentation:

• 1. Chronic dementing illness


• 2. Progressive cerebellar dysfunction

The condition is relentlessly progressive and patients usually die within a year of presentation.

Transmission:
The natural route of infection is not known, but CJD has been accidently transmitted by:
1. Corneal grafts - where the corneas were harvested from cadavers that died of CJD.
2. Growth hormone preparations, derived from human pituitary glands.
3. In two patients who received grafts of dura mater, prepared from cadavers, and
4. through electrodes used for electro-encephalography which had previously been used in a patient
with CJD.

The prion detected in the brains of patients with CJD is termed PrPCJD. The disease has also been
experimentally transmitted to chimpanzees and other primates. Following intracerebral inoculation,
the incubation period is 11-14 months. The disease can also be transmitted peripherally (IV, intra
peritoneal or intramuscular routes). But transmission is much less efficient and the incubation period
is much longer (many years).

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Decontamination procedures:
Prions are extremely resistent to inactivation by ultra-violet light, forrmaldehyde and heat.

Recommended methods of decontaminating infected material include:


1. Autoclaving for 4.5 hours (121oC, 15 p.s.i)
2. 1N NaOH followed by autoclaving for 1.5 hours

Gerstmann-Streussler Syndrome

Rare familial neurodegenerative disease which usually manifests in the third to the seventh decade of
life. The condition is both infectious and genetic: the predisposition is inherited as an autosomal
dominant condition; while, in addition, the disease can be transmitted to non human primates by intra
cerebral inoculation with brain tissue from cases of GSS.

Recent genetic studies have revealed that affected individuals have a point mutation in their PrPc
gene. The altered amino-acid sequence of the PrPC protein may make it more susceptible to
transformation to the abnormal conformation.

Fatal Familial Insomnia

Another rare familial disorder. Predisposed individuals also have a mutation in their PrPc gene.

Kuru

This is a transmissable prion disease found only in the Fore people of New Guinea. It first appeared
about 60 years ago and the incidence increased until the late 1950's. The disease is now known to
have been transmitted through ritual cannibalism: until the late 1950's it was the practice of women
and children to eat the brains and viscera of dead relatives, including those who had themselves died
of Kuru. Cannibalism stopped in 1957 and the incidence of Kuru has declined sharply since then.

Clinical features:
The incubation period varied from 4-20 years.
Patients presented with progressive cerebellar dysfunction. Death usually occurred within a year of
initial presentation.

Transmission studies:
Intra-cerebral inoculation of brain tissue from Kuru victims into non human primates leads to the
development of symptoms within two years.

CONTINOUS COURSE OF VIROLOGY

Retrovirus Disease

Lentivirinae:
Lentiviruses characteristically establish a persistent infection in the host and cause chronic wasting
disorders which are uniformly fatal. Members of this family that typically cause CNS pathology
include:

Virus Host Clinical Features


Visna-Maedi virus Sheep paralysis, wasting, ataxia
Caprine arthritis encephalitis virus (CAEV) Goats paralysis, wasting, ataxia

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HIV 1 and 2 humans Dementia, immunodefiscience

Pathology
Inflammatory cell infiltration, perivascular cuffing of blood vessels, demyelination, necrosis and
reactive gliosis.

Pathogenesis
The pathogenesis of the neuropathology is not very clear. Virus is probably introduced into the CNS
by infected monocytes which cross the blood brain barrier. Differentiation of the infected monocytes
into microglial cells is thought to trigger viral replication. None of these viruses appear to infect
neuro-ectodermal cells directly, and damage to brain tissue is therefore thought to occur indirectly by
cytokines released during inflammation. It is also thought that certain viral proteins, such as the gp160
of HIV, may be directly toxic to neurones.

In addition to the above ( where the neurological damage arises directly as a result of HIV infection)
CNS pathology in patients with AIDS may also be caused by opportunistic infectious agents such as:
JC virus (PML), HCMV, VZV as well as a number of bacterial and fungal diseases

Oncovirinae

HTLV 1 - Tropical spastic paraparesis

HUMAN RETROVIRUSES

Retroviruses infect a wide range of animal species


and cause a variety of diseases including:
tumours, wasting and auto-immune diseases, immunodeficiency syndromes and aplastic and
haemolytic anaemias.
Human retroviruses
Four human reteroviruses have been identified. All infect CD4 bearing cells. These were only
identified in the 1980's when it became possible to culture T-cells in vitro.

• HTLV1 - T-cell leukaemias/lymphomas


Tropical spastic paraparesis
• HTLV2 - No known pathology
• HIV 1 & 2 - AIDS

They are enveloped viruses, with an RNA genome. The name is derived from the fact that the virus
particle contains an RNA-dependent DNA Polymerase (Reverse transcriptase). This enzyme
converts the RNA genome into DNA, which then integrates into the host chromosomal DNA. The
reverse transcriptase is highly error prone and rapid genetic variation is a feature of this group.
The genus is divided into three sub families:
Oncovirinae - oncogenic - cause sarcomas and leukaemias in animals;
eg Rous Sarcoma Virus
Lentivirinae - slow progressive degenerative disorders
eg visnavirus of sheep, human immunodeficiency virus (HIV)
Spumavirinae - no pathology known

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Defective virus
Virus which has lost a gene that is essential for replication and can, therefore, only undergo
productive infection if the cell that is harbouring the virus is superinfected with a helper virus that
can supply the function of the lost gene.
Many oncogenic retroviruses are defective.

