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Diarrhoeal Viruses Slide Set
It had taken a long time before particular viruses were identified as agents in gastrointestinal diseases. The search for viruses causing diarrhoeal disease was unsuccessful initially because the viruses involved failed to grow in vitro. However, by the early 1970s, close to 100 serotypes from at least 6 different viral families had been recovered from stools by culture. However, there is little evidence that they are involved in the causation of GI disease ; Viruses could be recovered from the faeces of apparently healthy persons and an association between cultivable viruses and diarrhoea was rare. However it cannot be ruled out of hand that under the right circumstances, these cultivable viruses can show greater virulence in the gut and cause GI disease. Nevertheless, it is now apparent that these cultivable viruses make no more than a minor contribution to viral gastroenteritis. This was emphasized in the late 60s when the use of the oral Sabin vaccine became widespread which did not result in GI disease in the recipients.
With the use of electron microscopic techniques, a new group of viruses which were seen in faecal extracts, but cannot be grown routinely were discovered. Electron microscopic techniques were first applied to the faecal extracts of a school outbreak in Norwalk, Ohio. Acute and convalescent serum from cases were used in an immune electron microscopic technique. 3 years passed before the discovery of the next virus associated with diarrhoeal disease, the rotavirus. Rotaviruses and adenoviruses are regularly involved in endemic diarrhoea throughout the world, whereas epidemic diarrhoea is associated with small round viruses or small round structured viruses. Animal experiments have played an important role in the investigation of human disease. Diarrhoea may be induced in newborn animals by challenging them with viruses extracted from human faeces. Furthermore, naturally occurring diarrhoeal diseases are seen in animals which are caused by viruses related to those seen in human disease.
A. Viruses which will routinely grow in cell culture or newborn mice ;-
2. Coxsackievirus A
3. Coxsackievirus B
5. Enterovirus 68-71
B. Viruses discovered by electron microscopy, cannot be grown routinely.
b. Sappoviruses (classical caliciviruses)
5. Small Round Viruses
7. Breda like viruses
The evidence implicating viruses as causes of diarrhoea is variable. Some particles could no doubt be positively identified as viruses on morphological grounds alone eg. rotaviruses and adenoviruses. Moreover, these viruses are often present in the stools in large numbers. The smaller particles though ie. 20-35nm particles, present much greater difficulties. A good electron microscope is required in order to visualize them. It is probable that they are found more frequently by those who are more strongly motivated to do so. Moreover, astroviruses and caliciviruses often do not show their characteristic appearance on electron microscopy. Thus some astroviruses may be recorded as SRVs and some caliciviruses as noroviruses. The advent of PCR have dramatically increased our understanding of noroviruses and other diarrhoeal viruses.
Noroviruses and some strains of the sappoviruses appeared to be associated primarily with outbreaks. Rotaviruses, adenoviruses and astroviruses are associated with endemic diarrhoea. It was previously thought that rotaviruses were by far the most common cause of viral gastroenteritis. However, recent data suggests that noroviruses are the most common cause of outbreaks and endemic infection in all age groups. It is thought that noroviruses could account for up to 90% of all cases of gastroenteritis.
Virus infection in the gut induce the formation of systemic,
and possibly local antibodies. Experiments with Norwalk virus
showed that even the induction of systemic and local antibodies
did not prevent reinfection. It is uncertain though whether this
is a general phenomenon as poliovirus vaccine induces effective
protective local and systemic immunity. The actual role of
antibody in the gut is uncertain. Breast milk has been shown to
contain good levels of antibody but breast fed babies are no less
susceptible to infection but the proportion developing overt
symptoms appear to be less.
Belong to the family of Reoviruses
dsRNA viruses with cubical symmetry
virion 72nm in diameter
RNA contained in 11 separate ds RNA fragments
5 antigenic groups are known (A-E), most human strains belong to group A.
Unable to grow in routine cell cultures.
Electronmicrograph of rotavirus particle. (Courtesy of Linda M. Stannard, University of Cape Town)
The 11 RNA fragments of rotaviruses can be visualized easily by PAGE of faecal extracts. The banding patterns show considerable diversity but those of each antigenic group show general similarities within each group. The pattern of each strain is generally constant although variation through mutation appear through time. Other sources of variation in the patterns found arise through reassortment between strains and, following passage at high multiplicities of infection, through fusion of fragments. Human rotaviruses do not produce new infectious virus in routine cell cultures, the reasons not being fully understood. In several cell types, e.g. LLC-MK2, a partial replication takes place with the production of intracellular viral antigens. However, some animal strains can grow in cell cultures and some human strains can be adapted to grow in cell cultures by prior treatment with small amounts of trypsin. (This probably facilitates the release of the nucleic acid into the cells but the exact mode of action of trypsin is still not known.) Efficient techniques are now available for the direct cultivation of most rotaviruses in cell culture. The virus is pretreated with trypsin and trypsin is incorporated in the maintenance medium. Simian MA104 cells, primary AGMK or cynomologus MK cells in roller tubes are used and incubated at 37oC.
