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Assignment of Analytical microbiology

“NOROVIRUSES”

Assignment of Analytical microbiology “NOROVIRUSES” B.K. Kolita Kamal Jinadasa, M.Sc. Food Science and Technology

B.K. Kolita Kamal Jinadasa,

M.Sc. Food Science and Technology

Department of Food Science and Technology

University of Sri Jayawardanapura

Index No :…………………

Summary

Noroviruses are members of a group of viruses called caliciviruses also known previously as “Norwalk-like viruses.” Infection with norovirus affects the stomach and intestines, causing an illness called gastroenteritis, or “stomach flu”. In addition, noroviruses are not related to bacteria and parasites that can cause gastrointestinal illnesses. Norovirus is not a “new” virus, but interest in it is growing as more is learned about how frequently noroviruses cause illness in people. Norovirus infection causes gastroenteritis, which is an inflammation of the stomach and the small and large intestines. The symptoms of gastroenteritis are nausea, vomiting, and/or diarrhea accompanied by abdominal cramps. Some people also complain of headache, fever/chills, and muscle aches. Symptoms usually last for 1 or 2 days. Symptoms usually begin 24 to 48 hours after ingestion of the virus, but can appear as early as 12 hours after exposure. Noroviruses are found in the stool or vomit of infected people. People can become infected with the virus in several ways. Food and drinks can very easily become contaminated with norovirus because the virus is so small and because it probably takes fewer than 100 norovirus particles to make a person sick. Most people recover completely within 1 to 2 days, with no long-term complications of norovirus illness. However, persons who are unable to drink enough liquids to replace those lost with vomiting and/or diarrhea may become dehydrated and require special medical attention.

However reported of Norovirus influence is less in Sri Lanka. Therefore, available information and attention are also very less. But it can happen, and have to chance and out comes here, therefore I thought to study and find out the information about Norwalk like virus.

Index

Page No:

General Identification

01

Noro virus

07

Identification

10

References

12

Table list:

Table 1: Size and genome content of viruses, E.coli and a human cell

03

Table 2: Difference between viruses and cells

05

Table 3: Major food borne illness (1999)

09

Table 4: mean incidence for reported cases in New Zealand by pathogen, 2005

09

Table 4: Similarities and difference between Norovirus and Rotavirus

11

List of figures:

Figure 1: Morphology of virus

06

Figure 2: Transmission electron micrograph of Norovirus. The bar = 50 nm

08

Figure 3: Oyster accumulates with Noro Virus

10

Viruses:

General characteristics

Viruses are nonliving agents that are associated with all forms of life, including all members of eubacteria, archaea, and eukarya. These virus particles consist of nucleic acid surrounded by a protection protein coat, as a capsid. According to several features viruses distinguish from cells. The most obvious feature is small in size. Viruses are normally 100 fold to 1000 fold smaller than fold smaller than the cell they infected.

The sizes of the longest viruses are same to the smallest bacteria cells. Viruses contain very few nucleic acid (very few gene), because their smaller size.

 

Size

Genome content

(Diameter)/nm

Smallest virus

20

4-5 gene

Largest virus

300

150-200 gene

E. coli cell

1,000

3,000 gene

Human cell

20,000

<60,000

Table 1: Size and genome content of viruses, E.coli and a human cell

Viral genome is different from cells. Viruses contain either RNA or DNA. Viruses are not containing both DNA and RNA. Viruses are called as RNA viruses or DNA viruses depending on the type of nucleic acid. The nucleic acid can occur one or several different forms. This feature is a characteristic of the family of virus. Viruses include single stranded DNA or double stranded DNA and single stranded RNA or double stranded RNA.

Viruses lack of cellular components. This is one characteristic differ from cells. They can’t generate energy and synthesize many different proteins, because of lack of cellular components. Therefore viruses contain are generally involved in to cells when viruses entry to the cells. Then start to replicate their own nucleic acid. Therefore viruses can only multiply inside living cells. They use cell structures and enzymes to support their own multiplication.

Viruses have two distinct phases in their life cycle. They multiply only within cells other than outside of cells they are metabolically inert. Inside susceptible cells they can use metabolic machinery and pathways of their host cells to produce new virones, as the second phase of life cycle. When there genome only enter the host cells viruses can reproduce themselves. This is a character differ from cells.

If viruses cannot multiply within living cells they are died. Virus differences from living cells can be seen following table.

 

Viruses

Living cells

Growth

Grow only within cells, outside cell metabolically inert

Most free living

Nucleic

acid

Contain either DNA or RNA, never both

Always contain both DNA and RNA

content

Enzyme content

Have very few if any enzymes

 

Have many enzymes

Cell components

Lack

ribosome

and

enzymes

to

Contains ribosome and enzymes for generation of energy

generate energy

 

Table 2: Difference between viruses and cells

Regarding their morphology different species of viruses have different shapes. The shapes are polyhedral, helical and combination of these two shapes. Sometimes polyhedral appear to spherical shape when viewed with electron microscope. Most viruses are very complex structure according to host cells.

