Laboratory Diagnosis

1
 Category of Sample

• Blood, Urine, Stool, nasal washing, nasal

swab , throat swab, saliva , sputum, rectal
swab, vesicle fluid( scraping or swab),
tissue ,brain biopsy, cerebrospinal fluid, et
al.

2
 Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)]
• Detection of viral genetic material
• Serologic procedures

3
 Microscopy Identification

• Light microscopy
• Fluorescent microscopy
• Electron microscopy

4
 Light microscopy

• Characteristic CPE
• Inclusion Bodies

5
• Cell death
Cell rounding
Degeneration
Aggregation
Loss of attachments to substrate
• Characteristic histological changes:inclusion
bodies in the nucleus or cytoplasm, margination of
chromatin
• Syncytia: multinucleated giant cells caused by virus-
induced cell-cell fusion

6
 Fluorescent microscopy
• Fluorescent-antibody staining

7
 Electron microscopy

• Direct detection : Human rotavirus; HAV;

HBV; Smallpox virus; Herpes virus.
• Immune Electron microscopy: Human
rotavirus; HAV;

8
 Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)]
• Detection of viral genetic material
• Serologic procedures

9
Viral isolation and Identification

• Viral Growth and Cell culture

• Viral Detection
• Viral Identification
• Interpretation of culture results

10
 Systems for the Propagation of
Viruses
• People
• Animals: cows, chickens, mice,rats, suckling mice
• Embryonated eggs
• Organ and tissue culture
Organ culture
Primary tissue culture
Cell lines: diploid
Tumor or （ immortalized ） cell line
11
 Viral detection

• CPE
• Hemadsorption
• Interfere
• Metabolize of cell

12
 TCID50
(Tissue culture infective dose)
• TCID50 is defined as that dilution of virus
which will cause CPE in 50% of a given batch
of cell culture

• TCID50= log10 of highest dilution giving 100%CPE +1/2 – (total

number of test units showing CPE)/ (number
of test units per dilution)

13
 Viral identification
• Complement fixation ：
• Hemagglutination inhibition
• Neutralization
• Immunofluorescence ( direct or indirect)
• Latex agglutination
• In situ EIA
• ELISA
• RIA(radioimmuno
14
 Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)
• Detection of viral genetic material
• Serologic procedures

15
 Detection of viral proteins
( antigens and enzymes)
• Antigen detection ( ELISA, RIA, Western blot)
• Hemagglutination and hemadsorption
• Enzyme activities( reverse transcriptase)
• Protein patterns( electrophoresis )

16
 Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)]
• Detection of viral genetic material
• Serologic procedures

17
Detection of viral genetic material

• PCR ( Polymerase chain reaction)

• RT-PCR (Reverse transcriptase polymerase chain
reaction)
• Southern （ DNA), Northern(RNA), and dot blots
• DNA genome hybridization in situ(cytochemistry)
• Electrophoretic mobilities of RNA for segmented RNA
viruses( Electrophoresis)
• Restriction endonuclease cleavage patterns

18
 Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)]
• Detection of viral genetic material
• Serologic procedures

19
 Serologic procedures
• If the antibody titer in the convalesent-phase
serum sample is at least 4-fold higher than the
titer in the acute-phase serum sample, the patient
is considered to be infected.
• In certain viral diseases, the presence of IgM
antibody is used to diagnose current infection
• Other nonspecific serologic tests are available

20
 Serologic procedures
• Complement fixation ：
• Hemagglutination inhibition
• Neutralization
• Immunofluorescence ( direct or indirect)
• Latex agglutination
• In situ EIA
• ELISA
• RIA
21
Viruses Diagnosed by Serology

• Epstein-Barr virus
• Rubella virus
• Hepatitis A, B, C, D, and E viruses
• HIV
• Human T-cell Leukemia virus
• Arboviruses ( Encephalitis viruses)

22
 Prevention
• Successes of the Past
• Possibilities for the Future

23
Active immunization

Vaccines

24
Overview of Active immunization
• Active immunization - administration of
antigen resulting in production of a specific
immune response with immunologic
memory. Response may be cellular or
humoral or both.
 Natural immunity - to diseases you have
caught and successfully fought
 Artificial immunity – Vaccination(vaccines)