Endogenous reteroviral sequences


Defective retroviruses which integrate into the host genome and are passed down from generation to
generation. Two percent of the human genome is made up of endogenous retroviral sequences.

Genome organization The virus has a diploid genome (2 copies of RNA genome per virus particle).
The genome codes for at least three genes: gag, pol and env.

• gag - codes for the core proteins, structural virion components


• pol - reverse transcriptase with integrase and protease
• env - envelope glycoprotein

LTR -Long terminal repeat - is a regulatory sequence at each end of the genome.

Regulation genes : tat, vjf vpu and nef

LTR - gag - pol - env - (onc) - LTR

Their presence allows integration into the host chromosome and controls gene expression

onc - (see oncogenes)

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Life cycle of lentiviruses

Oncogenes

Viruses of the Oncovirinae subfamily contain an extra gene termed, 'onc'. These genes are called
oncogenes because their expression in the virus infected cell is associated with tumour production.
These genes were originally acquired from the host cell itself. The cellular gene was picked up during
integration of the virus genome into the host DNA, way back in evolution. Most oncogenes code for
proteins with growth promoting properties and their expression can lead to uncontrolled proliferation
of the infected cell and tumour development.

HTLV 1 & 2

Human T-Cell Lymphotrophic Viruses,

belong to the sub-family Oncovirinae.

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HTLV1

Clinical features
1. T-cell leukaemia/lymphoma. Aggressive tumour of CD4 cells which infiltrates skin and brain.
Tumours are only produced after a prolonged latent period. The virus does not contain an oncogene
and it is believed that the malignant change is the result of interruption and disregulation of host
DNA, by viral genome insertion. Less than 1% of HTLV 1 infected individuals develop this
malignancy.

2. Tropical spastic paraparesis. Aggressive non-demyelinating spastic paraparesis.

Epidemiology
Seroprevalence is high in South West Japan, the Caribbean and parts of West Africa. In high
incidence areas up to 10% of adults may be infected. The seroprevalence increases with age and
family clustering of infection is common. Spread occurs through blood transfusion and sexual
intercourse. Mother to child transmission through breast feeding has also been shown.

Laboratory Diagnosis
HTLV 1 specific antibody, ELISA.

HTLV 2

The virus shares extensive nucleic acid homology with HTLV 1. It was first isolated from a patient
with hairy cell leukaemia, but no specific pathology has been attributed to it.

HIV 1 and 2

Human Immunodeficiency Viruses

Background:-
In the spring of 1981, previously healthy homosexual men in New York and Los Angeles were found
to be suffering from severe immunodeficiency states associated with severe opportunistic infections
and rare malignancies.

At this time an infectious aetiology was suspected especially when it was perceived the disease could
be transmitted by blood transfusions and blood products. (Haemophiliacs)

In 1983, a new retrovirus termed LAV (now called HIV 1) was isolated from the T-cells of a patient
with persistent generalised lymphadenopathy.

In 1986, a second closely related virus, termed HIV 2 was isolated from a patient from West Africa
with acquired immunodeficiency syndrome, AIDS.

Currently, about 41 million people are believed to be infected, world-wide; 2/3 of these are in Africa
(see figure 2). HIV1 is the major cause of the AIDS pandemic; HIV2 is of lower virulence and
infection has largely remained confined to West Africa and spread slowly to Central Africa

Origin
AIDS appears to be a new disease. The origin of the virus is obscure but it is probable that infection
arose in Africa. Retrospective screening of stored sera has shown that the earliest known infection

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occurred in Kinshasa, Zaire, in 1959.


HIV is believed to have evolved from related viruses that infect African monkeys, namely the simian
immunodeficiency viruses (SIV) (which cause a similar disease to AIDS in monkeys). HIV 2 is
more closely related to SIV than HIV 1. It is not known how the original transfer of virus from
monkey to man took place. These viruses mutate readily which explains the rapid subsequent
divergence.(figure 4)

Spread(figure 3)

Epidemiology - South Africa


HIV was introduced into South Africa from two sources:
a) Into the homo-sexual community - from North America and Europe,and
b) into the hetero-sexual community - from north of our borders by truck drivers, returning exiles,
migrant labourers.

A survey, conducted in October/November 1993, of women attending ante natal clinics in different
parts of the country has shown that the prevalence of HIV infection varies from 1.33% in the Cape to
9.62% in KwaZulu/Natal. ( prevalence of 25% in inf South africa)

Transmission
Infection is transmitted in a manner identical to that of hepatitis B.
1.) Blood products

• Blood transfusions
• Intra-venous drug abusers - sharing of needles
• Health care workers:
needlestick injuries - risk approximately 0.36% (depends on extent of the injury)
muco-cutaneous exposure - no sero-conversion incidents have been reported

2.) Organ transplants


3.) Sexual intercourse
Both homosexual and hetero-sexual exposure
Increased risk of transmission if partners have other sexually transmitted diseases

4.) Vertical Transmission


10-40% of babies born of HIV-infected mothers will be infected.

