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Diarrhoeal Viruses Slide Set
Norwalk virus was the first fastidious enteric virus to be discovered, following an outbreak affecting both children and adults in a primary school in Norwalk, Ohio in 1968. Bacterial-free filtrates of stools collected induced similar illness in human volunteers. Virus like particles were seen under EM by Kapikan in 1972, immune electron microscopy was used. In subsequent years a number of outbreaks in summer camps, cruise ships and other gatherings have been associated with this virus. Antibodies against this virus are acquired later in life, indicating a less widespread distribution, in the USA at least. At the age of 50, only 50% of the population surveyed had antibodies. In a similar study in Bangladesh, antibodies were acquired earlier and the pattern was closer to that of rotaviruses in that the majority of the population had acquired antibody by the age of 3. Morphologically similar viruses have been found in Europe and elsewhere are were called small round structured viruss (SRSV). Progress in the understanding of Norwalk-like viruses had been excruciatingly slow until complete sequences of norovirus strains became available in 1995. Norwalk-like viruses or SRSVs are now firmly established in the family of Caliciviridae. Sensitive RR-PCR techniques became available and it was only then that the true extent of Norovirus infections was realized. It is now generally accepted that Noroviruses are the most common viral gastroenteritis agent, well surpassing rotaviruses.
Norwalk virus was originally described as parvovirus-like and 28nm in diameter. On electron microscopy, its surface has a ragged structured appearance. this was originally thought to be antibody against the virus binding to the surface as the first electron micrographs were obtained by immune electron microscopy. However, it is mow thought that the ragged outer edge is part of the surface of the virus particle. If so, the true mean diameter of the Norwalk virus particle is at least 33nm. The virus is thought to have a genome consisting of positive stranded ssRNA. Norwalk virus has not been shown to grow in cell culture with or without the addition of trypsin. Even partial growth has not been described. When present in the stool, the virus is difficult to detect by electron microscopy as the concentration of the virus in stool culture is much lower than that of rota and adenoviruses. The difficulties of finding Norwalk by electron microscopy have resulted in most infections being diagnosed by antibody tests. however, such tests will only diagnose infections by Norwalk virus itself and other similar viruses if they share a common group antigen. There is some cross-reactivity between Norwalk and Montgomery County viruses but the Hawaii agent appears to be antigenically distinct.
Noroviruses belong to the family off caliciviridae. They have similar sizes and buoyant
densities, and have a major protein of very similar molecular
weight to that of caliciviruses. There is increasing serological
evidence of cross-reaction between strains of calicivirus and
SRSV. Recently, nucleotide sequences for several NLVs have
become available which should greatly facilitate research on
these viruses. On the basis of genetic analysis, there appears to
be two main genotypes of Norwalk-like viruses, each with a large
number of subtypes. With a dramtic increase in sensitivity
compared to EM, PCR assays are now being increasingly used
for the diagnosis and investigatrion of outbreaks caused by
Electronmicrograph of Norwalk-like virus particles. Note that unlike astroviruses, it is extremely rare to find such as large number of virus particles congregated together like this.
Phylogram of Norwalk-like Viruses using the capsid sequences
Noroviruese are distributed globally, with genogroup II being more common than genogroup I viruses. They are associated with both epidemic outbreaks and sporadic cases of gastroenteritis. Outbreaks occur regularly in institutions such as nurseries, hospitals, nursing homes, restaurants, and cruise ships. It is now clear that noroviruses accounts for more endemic infections than epidemic infections. Recent data suggests that they cause at least as much or even more cases of gastorenteritis in young children than Rotaviruses. Norovirus infections are more likely to occur in winter months, hence the nickname "winter vomiting disease", although this depends on the individual country. Highly infectious, aerosol spread possible as well as faecal-oral spread. Associated with consumption of shellfish, other contaminated foods such as salads, and faecally contaminated water.
Association with Disease
A small series of volunteer experiments confirmed that the virus was capable of causing disease in adults. Half the volunteers challenged developed illness although none were severely ill. These volunteers were rechallenged between 27 and 41 months later and those who were ill on the first occasion were ill again. Virus was detected in the faeces of those challenged. The volunteers developed both serum and secretory antibodies in the gut and it is surprising that this appeared to confer no protection. Norwalk virus has been associated almost exclusively with outbreaks of diarrhoea and vomiting. Vomiting appears to be a more prominent feature of infection than with rota and adenoviruses. There is usually little or no pyrexia and the diseases is usually mild with spontaneous recovery. Outbreaks have been associated with contaminated seafood such as oysters. Person to person spread is probably a component of almost all outbreaks involving SRSVs. Outbreaks in institutions have an epidemic curve typical of person to person spread. Faecal oral spread is probably the major means of transmission. However other routes are possible such as through environmental contamination through vomiting and possibly through the airbourne route. Outbreaks of SRSV have also been associated with exposure to recreational water such as swimming in contaminated lakes. Occasionally SRSVs are responsible for sporadic cases of gastroenteritis.
1. RT-PCR - have become the major means of diagnosing norovirus infection. It is especially useful in the investigation of outbreaks. At present, only in-house methods are available that uses primers directed against the polymerase region of noroviruses. The amplicon could then be sequenced for epidemilogical analysis.
2. Electron Microscopy - classical means of diagnosis of Norovirus infection, the viruses are non-cultivable. Require samples with large number of virus particles and also highly skilled observers.
3. Antigen Detection – a number of RIAs and EIAs had been developed using norovirus-specific polyclonal or monoclonal IgG. However, none are shown to detect all strains of noroviruses and are not as sensitive as RT-PCR
4. Antibody Detection – a number of serological assays are available for the detection of rising titres of IgG or IgM using recombinant capsid proteins.