Rotaviruses possess group, subgroup and serotype antigens. The majority of human and animal and animal strains possess the same group antigen but a minority of human and animal strains belong to the other 4 groups. The 5 groups are designated A to E. Group A viruses have been further subdivided into subgroups I and II, again based on VP6. Most human strains belong to group A but human strains belonging to groups B and C have been identified which is associated with outbreaks of gastroenteritis. Group B strains are rare outside China. Group A strains have 9 serotypes, of which types 1-4, 8 and 9 have been found in man. The others being found in animals. The group specificity is located on the outer surface of the 60nm rough particle. The outer smooth capsid layer of complete particles is composed of products of 3 RNA segments coding for VP4, VP7 and VP11 respectively. VP7 has serotype specificity and is thought to be the main neutralizing antigen but VP4 is also involved in neutralization. Serotypes are defined by neutralization tests whilst subgroups are defined by ELISA or IAHA.
G serotype specificity refers to VP7, while P serotype specificity refer to VP4. Fourteen G types have been defined, of which 10 had been isolated from two or more animal species. However only four G types, G1-G4, are important causes of diarrhoea in infants worldwide. To date, 10 P types had been characterized serologically. G1, G3, and G4 are usually associated with P8, and G with P4. However in India, a high percentage of the strains were of G9 and P8, and in Brazil, G5 and P8. This suggests that reassortment between common and uncommon serotypes can arise in some settings and thus leading to unusual diversity.
Rotaviruses account for around half a million deaths in infants in developing countries per year. Even in advanced countries such as the US, it accounts for up to 500 deaths per annum there. Rotavirus outbreaks appear at regular outbreaks in the winter in temperate climate. In different years, the prevalence of different serotypes varies dramatically. There is an unpredictable change in serotype prevalence in different areas and time. G1-G4 serotypes account for 95% of all isolates, of which G1 seems to prevail in most areas. The following are thought to be responsible for the changing serotypes in a population
Rotaviruses are mainly endemic, being involved only occasionally in outbreaks. Rotaviruses are by far the largest cause of diarrhoea in children, accounting for 50-80% of cases. However group B rotaviruses are regularly involved in outbreaks in China. Subclinical infections are common and no clinical syndrome can be assigned to rotaviruses. Symptoms of diarrhoea, fever and vomiting are common. The site of infection is principally the upper small bowel, the evidence having been derived from animal studies. Respiratory symptoms and signs have been noted at the same time as rotaviruses have been found in the faeces but the virus have yet to be isolated from the nasopharynx. The virus has also been reported in the vomitus. Rotaviruses are responsible for half a million deaths in developing countries per year. Rotavirus infections are thought to become more frequent as the weather becomes colder in temperate regions. in tropical areas, the fluctuations have been linked with periods of low humidity. Hospital acquired infections are common, particularly in neonatal nurseries. Experiments with cultivable human and bovine strains have shown rotaviruses to be rather durable and resistant to disinfection processes. Prevention of spread of infection in wards must depend on scrupulous cleanliness.
Animal Parallels - virtually any animal species can experience rotavirus infection which is associated with diarrhoea. The opportunity for strains to cross species barrier makes duel infections theoretically possible. Reassortment of genes can occur, leading to hybrid strains having individual RNA segments from either parent.
Immunity - seroconversion occurs following infection
with rotaviruses in both animals and man. Worldwide studies have
shown that 80% of the population has antibody from the age of 3
onwards. Only a small minority of these have had disease severe
enough to merit medical attention. Possession of antibodies does
not necessarily prevent reinfection but the chances of overt
disease are reduced. Some correlation between immunity and the
level of serum antibodies have been reported.
D. Development of rotavirus vaccines
The aim of a rotavirus vaccine would be to prevent severe rotavirus gastroenteritis during the first 2 years of life. Although reinfections with rotaviruses are common, it appears that homotypic immunity is effective against diarrhoeal illness. There are at least 4 epidemiologically important human rotaviruses (serotypes 1, 2, 3 and 4) and that means that effective coverage against disease may require a quadravalent vaccine. The most evaluated approach to the development of a vaccine is the Jennerian approach where a live animal virus is used. The results of the trials of these vaccines ranges from very encouraging to disappointing. Whilst the vaccine was effective in protecting the children against infection by rotavirus of the same serotype, it was ineffective against another serotype. Other approaches to the production of a vaccine based on molecular biology methods are being tried, such as the use of synthetic viral proteins.