Infection may occur


in utero
during birth
post-natally, through breast feeding
NB! THERE IS NO EVIDENCE TO SUGGEST THAT HIV CAN BE TRANSMITTED BY:
Insects
Casual contact saliva, kissing
sharing of eating and drinking utensils

Clinical features

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Clinical categories
Catégorie Cliniques
+
Nombre de LT CD4 A B C
- Asymptomatique SIDA
- Primoinfection ou - Symptomatique sans
lymphadénopathie ; critère (A) ou (C)
généralisée
>500/mm3 A1 B1 C1
200-499/mm3 A2 B2 C2
<200/mm3 A3 B3 C3

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Primary infection
About 90% of patients develop a flu-like illness which co-incides with seroconversion, between 2 and
4 weeks post exposure. Symptoms include, fever, night sweats, sore throat, lymphadenopathy,
diarrhoea. The illness is self limiting.

Asymptomatic phase
Of variable duration, from 2 to 10 years. Patients are clinically well, but infectious.

Prodromal phase
This period is heralded by the insidious onset of a variety of prodromal disorders, including: weight
loss, fever, persistant lymphadenopathy, oral candidiasis and diarrhoea. These symptoms precede the
progression to AIDS.

Acquired Immunodeficiency Syndrome (AIDS)

Syndrome with the following features:


1) Constitutional disease: fever, diarrhoea, weight loss, skin rashes
2) Neurological disease: dementia, myelopathy, peripheral neuropathy
3) Immunodeficiency: Increased susceptibility to opportunistic infections: (see table 1)
4) Rare malignancies: Kaposi sarcoma, oral hairy leukoplakia, lymphomas.

Kaposi sarcoma - is a tumour of endothelial cells. Prior to the AIDS epidemic, this tumuor was rare
and only found in middle aged African and Mediterranean Jewish men, in whom it was an indolent
condition. AIDS patients develop a disseminated highly aggressive form of the disease.

Paediatric Infection
Following infection in the perinatal period, babies may develop a progressive illness in the first few
months of life (No latent period). Clinical features include: Failure to thrive, diarrhoea,
lymphadenopathy, susceptibility to opportunistic infections hepato-splenomegaly, lymphoid
interstitial pneumonia and parotitis.

Pathogenesis

HIV infects CD4 cells

Disseminated infection

Specific immune Response


Antibody
Cell mediated immunity

Clearance of most virus

Some persistence
a) Gradual loss of CD4 cells
b) Destruction of microenvironment of lymphoid tissue

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IMMUNO-DEFICIENCY

Cell tropism: CD4+ T cells, Macrophages

Destruction of CD4 cells occurs through the following mechanisms:


direct lysis of infected cells
syncytium formation

Infection of precursor cells and destruction of microenvironment

Impairment of CD4 cell function

OPPORTUNISTIC INFECTIONS

a) Parasites
- Pneumocistis carinii causative agent of a atypical pneumonia
- Toxoplasma gondii form cerebral abcess
- Cryptosporidies which cause diarrhea
- Isospora belli rsponsible of recidive diarrhea
- Leishmania cause skin and mucous gravis lesions

b) Fungi
- Crypotococcus neoformans caustive meningitis agent by immyunocompromised patients
- Aspergillus that mainly cause pulmonary diseases
- Histoplasma responsible of histoplasmosis
- Candida albicans responsible of oropharyngial and intestinal candidosis

c) Virus
-Cytomégalovirus causative agent of retinitis and colistis
-les Herpes simplex virus cause classical herpetic lesions
- le VZV cause a zona

d) bacteria
-Mycobactérium tuberculosis often multiresistant causative agent of pulmonary and extra pulmonary
infections
- complexe avium-intercellularaea of atypical mycobacterium
-les salmonella non typhi

On this listing we can add :


- Kaposi sarcoma
- a cerebral lymphoma
-HOGDKIN disease
- buccal and anal carcinoma

LABORATORY DIAGNOSIS

Serology
IgG develops 4-6 weeks post exposure and remains detectable for life. Its presence in serum therefore
indicates infection.
Exception: Uninfected infants of HIV positive mothers

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Direct detection of virus


p24 antigen ELISA
culture from PBMC's
PCR

§5.Les diagnostics des infections a VIH

En pratique courante, on recherche tout d’abord à dépister et confirmer la séropositivité aux


VIH ou séroconversion c-a-d apparition d’anticorps anti-VIH
Cela se fait par plusieurs techniques et en suivant divers algorithmes
Le dépistage se fait :
-par des analyses du sang entier sur bandelettes ou en cassettes : ce sont les tests
rapides basée sur l’immunochromatographie
-par analyse du sérum en ELISA ou tests immunoenzymatique sur phase solide

Ces techniques dites de première intention devront être confirmés SI LE RESULTAT EST
POSITIF soit par un deuxième tests , soit par une technique de confirmation du type Immunoblotting
,ou de Western_Blot
Avant la séroconversion on peut détecter l’antigénémie p 24-25 par ELISA qui signe la primo-
infection

On peut aussi cultiver les échantillons sur co-culture de lymphocytes et rechercher les effets
cytopathiques des VIH qui sont la formation de syncytium ( fusion des cellules infectées avec
polycaryocytose ou présence de nombreux noyaux intercellulaire) . La culture des prélèvements
biologiques reste l’apanage des laboratoires de référeence
Microplaque d’ ELISA

Principe d’ ELISA

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Western-Blot
Test de confirmation d’une séropositivité dépistée (par ELISA ou tests rapides)
En résume :

• Il faut : Première étape: le test de dépistage


– Test sensible: pour le premier dépistage
Permet d’éviter des résultats faussement positifs
UN RESULTAT NEGATIF EST RENDU COMME TEL
UN RESULTAT POSITIF DOIT ETRE CONFIRME