25
 Attributes of a good vaccine

• Ability to elicit the appropriate immune response for

the particular pathogen
• Long term protection ideally life-long
• Safety vaccine itself should not cause disease
• Stable retain immunogenicity, despite adverse
storage conditions prior to administration
• In-expensive

26
 LIVE VACCINES
• Live attenuated organism
• Heterologous vaccines
• Live recombinant vaccines

• Attributes – live vaccines 27

Live attenuated organism
• Organisms whose virulence has been
artificially reduced by in vitro Culture under
adverse conditions, such as reduced
temperature.

28
Heterologous vaccines
• Closely related organism of lesser virulence,
which shares many antigens with the virulent
organism. The vaccine strain replication in
the host and induces an immune response
that cross reacts with antigens of the virulent
organism.
• Vaccinia virus /cowpox virus--- Variola virus

29
 Live recombinant
• Vector
1. bovine vaccine
2. BCG

30
 Advantages of Attenuated
Vaccines 2-1
•Both cell mediated immunity and antibody response
•Activates all phases of immune system. Can get
humoral IgG and local IgA
•Raises immune response to all protective antigens.
Inactivation may alter antigenicity.
•More durable immunity; more cross-reactive
•Immunity is long lived
•Single dose

31
Advantages of Attenuated
Vaccines 2-2
• Low cost

• Quick immunity in majority of vaccinees

• In case of polio and adeno vaccines, easy

administration

• Easy transport in field

• Can lead to elimination of wild type virus from

the community 32
Disadvantages of Live Attenuated Vaccine

• Mutation; reversion to virulence (often frequent)

•Spread to contacts of vaccinee who have not
consented to be vaccinated (could also be an
advantage in communities where vaccination is not
100%)
• Spread vaccine not standardized--may be back-
mutated
• Poor "take" in tropics
• Problem in immunodeficiency disease (may spread to
these patients)
33
 Killed vaccines
• 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.
34
 Killed vaccines
• Inactivated organism: rabies virus;
epidmic type B encephalitis virus.
• Subunit Vaccines: Influenza virus( HA and
NA)
• Recombinant proteins: HBV

35
Advantages of inactivated
vaccines
• Gives sufficient humoral immunity if
boosters given
• No mutation or reversion
• Can be used with immuno-deficient patients
• These vaccines tend to be able to
withstand more adverse storage
conditions,Sometimes better in tropics

36
Disadvantages of inactivated
vaccines
• Many vaccinees do not raise immunity
• poor, only antibody, no cell immediated
immune response
• response is short-lived and multiple doses are
needed
• No local immunity (important)
• Inactivated, therefore can not replicate in the
host and cause disease
• Failure in inactivation and immunization with
virulent virus
• Expense: Expensive to prepare
37
 New Methods
Selection of attenuated virus strain
• Varicella
• Hepatitis A
Use monoclonal antibodies to select for virus with altered surface
receptor
• Rabies
• Reo
Use mutagen and grow virus at 32 degrees. Selects for
temperature-sensitive virus. Grows in upper respiratory tract but not
lower
• ‘flu (new vaccine)
38
• respiratory syncytial virus
 New Methods
Passage progressively at cold temperatures
TS mutant in internal proteins
Can be re-assorted to so that coat is the strain that is
this years flu strain

39
 PB2
PB1 X PB2
PB1
PA
PA
HA HA
NA NA
NP NP
M M
NS NS

PB2
PB1
Attenuated Donor PA New Virulent
Master Strain HA Antigenic Variant
NA Strain
NP
Attenuated Vaccine Strain: M
Coat of Virulent strain with NS
Virulence Characteristics of
Attenuated Strain
40
 New Methods
Deletion mutants
• Suppression unlikely (but caution in HIV)

• Viable but growth restrictions

Problems
• Oncogenicity in some cases (adeno, retro)

41
 New Methods

• Recombinant DNA
•Single gene (subunit)