Shape of the protein capsule is determined the shape of viruses. Each capsid contains many identical protein subunits call capsomers. The viral capsid and nucleic acid tightly packed with the protein coat is called nucleocapsid. In many viruses the capsid is the only outer coat. These viron are called necked or non enveloped capsid virus. Some viruses which infect human other animals have and additional lipid membrane or envelop. The envelop consists of layer of lipids as similar of the cytoplasmic membrane of the cell. Inside the lipid envelop contain matrix protein. Attachment protein called spikes help to involve in attaching the host cells.

Figure 1: Morphology of virus. Major types of relationships betw een virus and the host

Figure 1: Morphology of virus.

Major types of relationships between virus and the host cells they infect. These relationships are found in both bacterial and animal host cells. It can summarize as bellows.

Virion

and animal host cells. It can summarize as bellows. Virion Host cell Infectio Disease of host

Host cell

Infectio

Disease of host cell

as bellows. Virion Host cell Infectio Disease of host cell Genetic alternation of host Productive response
as bellows. Virion Host cell Infectio Disease of host cell Genetic alternation of host Productive response

Genetic alternation of host

Productive response (More virus produced)

Release of virion- non lysis of cells

virus produced) Release of virion- non lysis of cells Latent state (Nucleic acid of virus becomes

Latent state (Nucleic acid of virus becomes part of host cell

Lysis of cells- release of virions Host cell dead
Lysis of
cells- release
of virions
Host cell
dead

Host cell multiplies- continuous release of virions

Host cell modified and multiplies

Noro viruses:

Noro viruses are group of viruses that can cause stomach flu or gastro enteritis in people. The tem noro viruses were recently approved as the official name for this group of viruses. This noro viruses are called in several names as Nor-walk like viruses (NLV), calci viruses (Noro viruses belong to claciviridae family), and small round structure viruses (SRSVs). Noro viruses are belonging to group of RNA viruses and belong to the genus Norovirus.

This group of viruses caused an outbreak of gastroenteritis in a school in Norwalk, Ohio in 1968. Therefore they are called Norwalk viruses.

There are at least five norovirus genogroups. They are GI, GII, GIII GIV and GV. They can be divided into at least 25 genetic clusters.

GI (five genetic clusters)

GII (>15 genetic clusters)

GIII (detected in pigs and cattle)

GIV

GV (recently detected in mice)

Among the genogroups GI, GII and GIV strains in feed only humans. They cause acute gastroenteritis. Examples of GI clusters include Norwalk virus (GI/1), Southampton virus (GI/2), and Desert Shield virus (GI/3); examples of GII clusters include Hawaii virus (GII/1), Snow Mountain agent (GII/2), and Toronto virus (GII/3); and an example of a GIV cluster is Ft. Lauderdale virus (GIV/1)

Norovirus contain positive sense RNA genomes of approximately 7.5 kbp. Major structure protein (VP1) is about 58-60 kDa and a minor capsid protein is VP2. Using an electron microscopy, the virus particles can be identified an amorphous surface structure. The size of the virus particles are between 27-38 nm.

Figure 2: Transmission electron microgra ph of Norovirus. The bar = 50 nm Norovirus are

Figure 2: Transmission electron micrograph of Norovirus. The bar = 50 nm

Norovirus are resistant to environment changes. They can survive in up to 10 ppm chlorine,

present in public water systems. They can bare freezing, can survive temperature as high as

140 0 F (60 0 C). They have been associated with illness after steamed in shellfish.

Norovirus are a common cause of acute gastroenteritis. Noroviruses occur all over the world but data are not enough based on annual medicine of disease. A research conducted in the Netherlands found that the incidents of gastroenteritis was 79.7 per 10,000 person year and Norwalk like virus were isolated from 5% of patient who participated in the study. A study conducted in England small round structured viruses were found in 6% to 7% of stools from patient with gastrointestinal illness. Norovirus are common cause to of spread diarrhea. Among 124 travelers to Mexico who had acute diarrhea, noroviruses were the second most commonly identified pathogen in diarrheal stool samples (21/124, 17%). This amount exceeded only by enterotoxigenic Escherichia coli (50/106, 47%).

The incidence of norovirus GII/IV strains has been increased during the period from July 2002 to June 2004 in the USA. This is and same to what occurred in England, Wales, Germany and Netherlands in 2007. The incidence of norovirus of GII strains has increased in other countries like Ireland, Hungary, Japan, New Zealand and Australia. In these countries, molecular sub typing demonstrated a preponderance of GII strains, although the specific sequoias varied somewhat. The Virus Reference Department at the Health Protection Agency's Centre for Infections in the United Kingdom recently reported that an increasing proportion of norovirus outbreaks have been caused by a new variant of the genogroup II.4 (Bristol/1993/UK [Grimsby]) strain. Changes have been conserved amino acid by revealed by sequencing of capsid compared with other strains of genotype.

The hunter 284 strain had been the predominant strains until the end of 2005. The new variant was found in December 2005. This new variant has been reported in the Netherlands, France and Denmark. Strains from two outbreaks on cruise ships have been identified as the new variant.