• Seconde étape: la confirmation du test positif


– Test spécifique: pour la confirmation du premier test (positif, douteux ou discordant)
• Permet d’éviter des résultats faussement positifs
• Vérifier que les AC détectés par le test de depistage sont spécifiques au VIH
a/La confirmation d’un test rapide se fait par un deuxième test rapide de marque différente exp : 1er
test rapide Unigold* ; 2eme test rapide Capillus* ;

b/La confirmation d’un ELISA(positif, douteux ou discordant) se fait par un autre Elisa de principe
différent(1er ELISA en sandwich, 2eme ELISA en compétition) Sinon employer
le Western-Blot un test d’immunoempreintepermettant de détecter TOUS LES ANTIGENES
VIRAUX

Exemple d’algorithme de dépistage de la séropositivité

1ere situation sérologique


Test rapide 1 : positif
Test rapide 2 : positif
REPONDRE RESULTAT POSITIF ET/OU TEST DE CONFIRMATION (si disponible)

2eme situation sérologique


Test rapide 1 : positif
Test rapide 2 : négatif
Test rapide 3 : positif ou negatif
LE RESULTAT EST CELUI DU TEST RAPIDE 3

3eme situation sérologique


Test rapide 1 : négatif
RENDRE LE RESULTAT NEGATIF SANS CONFIRMATION

NB : les tests rapides 1, 2 et 3 sont de marques différentes

§6 Traitements des infections a VIH et prévention

On prévient et on traite les maladies opportunistes. ( bactrim pour prevenir de la toxoplasmose et


d’une pneumonie a Pneumocystis)
On met le patient séropositif sur ARV ou antirétroviraux suivant certains critères clinicobiologiques
en particulier son état clinique, sa charge virale plasmatique et le taux de ces lymphocytes CD4+.
Les ARV actuellement utilisés sont des inhibiteurs de la transcriptase inverse (polymérase permettant
la transcription de l’ARN génomique en ADN proviral) ; ainsi que des inhibiteurs des protéases qui
vont scinder les polypeptides codes par les différents gènes des VIH (gp 160 en gp 120 et gp 41 pour
le VIH 1)

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Avant et dans le suivi du traitement des PVVIH( personnes vivant avec le VIH) des bilans
biochimiques hématologiques, des enzymes hépatiques et autres sont pratiqués pour suivre comment
le patient supporte cette chimiothérapie antirétrovirale .
La posologie est une trithérapie (trois médicaments) à base des inhibiteurs des protéases et des
inhibiteurs de la transcriptase inverse (analogues ou non des nucléosides) ; la trithérapie est indiquée
pour empêcher les résistances aux ARV Ces médicaments sont sous formes génériques ou souvent
combinés pour assurer plus de compliance ou adhérence aux traitements proposes.
Plusieurs stratégies thérapeutiques sont proposées.
En général on est mis sous ARV à partir d’un taux de lymphocytes T CD4 inférieur à 3OO par µl
de sang.
La trithérapie peut être constituée des associations suivantes :
- 2 inhibiteurs de la transcriptase inverse nucléosidiques +un inhibiteur des protéases
- 2 inhibiteurs de la transcriptase inverse nucléosidiques + 2 inhibiteurs des protéases
- 2 inhibiteurs de la transcriptase inverse nucléosidiques + 1 inhibiteur de la transcriptase
inverse non nucléosidique
- 3 inhibiteurs de la transcriptase inverse nucléosidiques
- 3 inhibiteurs de la transcriptase inverse non-nucléosidiques

Principales Classes

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Inhibiteurs de la Reverse Transcriptase Inhibiteurs de la Protéase

Non Nucléosidiques Nucléosidiques Anti protéases


NNRTIs NRTIs Pis

nevirapine (NVP) Lamivudine(3TC) Indinavir (IDV)


delavirdine(DLV) stavudine (d4T) saquinavir (SAQ)
efavirenz (EFV) zidovudine (AZT lopinavir (LPV)
didanosine (ddI) ritonavir (RTV)
abacavir (Abv) amprenavir (ATV)
nelfinavir (NFV)

Une nouvelle classe d’ARV va bientôt être mise sur le marché à savoir des anti- intégrases qui
inhibent l’intégration de l’ADN proviral au génome de la cellule parasitée.

La prévention repose sur des attitudes individuelles de responsabilité. Il faut protéger les rapports
sexuels en utilisant un codom (capote) en particulier si ce sont des rapports avec de multiples
partenaires ; s’abstenir si on le peut ; arrêter le vagabondage sexuel.
Le personnel de santé, en particulier ceux qui sont en rapport avec des prélèvements biologiques,
devra se protéger :
-en portant toujours des gants (ainsi qu’un masque et des lunettes protectrices) surtout lors
des prélèvements
-ne pas recapuchonner les aiguilles utilisées pour les prélèvements
-toujours respecter les bonnes pratiques des laboratoires ;
La prévention des personnes violées :par une monothérapie (AZT) ; de même que la femme enceinte
séropositive : traitement à l’AZT et délivrance haute par césarienne.

VACCINE PROSPECTS

There is no effective vaccine available for HIV. Attempts have been made to develop a vaccine,
using:

• purified viral envelope glycoproteins, gp120 or 160


• whole inactivated virus
• live attenuated HIV strains (lacking certain genes)
• live recombinant virus vectors, expressing HIV proteins.