S-antigen mRNA
Hepatitis B
vaccine
cDNA
raised in yeast
Express plasmid

S-antigen mRNA
protein 42
Single gene (subunit) - problems
• Surface glycoprotein poorly soluble -
deletion?
• Poorly immunogenic
• Post-translational modifications
• Poor CTL response

43
 Single gene (subunit) in
expression vector
Vaccinate with live virus
Canary Pox
• Infects human cells but does not replicate
• Better presentation
• CTL response
Vaccinia
Attenuated Polio
Being developed for anti-HIV vaccine 44
 New Methods

Chemically synthesized peptide

• malaria
poorly immunogenic

45
 New methods
Anti-idiotype vaccine

Virus

epitope
antibody

Antibody
with epitope
binding site

46
 Anti-idiotype vaccine cont

Anti-
idiotype Make antibody
antibody
against antibody
idiotype
antibody Anti-idiotype
antibody mimics the
epitope 47
 Anti-idiotype antibody cont 2
Use anti-idiotype antibody as
injectable vaccine

Anti-idiotype Use as vaccine

antibody
Binds and
neutralizes virus
Anti-anti-idiotype
antibody
Anti-anti-idiotype
antibody Anti-anti-idiotype
Antibody to anti-idiotype antibody
antibody
48
 New Methods
New “Jennerian Vaccines”
• Live vaccines derived from animal strains of
similar viruses
• Naturally attenuated for humans
Rotavirus: Monkey Rota
80% effective in some human populations
Ineffective in others
49
Due to differences in circulating viral serotypes
 New Methods
New Jennerian Vaccines
Bovine parainfluenza Type 3
Bovine virus is:
• Infectious to humans
• Immunogenic (61% of children get good
response)
• Poorly transmissable
•Phenotypicaly stable 50
 New Methods
Second Generation Jennerian Vaccines
Rotavirus
11 segments of double strand RNA
Two encode:
• VP4 (hemagglutinin) Elicit neutralizing
antibodies
• VP7 (glycoprotein)
Co-infect tissue culture cells reassortment
•10 segments from monkey rotavirus
• 1 segment outer capsid protein of each of four major rotavirus strains
51
Efficacy >80%
 Vaccines
• 1796 Jenner: wild type animal-adapted
virus
• 1800’s Pasteur: Attenuated virus
• 1996 DNA vaccines

The third vaccine revolution

52
 DNA vaccines
• DNA vaccines are at present experimental ,
but hold promise for future therapy since they
evoke both humoral and cell-mediated
immunity, without the dangers associated
with live virus vaccines

53
 DNA Vaccines
Gene for
antigen

Muscle cell
plasmid

Muscle cell expresses

protein - antibody made
54
CTL response
 DNA Vaccines
• Plasmids are easily manufactured in large amounts
• DNA is very stable
• DNA resists temperature extremes so storage and
transport are straight forward
• DNA sequence can be changed easily in the laboratory.
This means that we can respond to changes in the
infectious agent
• By using the plasmid in the vaccinee to code for antigen
synthesis, the antigenic protein(s) that are produced are
processed (post-translationally modified) in the same way
as the proteins of the virus against which protection is to
be produced. This makes a far better antigen than
purifying that protein and using it as an immunogen.
55
 DNA Vaccines
• Mixtures of plasmids could be used that encode many
protein fragments from a virus/viruses so that a broad
spectrum vaccine could be produced

• The plasmid does not replicate and encodes only the

proteins of interest

• No protein component so there will be no immune response

against the vector itself

• Because of the way the antigen is presented, there is a CTL

response that may be directed against any antigen in the
pathogen. A CTL response also offers protection against
diseases caused by certain obligate intracellular pathogens56
(e.g. Mycobacterium tuberculosis)
 DNA Vaccines

Possible Problems

• Potential integration of plasmid into host

genome leading to insertional mutagenesis

• Induction of autoimmune responses (e.g.

pathogenic anti-DNA antibodies)

• Induction of immunologic tolerance (e.g. where

the expression of the antigen in the host may lead
to specific non-responsiveness to that antigen) 57
 DNA Vaccines
DNA vaccines produce a situation that reproduces a virally-
infected cell
Gives:
• Broad based immune response
• Long lasting CTL response
Advantage of new DNA vaccine for flu:
CTL response can be against internal protein
In mice a nucleoprotein DNA vaccine is effective against a range of viruses
with different hemagglutinins 58
 Adjuvants
• Certain substances, when administered
simultaneously with a specific antigen, will
enhance the immune response to that
antigen.