A rotavirus vaccine (Rotashield, Wyeth) was licensed in the USA but was later withdrawn by manufacturer because of suspected association with intussuception. The Wyeth rhesus rotavirus tetravalent vaccine consists of a mixture of four viruses (a rhesus rotavirus and three human-rhesus reassortants) that together include the four G types most commonly found in the US. A rotavirus vaccine is being developed by Merck which is a mixture of four human-bovine reassortants that include three of the four major G types and one human P type. Trials in the US showed that both the Merck and Wyeth were safe and gave 50-60% protection against all rotavirus disease and >80% protection against severe dehydrating diarrhoea. However trials in S America indicate that protection may be somewhat less in those countries. It is hoped that eventually, rotavirus vaccines will come into general use, resulting in massive cost savings in industrialized countries (there are up to 20000 hospital admissions per year in the UK), and marked reduction in mortality in developing countries. The ultimate goal would be inclusion into the WHOs expanded program for immunization. There is still no good immune correlate of protection for rotavirus disease. No correlate of immunity could be identified on the basis of seroconversion to any antibody measured.
As of April 2008, the US FDA has approved two oral rotavirus vaccines. The Rotarix (GlaxoSmithKline) vaccine is given in a two-dose series to infants from 6 to 24 weeks of age, and the RotaTeq (CSL Limited/Merck and Co, Inc) is given as 3 doses.
Two types of adenoviruses may be recovered from the stools, the conventional cultivable types,and the fastidious types which do not grow in routine cell culture. Apart from the failure to propagate in cell cultures which are normally permissive for adenoviruses, these fastidious strains show no obvious differences from those isolated in cell culture from both the respiratory tract and the gut. PAGE of the stool extracts containing adenoviruses detectable by electron microscopy reveal a single band corresponding to intact DNA. The strain(s) present can be characterized by a restriction digest. The classical adenoviruses can be put into 5 subgenera (A-E) on the basis of broadly similar patterns. By the same criteria, the fastidious serotypes 40 and 41 belong to a new subgenus F. The fastidious adenoviruses do not grow in routine cell culture. Some of these fastidious viruses will grow in cell lines such as HEK-293. In other cell types, a partial cycle of replication takes place where some virus antigens can be detected by immunofluorescence. As with rotaviruses, neither the reason for failure to produce mature virus is known nor the factor provided by these permissive cell types which allows complete virus to be produced have been identified. Very large numbers indeed of fastidious adenoviruses may be found in stool extracts. The fastidious adenovirus possess the same group antigen of all human adenoviruses. 2 new serotypes (40 and 41) have been identified in the strains which will replicate in only on one of the fastidious cell types.
Electronmicrograph of adenovirus particle with pentons fibers clearly visable. (Courtesy of Linda M. Stannard)
Association with disease
It is common to find adenovirus in the stools of children by electron microscopy. It has been estimated that up to 6-8% of children contain concentrations detectable by electron microscopy, but this is not confined to diarrhoea stools. Asymptomatic and prolonged excretion of adenoviruses has been found in babies, which parallels the prolonged excretion from the respiratory tract. Adenoviruses have also been associated with common source outbreaks where it has been the only potentially pathogenic microorganism present. No fastidious adenoviruses have been recovered from the respiratory tract.
Little is known about immunity to fastidious adenoviruses in man. Recent seroepidemiological surveys reported a high prevalence of antibodies against strains 40 and 41 in the community and these are acquired in early childhood.
The structure of astroviruses is unique amongst human viruses and they have been found so far only in the gut. They are so called because of their surface has a star-shaped configuration, which is not seen on all particles. Astroviruses are spherical particles with a mean diameter of 28 nm. Its structure is unknown but is unlikely to be based on an icosahedron, with which the 6 pointed star is incompatible. Animal astroviruses have a positive stranded RNA. 5 serotypes of human astroviruses have now been described by neutralization tests. There is no group antigen.
Electronmicrograph of astrovirus particles with the five pointed star structure clearly visible on the surface.
Association with disease
Astroviruses, like rota and adenoviruses may be found in the stool in large numbers. They may also be found in the stools of normal babies but more frequently in the stools of those suffering from diarrhoea. They are often hard to recognize under electron microscopy and thus they are probably considerably under- reported. Volunteer studies demonstrated that adults can be infected with the development of diarrhoeal symptoms in some cases. In addition, the majority seroconverted. However, this does not mean they have a significant role in causing diarrhoeal diseases in babies and young children. Overall it is still uncertain whether and to what extent astroviruses play a role in the causation of diarrhoeal diseases although on balance, the evidence tilts towards a causative role.
Immunity and its transfer
Antibody is induced following infection but it is uncertain whether it confers protection. A survey carried out in the UK showed that by the age of 3, the majority of children have acquired antibody against astroviruses. This pattern of antibody acquisition is similar to rotaviruses, suggesting that infection is common and that many such infections are unrecognized.
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.
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.
A. 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