A major difficulty is the fact that neutralizing antibody in the serum does not protect the host from
infection with HIV: Possible reasons for this include:

• Antibody enhancement of infection


• Rapid virus mutation may result in variation of envelope antigens (escape mutants)
• HIV can infect cells in sites that are sequestered from antibody
• Host may be infected by whole virus-infected cells

POX VIRUSES

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INTRODUCTION

A range of pox viruses cause febrile illnesses in man and animals with a prominent vesicular rash.
The most prominent of them was smallpox virus (variola) which caused a severe disease in man but
which has now been eliminated by intensive international vaccination.
Current interest in poxviruses centers around their possible use as vaccine vectors.

THE VIRUS

Smallpox belongs to the Orthopox virus genus. Poxviruses are very large, brick-shaped viruses about
300 x 200 nm (the size of small bacteria). They have a complex internal structure - a large double-
stranded DNA genome (about 200 kbp in size) is enclosed within a "core" that is flanked by 2 "lateral
bodies". The surface of the virus particle is covered with filamentous protein components, so that the
particles have the appearance of a "ball of knitting wool". The entire particle is enclosed in an
envelope derived from the host cell membranes. Most poxviruses are host-species specific, but
vaccinia is a remarkable exception.

True pox viruses are antigenically rather similar, so that infection by one elicits immune protection
against the others.
Laboratory diagnosis of pox viruses may be undertaken by electron microscopy of negatively stained
vesicle fluid or lesion material. Some pox viruses can be cultured on the chorio-allantoic membrane of
chick embryos, where they form pocks, and some can be isolated by cell-culture.

HUMAN INFECTION

Vaccinia - a virus strain which has been used for immunisation against smallpox.
It's origins are not known but it seems to be a genetically distinct type of pox virus which grows
readily in a variety of hosts. In man it causes a localised pustule with scar formation. In
immunocompromised persons or eczematous persons it sometimes caused a severe generalised
vaccinia infection. Routine immunisation of all children in RSA stopped in about mid 1980's. (Last
case of smallpox in RSA was on Transvaal-Botswana border in 1971.)

Cowpox - is acquired by humans usually by milking cows; it then manifests as ulcerative lesions
(sometimes called "milkers nodules") on the hands of dairy workers. It was noted to protect against
smallpox and was used by Jenner as a vaccine strain to protect persons against smallpox. Despite its
name, rodents are the main reservoir of cowpox; it spreads secondarily to cows and domestic cats.

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Molluscum contagiosum - is a minor infectious warty papule of the skin with a central umbilication,
transferred by direct contact, sometimes as a veneral disease.
Click here to see electron micrograph

Monkey pox - is a rare smallpox like disease of children in central Africa. It is acquired from
monkeys or wild squirrels, but does occasionally spread from man to man in unvaccinated
communities. Antigenically cross-reacts with other poxviruses. Sick monkeys have not been
identified, but apparently healthy animals have antibodies.

Pseudocowpox - occurs worldwide and is a disease primarily of cattle. In humans it causes non-
ulcerating "milker's nodes".

ORF - a worldwide occupational disease associated with handling sheep and goats afflicted with
"scabby mouth". In humans it manifests as a single painless, papulo-vesicular lesion on the hand,
forearm or face.

Historical Information regarding


SMALLPOX (the only disease that has been globally eradicated)

Source : only from close contact with infected persons. No animal reservoir or vector. No latency in
man.

Spread : by close contact, eg. within households, via droplet infection of pharyngeal secretions
(ulcers in pharynx).

Incubation : 10 - 12 days.

Major illness : Rather similar to severe chicken pox (varicella). Abrupt onset of fever and prostration
with a macular rash on the third day (head, limbs, hands and feet rather than trunk, including palms
and soles). Progressed to vesicles which become pustular, ulcerated, scabbed, healed with scarring
("pock marked face"). Also inside mouth. 16 to 30% mortality. Milder subtypes were also described.

EPIDEMIOLOGY

As result of an unprecedented international WHO campaign of 'seek and contain' by vaccination, the
last natural case of smallpox was diagnosed in 1977. Despite intensive searching, no other cases
have been detected in 17 years. In 1994, smallpox virus remains only in laboratory deepfreezers in
Moscow and Atlanta, USA, awaiting a politico-scientific decision for final extinction. It's genetic
sequence information is stored in computer memory banks.

POX VIRUSES AS VACCINE VECTORS

Certain poxvirus strains which cause localised lesions or abortive infections in mammals (vaccinia) or
birds (avipox) are currently being studied as possible recombinant vaccine vectors for use in man and
domestic animals.

Genes coding for immunising antigens of a variety of pathogens may be inserted into the large
poxvirus genome, and some undesirable genes of the pox virus may be excised. A vaccinia-based
rabies vaccine has been successfully used on wild foxes (via bait) in Europe. Half-a-dozen others are
in clinical trial.

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GA Keen, (JULY 1998)

ANTI-VIRAL THERAPY

ANTI-VIRAL CHEMOTHERAPY

Antiviral Agents

Antiviral drugs are available to treat only a few viral diseases. The reason for this is the fact that viral
replication is so intimately associated with the host cell that any drug that interferes significantly with
viral replication, is likely to be toxic to the host.

Two useful antivirals are:


the nucleoside analogues and
the interferons
but there are other targets in the different stages of intracellular viral growth which show potential
for antiviral chemotherapy.