Management and Prevention
There is no specific antiviral therapy for norovirus infections. Most cases are mild with rapid spontaneous recovery. Severe cases may required admission of hospital to ensure that the patient is not dehydrated. Outbreaks of noviruses in institutions such as hospitals, schools, nurseries and cruise ships are notoriously difficult to contain. It is essential that all vomitus and diarrhoea be removed quickly and the surrounding areas disinfected scrupulously. There are programs in several countries to screen for noroviruses in imported shellfish although there is no consensus as to how useful this is. There is a possibility of developing vaccines against noroviruses using recombinant capsid proteins. However, this is complicated by the high variability and high mutation rate of norovirus strains. Also, it is not clear whether there would be great demand for a vaccine against a virus that normally causes a very mild disease.
A. Caliciviruses (Sapoviruses)
Caliciviruses have been known to infect pigs, cats, sea-lions and fur-seals but it was not until 1976 that a calicivirus was fond in human faeces. The human calicivirus is morphologically indistinguishable from these animal strains. However, human calicivirus strains are antigenically distinct from animal strains. Human caliciviruses contain a single positive stranded RNA genome. and are about 33nm in diameter. The virus shows icosahedral symmetry, the face and the apices of the icosahedron are represented by cupped hollows, of which there are 32. This gives a virus a very characteristic appearance. However, not all the particles in the stool show the characteristic hollows and these particles can resemble Norwalk agents and SRSVs very closely. 4 serotypes of human calicivirus have been recognized, 3 in the UK, and 1 in Japan. No group antigen has been detected.
Electromicrograph of classical calicivirus particles. Note the charcteristic cupped shaped depressions on the surface
3 of the 4 human serotypes described are associated with outbreaks of diarrhoea and vomiting. Vomiting being a more prominent feature than diarrhoea and in this way and the epidemic nature of the viruses, infection by human caliciviruses resemble that by Norwalk agents. Caliciviruses have also been reported infrequently in the stools of normal babies. However it is not an easy virus to find and considerable under reporting may have taken place. Antibody against caliciviruses may be no more protective than with Norwalk, a further point of similarity with Norwalk agents. Volunteer studies in the UK and USA have shown the viruses to be capable of inducing disease in adult volunteers. The incubation period being 24 - 72 hours with symptoms of N+V, abdo pain and diarrhoea. Virus is excreted in the faeces during the illness. A serological survey carried out in Japan showed that the majority had acquired antibody in the first three years of life. The pattern of acquisition of antibody closely parallels the acquisition of antibody to rotaviruses and astroviruses, suggesting this virus is widespread although few actual infections are diagnosed. With much of the attention focused on noviruses, research on sapoviruses have been comparatively lacking. Recent studies that had been carried out with sensitive RT-PCR shows that like noroviruese, sappovirus infections are much more common than previously thought. Hopefully, more definitive date will become available in the near future.
B. Small Round Viruses
Small round virus-like objects are often seen in human faeces.
SRVs show consideration variation in size. Some of the smaller
SRVs have a diameter of 22nm and are often hexagonal in outline
so that they resemble parvoviruses. In some instances,
adenoviruses may also be found in the same stool and thus these
are probably defective adenovirus-associated virus. However,
adenoviruses are not always detectable and it may well be in
those instances that one is dealing with an autonomous
parvovirus. Indeed, certain animal parvoviruses e.g. canine
parvovirus, can cause severe diarrhoeal disease in animals. SRVs
in the next larger size band are about 25nm in diameter and
resemble enteroviruses. It is uncertain whether these are new
strains of enteroviruses but these viruses do not grow in cell
culture. Larger SRVs may occasionally be seen but it is doubtful
whether these are viruses. Occasionally, cubic bacteriophages may
be misdiagnosed as SRVs. SRVs may be present in a considerable
number of stools of infants. However, when detectable, the
numbers are relatively small. They are more likely to be found in
common source outbreaks. Both SRVs and SRSVs have been associated
with common source outbreaks, of which a considerable proportion
is associated with the consumption of shellfish, particularly
oysters. The incubation period is 36-48 hours, following which
the patient develops vomiting and diarrhoea. At present, there
are no known animal parallels.
Coronavirus-like particles are occasionally seen in stool extracts. The particles seen in the stools differ from the human respiratory coronaviruses in morphology. The particles in the stools have projections that resemble pins with a narrow shaft whereas those seen in the respiratory tract have club-shaped surface projections. The identities of these particles as true viruses have yet to be proved. Most attempts to culture coronaviruses have failed although there was a single unconfirmed report of growth in fetal intestinal cells. These particles have been seen in the faeces of adults as well as children. Prolonged excretion following recovery is common. Most reports associate these viruses with endemic rather than epidemic diarrhoea. No volunteer experiments have been reported to date. Coronaviruses are well established as causes of diarrhoea in animals, particularly in swine.
Breda-like Agents ;- Virus-like particles resembling those previously described in the faeces in cows have been seen in the faeces of a few children. Although an association with diarrhoea in man has been reported, further information is needed.
Bacteriophages ;- The human gut contains vast numbers of bacteria which are potential hosts for bacteriophages. Like animal viruses, bacteriophages come in a variety of sizes and shapes. However, the tailed bacteriophage has a structure unique to this class of virus. It is possible for cubic bacteriophages to be mistaken for SRVs. Moreover, a possible role for these phages in the causation of human diarrhoea should not be totally overlooked.
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Diarrhoeal Viruses Slide Set