59
 Adjuvants in common use
• Aluminium salts
• Liposomes and immunostimulating complexes
• Complet Freund’s adjuvant is an emulsion of
mycobacteria, oil and water
• Incomplete Freund’s adjuvant
• Muramyl di-peptide
• Cytokines

60
Possible action modes of adjuvant

• 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

61
Smallpox

62
 Smallpox

• Variolation

•1% v. 25%
mortality
•Life-long immunity
• No drift or shift

63
 Smallpox
Vaccination
• Jenner 1796 : Cowpox/Swinepox

• 1800’s Compulsory childhood

vaccination
• 1930’s Last natural UK case
• 1940’s last natural US case
• 1958 WHO program
• October 1977: Last case
(Somalia) 64
 Smallpox • No animal reservoir
• Lifelong immunity

• Subclinical cases rare

• Infectivity does
not precede overt symptoms

• One Variola serotype

• Effective vaccine

• Major commitment by governments

65
 polio
• Killed virus vaccine(Salk, 1954)
• Live attenuated oral polio vaccine( Sabin,
1957)
• The inactivated Salk vaccines is
recommended for children who are
immunosuppressed.
•

66
 Polio Vaccine
Small RNA virus Some drift…but not too far as non-viable
Sabin attenuated vaccine
~ 10 cases vaccine-associated disease per year
• 50% vaccinees feces
• 50% contacts
• Vaccine-associated cases: revertants
• 1 in 4,000,000 vaccine infections paralytic polio
• 1 in 100 of wt infections
Scandinavia: Salk dead vaccine
• No gut immunity
67

•
 100
Reported cases per 100000 population Inactivated Cases per 100,000
(Salk) vaccine population United
10 States
Oral
vaccine
1

0.1

0.01

0.001
1950 1960 1970 1980 1990

68
 Total cases
Sweden and Finland
10000
Killed (Salk)
vaccine
Reported cases

1000

100

10

1
0
1950 1955 1960 1965 1970 1975
69
 Killed Live
512 (Salk) Serum IgG (Sabin) Serum IgG
Vaccine Vaccine
Reciprocal virus antibody titer

128

32
Serum IgM Serum IgM
Nasal IgA
Serum IgA
8

Serum IgA
2
Nasal and Duodenal IgA
duodenal IgA
1
48 96 48 96
70
Vaccination Days Vaccination
 Sabin Polio Vaccine
Attenuation by passage in foreign host
More suited to foreign environment and less suited to original
host
Grows less well in original host
Polio:
• Monkey kidney cells
• Grows in epithelial cells
• Does not grow in nerves
• No paralysis
•Local gut immunity (IgA)
71
Pasteur rabies vaccine also attenuated
 Salk Polio Vaccine
• Formaldehyde-fixed
• No reversion

72
 Polio Vaccine

Why use the Sabin vaccine?:

• Local immunity: Vaccine virus just like natural infection
• Stopping replication in G.I. Tract stops viral replication
TOTALLY
• Dead Salk vaccine virus has no effect on gut replication
• No problem with selective inactivation
• Greater cross reaction as vaccine virus also has antigenic drift
• Life-long immunity

73
 Measles
• Live attenuated virus grown in chick embryo fibroblasts,
first introduced in the 1960’s.
• Etiology: Measles virus
• Incubation: 8 to 12 days
• Clinical Manifestations: cough, coryza, conjunctivitis ,
erythematous maculopapular rash
fever ,Koplik Spots ,complictions include Encephalitis,
Pneumonia, and SSPE
• Treatment: Supportive