Stages in virus replication which are possible targets for chemotherapeutic agents:

• Attachment to host cell


• Uncoating - (Amantadine)
• Synthesis of viral mRNA - (Interferon)
• Translation of mRNA - (Interferon)
• Replication of viral RNA or DNA - (Nucleoside anologues)
• Maturation of new virus proteins (Protease inhibitors)
• Budding, release

Diseases for which effective therapy is available:

Herpes Simplex virus (Acyclovir)


Varicella-Zoster virus (Acyclovir)
Cytomegalovirus (Gancyclovir, Foscarnet)
AIDS (Zidovudine, Lamivudine[3TC], Protease inhibitors; in combination)
Respiratory Syncitial virus (Ribavirin)
Influenza (Amantadine)

Nucleotide analogues:

These are synthetic compounds which resemble nucleosides, but have an incomplete or abnormal
deoxy-ribose /or ribose group.
These compounds are phosphorylated to the tri-phosphate form within the infected cell. In this form,
the drug competes with normal nucleotides for incorporation into viral DNA or RNA.
Incorporation into the growing nucleic acid chain results in irreversible association with the viral
polymerase and chain termination.

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Interferons:

There are three classes: alpha- beta- and gamma-

The alpha and beta Interferons


are cytokines which are secreted by virus infected cells.
They bind to specific receptors on adjacent cells and protect them from infection by viruses.
They form part of the immediate protective host response to invasion by viruses.
In addition to these direct antiviral effects, alpha and beta interferon also enhance the expression of
class I and class II MHC molecules on the surface of infected cells, in this way, enhancing the
presentation of viral antigens to specific immune cells. Their presence can be demonstrated in body
fluids during the acute phase of virus infection.

Recombinant alpha and beta interferons are now available and have been used for the treatment of
Chronic hepatitis B and C virus infections.

However, side effects such as fever, malaise and weight loss have limited the use.

gamma Interferon (immune interferon)


is a cytokine secreted by TH1 CD4 cells.
Its function is to enhance specific T cell mediated immune responses.

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Mechanism of action of the interferons :

1. Enhancement of the specific immune response.


By increasing the expression of MHC class I molecules on the surface of infected cells, the
interferons increase the opportunity for specific cytotoxic T cells to recognise and kill
infected cells.
2. Direct antiviral effect
a) degradation of viral mRNA
b) inhibition of protein synthesis
Prevents the infection of new cells

Immunoglobulin Therapy

Passive Immunisation

Passive immunisation is the transfer of immunity to a host by means of immunoglobulins (preformed


antibodies). These immunoglobulins are typically prepared by cold ethanol fractionation as a 16%
solution of gammaglobulin from large pools of serum obtained from the blood donations of at least
1000 donors.

Immunoglobulin from immune individuals can be used as prophylaxis to prevent viral infections in
exposed, but non immune individuals. It works by binding to extra-cellular virions and preventing
them from attaching to and entering susceptible cells. The protective effect is short lived (up to three
months) because the antibodies are metabolised by the host.

"Normal" Immune globulin


This is a pooled product, prepared from the serum of normal blood donors. It contains low titres of
antibody to a wide range of human viruses. It is mainly used as prophylaxis against:

• hepatitis A virus infection,


• parvovirus infection, and
• enterovirus infections (in neonates).
• It may also be given as passive protection to HIV-infected babies.

Hyper-immune globulin
Immunoglobulin may be prepared from the serum of selected individuals who have high titres of
antibody to particular viruses.
Examples include:

• Zoster immune globulin


Prevention of Varicella in immunocompromised children and neonates.
• Human Rabies immunoglobulin
Post-exposure prophylaxis in an individual who has been bitten by a rabid animal.
• Hepatitis B Immune globulin
Non-immune individual who has been exposed to HBV.
• RSV Immune globulin
Treatment of respiratory syncitial virus infections in the very young.

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VACCINES

The introduction of vaccination has been one of the most decisive advances leading to
the dramatic downward trend in the incidence of many viral diseases.

The principle of vaccination is to induce a "primed" state in the vaccinated subject so


that, following exposure to a pathogen, a rapid secondary immune response is
generated leading to the accelerated elimination of the organism and protection from clinical disease.
Success depends on the generation of memory T and B cells and the presence in the serum of
neutralizing antibody.

Attributes of a good vaccine


1.Ability to elicit the appropriate immune response for the particular pathogen:
Tuberculosis - cell mediated response
most bacterial and viral infections - antibody
2. Long term protection
ideally life-long
3. Safety
vaccine itself should not cause disease
4. Stable
retain immunogenicity, despite adverse storage conditions prior to administration
5. Inexpensive

Types of Vaccine

Vaccines in general use include: LIVE vaccines; and KILLED vaccines

Vaccines are available for:

• Hepatitis B virus
• Hepatitis A virus
• Influenza
• Measles
• Mumps
• Polio
• Rubella
• Rabies
• Yellow Fever
• Varicella Zoster

INFORMATIONS ONLY

A. Live Vaccines

1. Live attenuated organisms

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Organisms whose virulence has been artificially reduced by in vitro culture under adverse
conditions, such as reduced temperature. This results in the selection of mutants which replicate
poorly in the human host and are therefore of reduced virulence. Replication of the vaccine strain in
the host reproduces many of the features of wild type infection, without causing clinical disease.
Most successful viral vaccines belong to this group.