In April 2006, 29% of all genotype II-4 strains were identified as new variant. The percentage had risen to 57% in May 2006. Every month the incidence of infections from the new strains has increased since January 2006. Such a seasonal pattern is unusual because norovirus out brakes normally decline by summer. Different seasonal patterns help to emergence new strains.

Annual nor-walk virus outbreak and incidents were not recorded in Sri Lanka. Foods borne illness are major problem to public health. It can be summarized as follows;

Pathogen

Cases

Hospitalization

Deaths

Salmonella

1,350,000

16,000

560

Campylobacter

2,000,000

10,500

100

Shiga-toxin, E.coli

110,000

950

26

Listeria

2,500

2,300

500

Norovirus

9,200,000

20,000

124

Toxoplasma

112,500

2,500

375

Table 3: Major food borne illness (1999)

At the recent publication of food focus magazine, New Zealand has indicated that Norovirus take important role for food borne diseases at year 2005.

 

Mean incidence

Salmonella

16,800

Yersinia

7,900

Campylobater

123,000

E. coli

340

Listeria

24

Norovirus

108,000

Table 4: mean incidence for reported cases in New Zealand by pathogen, 2005

Nausea, vomiting, diarrhea and stomach cramping are major symptoms of norovirus illness. Some people show symptom like low grade fever, chills, headache, muscle ache and tiredness.

The illness begins very quickly, it is very difficult to bare. The illness is last within one or two days. Children experience more vomiting than adults. Although people may feel very sick and vomit many times a day, norovirus disease is usually not serious. Most people recover within one or two days. They have no long term health effects related their illness. Some people can become dehydrated need special medical attention. 24-48 hrs after contaminated food or water is consumed, a mid and brief illness usually developed. The foods most often implicated in nor- walk out breaks are shellfish and salad ingredients. Eating raw or insufficiently steamed clams and oyster’s process and ill food handler cause a high risk for noro virus infections.

People can become infected with the virus and several ways, including;

1. Eating food or drinking liquids that are contaminated with Norovirus (water is the most common source of outbreaks).

2. Touching surface or objects contaminated with Norovirus, and then placing their hand in their mouth.

3. Having direct contact with another with person who is infected and showing symptoms.

another with pe rson who is infected and showing symptoms. Figure 3: Oyster accumulate with Noro

Figure 3: Oyster accumulate with Noro Virus

Still there is no antiviral medication to destroy norovirus and no vaccines to food prevent infection. Antibiotics can’t help to treat specific norovirus infections, because antibiotics work to fight bacteria not to virus.

Identification:

Using rapid antigen detection of norovirus in stool specimen is one identification method. Enzyme immunoassay or reverse transcriptase polymerase chain reaction is used for further identification, but not commonly used. A commercial EIA has been developed for detection of viral antigen in feces. The sensivity is only 55% when RT-PCR is the reference assay. Development of EIA serologic test helps to determines noro virus capsid genes. EIA tests are

used to characterize IgA, IgG, and IgM serologic responses. Electron microscope can confirm human noro virus infection, but required 106 to 107 virus particles per milliliter of stool for visualization. Immune electron microscopy (precipitation of virus with antibody before electron microscopy) can improve sensitivity by 1 to 2 orders of magnitude, but the technique is highly dependent on individual microscopics and virus can be masked if excess antibody is used. The technique is not used for routine screening. RT-PCR assays have become one of the principal means of diagnosing human noroviruses. RT-PCR technology is available at the CDC and most state public health laboratories. The following steps are involved:

Clinical stool (or vomitus) suspensions (10% to 50%) are made and extracted to purify nucleic acid and remove potential RT-PCR inhibitors.

Viral nucleic acid is purified.

Viral nucleic acid is reverse transcribed and amplified using viral specific primers.

Amplified DNA can be analyzed by gel electrophoresis, hybridization, or DNA sequencing to confirm the sequence.

Rotavirus gives the similarities and differences between Norovirus and the illnesses they produce; it can be summarized as below table.

 

Rota virus

Noro virus

Characteristics

70 nm diameter, double walled capsid; double-strained RNA; many strains cultivable

27 nm diameter, single strained, positive sense RNA, not cultivable

Incubation

24-48 hrs

18-72 hrs

period

Symptoms

Diarrhea, abdominal cramps, vomiting, 5-8 days

Abdominal cramps, vomiting, muscle and headaches; 24-48 hrs

Laboratory

Electron microscopy or ELISA of diarrheal stool for virus

Same

diagnosis

Treatment

No antiviral; occasionally intravenous fluid replacement for infants

Same

Table 4: Similarities and difference between Norovirus and Rotavirus

References:

Brett Michelle, 2006. Norovirus, international federation of infection control, volume 2.

Food focus, November2008, by food safety authority, New Zealand

Nester, Roberts, Pearsall, Anderson. Microbiology, a human perspective, 2 nd edition, chapter 13 th and 14 th .

Training manual of seafood safety and management, 2008. Post Harvest division, Agri & veterinary authority of Singapore.

noro virus/Virus - Wikipedia, the free encyclopedia.mht