74
 Mumps

Live attenuated virus developed in the 1960’s

•
MMR vaccine
•
Etiology: Mumps Virus
•
Incubation: 16 to 18 days
•
Clinical Manifestations:
•
swelling of the salivary glands
•
complications include Meningitis, Orchitis, Encephalitis, and Deafness
•

75
 rubella
• Live attenuated virus
• Etiology: Rubella Virus
• Incubation: 14 to 21 days
• Clinical Manifestations: Congenital , cataracts
• patent ductus arteriosus , deafness mental retardation ,
Postnatal mild disease , erythematous maculopapular
rash , postauricular lymphadenopathy transient
polyarthralgias

76
 Hepatitis B
• Two vaccines are in current use:
A serum derived vaccine
A recombinant vaccine
• Etiology: Hepatitis B Virus
• Incubation: 120 days (average)
• Clinical Manifestations: jaundice ; anorexia
• nausea and vomiting ; malaise
• complications include the development of a chronic
carrier state with a high risk for Hepatocellular Carcinoma
(liver cancer)
77
 Hepatitis A

• Formalin-inactivated , cell cultured-derived

virus,

78
 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.

79
 Rabies
No safe attenuated strain of rabies virus has yet
been developed for human. Vaccines in current
use include: a] The neurotissue vaccine
b] human diploid cell culture-
derived vaccine, which is much safer.
There are two situation where vaccine is given: a]
Post-exposure prophylaxis, followinf the bite of
a rabid animal, Hyperimmune rabies globulin
may also administered .
b] Pro-exposure prophylaxis is used for
protection of those occupation puts them at risk
of infection with rabies. 80
 Influenza

• New vaccines are produced every year

81
 Varicella-Zoster virus
• Not licensed vaccines

82
83
Passive Immunisation

84
 Modes of immunization
• Passive immunization - administration of antibody-
containing serum to provide immediate, but temporary
protection. Doesn't activate a lasting specific immune
response.

85
 Natural
• Provides immunity for diphtheria, tetanus,
streptococcus, rubeola (red measles), rubella
(German measles), mumps, polio, and
others.

86
 Artificial
• Often used as antitoxins for things such as
black widow spider and snake bites, botulism,
and tetanus. Important for some infectious
diseases such as rabies, since it provides
immediate protection rather than waiting the
7-10 days for a protective response to
develop from active immunization.

87
 Immunoglobulin

• “Normal”Immune globulin
• Hyper-immune globulin

88
 “Normal”Immune globulin

Low titres of antibody to a wide range

of human viruses
• Hepatitis A virus infection
• Parvovirus infection
• Enterovirus infections (in neonates)
• HIV-infected babies

89
 Hyper-immune globulin
--- high titres of antibody to particular viruses
• 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 individal
who has been exposed to HBV
• RSV immune globulin: treatment of respiratory
syncitial virus infections in the very young

90
Antiviral Therapy

91
 Antiviral Therapy

• Antiviral chemotherapy
• Interferon
• Gene therapy
• Chinese Herbs

92
Antiviral chemotherapeutic 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

93
 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
94
95
 Diseases for which effective
therapy is available
• AIDS:
Zidovudine 叠氮胸苷 + Lamivudine 拉米夫定 + protease
inhibitors
• Influenza: Amantadine
• Herpes simplex virus: Acyclovir
• Varicella-Zoster virus: Acyclovir
• Cytomegalovirus : Gancyclovir 更昔洛韦 , Foscarnet
膦甲酸
• Respiratory syncitial virus: Ribavirin 利巴韦林
96
 Nucleotide analogues
• Nucleotide analogues competes with
normal nucleotide for incorporation into
viral DNA or RNA.

97
 Interferon
• Direct antiviral effect ( prevents the infection
of new cells) by a) degradation of viral
mRNA, and b) inhibition of protein synthesis
• Enhancement of the specofic
immuneresponse by increasing the
expression of MHC class I molecules on the
surface of infected cells, the interferons
increase the opportunity for specifif cytotoxic
T cells to recognise and kill infected cells
• Chronic hepatitis B and C virus
98
 Chinese Herbs

• 板蓝根、大青叶、苍术、艾叶。
• 双黄连

99