The immune response is usually good - when the virus replicates in the host cells, both antibody as
well as cell mediated immune responses are generated and immunity is generally long lived. Often,
only a single dose is needed to induce long term immunity.

Potential drawbacks to these vaccines include:


the danger of reversion to virulence and
the possibility of causing extensive disease in immunocompromised individuals.

2. Heterologous vaccines

Closely related organism of lesser virulence, which shares many antigens with the virulent organism.
The vaccine strain replicates in the host and induces an immune response that cross reacts with
antigens of the virulent organism. The most famous example of this type of vaccine is vaccinia virus:
Both cowpox virus and vaccinia virus are closely related to variola virus, the causitive agent of
smallpox. The eighteenth centuary physician, Edward Jenner observed that milkmaids who had been
infected with cowpox virus were immune to smallpox. Widespread use of vaccinia virus as a vaccine
has lead to the world-wide eradication of smallpox.

3. Live recombinant vaccines

It is possible, using genetic engineering, to introduce a gene coding for an immunogenic protein from
one organism into the genome of another (such as vaccinia virus). The organism expressing a foreign
gene is called a recombinant. Following injection into the subject, the recombinant organism will
replicate and express sufficient amounts of the foreign protein to induce a specific immune response
to the protein.

Attributes - live vaccines

Good immune response

• Both Cell Mediated Immunity and antibody responses.


• Immunity is long lived
• Single dose

Safety

• Danger of reversion to virulence, or


• Severe disease in immunocomprised

Stability

• Organisms in the vaccine must remain viable in order to infect and replicate in the host
• Vaccine preparations are therefore very sensitive to adverse storage conditions
• Maintenance of the cold chain is very important.

Expense

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• Cheap to prepare

Killed (inactivated) vaccines

When safe live vaccines are not available, either because attenuated strains have not been developed
or else because reversion to wild type occurs too readily, it may be possible to use an inactivated
preparation of the virulent organism to immunize the host.

The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or
formaldehyde. These vaccines are not infectious and are therefore relatively safe. However, they are
usually of lower immunogenicity and multiple doses may be needed to induce immunity. In addition,
they are usually expensive to prepare.

Subcellular fractions

When protective immunity is known to be directed against only one or two proteins of an organism, it
may be possible to use a purified preparation of these proteins as a vaccine. The organism is grown in
bulk and inactivated, and then the protein of interest is purified and concentrated from the culture
suspension. These vaccines are safe and fewer local reactions occur at the injection site. However, the
same disadvantages of poor immunogenicity and the need for multiple boosters applies.

Recombinant proteins

Immunogenic proteins of virulent organisms may be synthesized artificially by introducing the gene
coding for the protein into an expression vector, such as E-coli or yeasts. The protein of interest can
be extracted from lysates of the expression vector, then concentrated and purified for use as a vaccine.
The only example of such a vaccine, in current use, is the hepatitis B vaccine.

Attributes - Killed vaccines


Immune response

• poor; only antibody - no cell immediated immune response.


• response is short-lived and multiple doses are needed.
• may be enhanced by the incorporation of adjuvants into the vaccine preparation (see below)

1. Safety

• Inactivated, therefore cannot replicate in the host and cause disease.


• Local reactions at the site of injection may occur.

2. Stability

• Efficacy of the vaccine does not rely on the viability of the organisms.
• These vaccines tend to be able to withstand more adverse storage conditions.

3. Expense

• Expensive to prepare.

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Adjuvants

Certain substances, when administered simultaneously with a specific antigen, will enhance the
immune response to that antigen. Such compounds are routinely included in inactivated or purified
antigen vaccines.

Adjuvants in common use:

1. Aluminium salts

• First safe and effective compound to be used in human vaccines.


• It promotes a good antibody response, but poor cell mediated immunity.

2. Liposomes and Immunostimulating complexes (ISCOMS)

3. Complete Freunds adjuvant is an emulsion of Mycobacteria, oil and water

• Too toxic for man


• Induces a good cell mediated immune response.

4. Incomplete Freund's adjuvant as above, but without Mycobacteria.

5. Muramyl di-peptide

• Derived from Mycobacterial cell wall.

6. Cytokines

• IL-2, IL-12 and Interferon-gamma.

Possible modes of action:

• By trapping antigen in the tissues, thus allowing maximal exposure to dendritic cells and
specific T and B lymphocytes.
• By activating antigen-presenting cells to secrete cytokines that enhance the recruitment of
antigen-specific T and B cells to the site of inoculation.

DNA Vaccines

DNA vaccines are at present experimental, but hold promise for future therapy since they will evoke
both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines.

The gene for an antigenic determinant of a pathogenic organism is inserted into a plasmid. This
genetically engineered plasmid comprises the DNA vaccine which is then injected into the host.
Within the host cells, the foreign gene can be expressed (transcribed and translated) from the plasmid
DNA, and if sufficient amounts of the foreign protein are produced, they will elicit an immune
response.

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Vaccines in general use

Measles

Live attenuated virus grown in chick embryo fibroblasts, first introduced in the 1960's. Its extensive
use has led to the virtual eradication of measles in the first world. In developed countries, the vaccine
is administered to all children in the second year of life (at about 15 months). However, in developing
countries, where measles is still widespread, children tend to become infected early (in the first year),
which frequently results in severe disease. It is therefore important to administer the vaccine as early
as possible (between six months and a year). If the vaccine is administered too early, however, there is
a poor take rate due to the interference by maternal antibody. For this reason, when vaccine is
administered before the age of one year, a booster dose is recommended at 15 months.

Mumps

Live attenuated virus developed in the 1960's. In first world countries it is administered together with
measles and rubella at 15 months in the MMR vaccine. This is not a legal requirement in South
Africa.

Rubella

Live attenuated virus. Rubella causes a mild febrile illness in children, but if infection occurs during
pregnancy, the foetus may develop severe congenital abnormalities. Two vaccination policies have
been adopted in the first world. In the USA, the vaccine is administered to all children in their second
year of life (in an attempt to eradicate infection), while in Britain, until recently, only post pubertal
girls were vaccinated. It was feared that if the prevalence of rubella in the community fell, then
infection in the unimmunized might occur later - thus increasing the likelihood of infection occurring
in the child-bearing years. This programme has since been abandoned in Britain and immunization of
all children is the current practice.

Polio

Two highly effective vaccines containing all 3 strains of poliovirus are in general use:

• The killed virus vaccine (Salk, 1954) is used mainly in Sweden, Finland, Holland and
Iceland.
• The live attenuated oral polio vaccine (Sabin, 1957) has been adopted in most parts of the
world; its chief advantages being: low cost, the fact that it induces mucosal immunity and the
possibility that, in poorly immunized communities, vaccine strains might replace circulating
wild strains and improve herd immunity. Against this is the risk of reversion to virulence
(especially of types 2 and 3) and the fact that the vaccine is sensitive to storage under adverse
conditions.
• The inactivated Salk vaccine is recommended for children who are immunosuppressed.

Hepatitis B

Two vaccines are in current use: a serum derived vaccine and a recombinant vaccine. Both contain
purified preparations of the hepatitis B surface protein.

The serum derived vaccine is prepared from hepatitis B surface protein, purified from the serum of
hepatitis B carriers. This protein is synthesised in vast excess by infected hepatocytes and secreted
into the blood of infected individuals. A vaccine trial performed on homosexual men in the USA has
shown that, following three intra-muscular doses at 0, 1 and 6 months, the vaccine is at least 95%
protective.

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A second vaccine, produced by recombinant DNA technology, has since become available.
Previously, vaccine administration was restricted to individuals who were at high risk of exposure to
hepatitis B, namely: infants of hepatitis B carrier mothers, health care workers, homosexual men and
intravenous drug abusers. However, hepatitis B has been targetted for eradication , and since 1995 the
vaccine has been included in the universal childhood immunization schedule. Three doses are given;
at 6, 10, and 14 weeks of age. As with any killed viral vaccines, a booster will be required at some
interval (not yet determined, but about 5 years) to provide protection in later life from hepatitis B
infection as a venereal disease.

Hepatitis A

A vaccine for hepatitis A has been developed from formalin-inactivated , cell culture-derived virus.
Two doses, administered one month apart, appear to induce high levels of neutralising antibodies.
The vaccine is recommended for travellers to third world countries, and indeed all adults who are not
immune to hepatitis A.

Yellow Fever

The 17D strain is a live attenuated vaccine developed in 1937. It is a highly effective vaccine which is
administered to residents in the tropics and travellers to endemic areas. A single dose induces
protective immunity to travellers and booster doses, every 10 years, are recommended for residents in
endemic areas.

Rabies

No safe attenuated strain of rabies virus has yet been developed for humans. Vaccines in current use
include:

• The neurotissue vaccine - here the virus is grown in the spinal cords of rabbits, and then
inactivated with beta-propiolactone. There is a high incidence of neurological complications
following administration of this vaccine due to a hypersensitivity reaction to the myelin in the
preparation and largely it has been replaced by
• A human diploid cell culture-derived vaccine (also inactivated) which is much safer.

There are two situations where vaccine is given:


a) Post-exposure prophylaxis, following the bite of a rabid animal:
A course of 5-6 intramuscular injections, starting on the day of exposure. Hyperimmune rabies
globulin may also administered on the day of exposure.

b) Pre-exposure prophylaxis is used for protection of those whose occupation puts them at risk of
infection with rabies; for example, vets, abbatoir and laboratory workers.
This schedule is 2 doses one month apart ,and a booster dose one year later. (Further boosters every 2-
3 years should be given if risk of exposure continues).

Influenza

Repeated infections with influenza virus are common due to rapid antigenic variation of the viral
envelope glycoproteins. Antibodies to the viral neuraminidase and haemagglutinin proteins protect the
host from infection. However, because of the rapid antigenic variation, new vaccines, containing
antigens derived from influenza strains currently circulating in the community, are produced every
year.
Surveillance of influenza strains now allows the inclusion of appropriate antigens for each season.The
vaccines consist of partially purified envelope proteins of inactivated current influenza A and B
strains.

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Individuals who are at risk of developing severe, life threatening disease if infected with influenza
should receive vaccine. People at risk include the elderly, immunocompromised individuals, and
patients with cardiac disease. In these patients, protection from disease is only partial, but the severity
of infection is reduced.

Varicella-Zoster virus

A live attenuated strain of varicella zoster virus has been developed. It is not licensed in South Africa
for general use, but is used in some oncology units to protect immuno-compromised children who
have not been exposed to wild-type varicella zoster virus. Such patients may develop severe, life
threatening infections if infected with the wild type virus.

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