Diarrhoeal Viruses Slide Set

Diarrhoeal Viruses

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 ;-

Virus Family

1. Poliovirus
2. Coxsackievirus A
3. Coxsackievirus B
4. Echovirus
5. Enterovirus 68-71
6. Adenovirus
7. Reovirus

B. Viruses discovered by electron microscopy, cannot be grown routinely.

1. Rotavirus
2. Adenovirus
3. Astrovirus
4. Caliciviruses
    a. Noroviruses
    b. Sappoviruses (classical caliciviruses)
5. Small Round Viruses
6. Coronaviruses
7. Breda like viruses
8. Bacteriophages

Pathogenesis

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.

Host Immunity

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.

Diagnosis
 

1. Electron microscopy - This is the only catch-all method currently available. Faecal extracts, preferably after undergoing concentration, are stained and placed onto grids. For the virus to be recognizable, it should be present in quantities exceeding 106 /ml. All diarrhoeal viruses were first discovered by electron microscopy. The drawback for electron microscopy is its cost and the requirement for trained staff. The sensitivity and specificity of EM may be enhanced by the use of immune electron microscopy techniques.

2. PAGE - Stool extracts may contain levels of viral nucleic acid high enough to be demonstrated by PAGE. Many types of viruses can be recognized eg. rotaviruses have 11 segments which could be recognized readily, as can a single line corresponding to the double stranded DNA for adenoviruses. It may be possible to apply restriction endonucleases to further characterize the DNA or RNA. There is no reason why other viruses cannot be demonstrated by this method. This method may eventually be considered as a cheaper alternative to EM as a catch-all method for the detection of diarrhoeal viruses.

3. Culture - None of the fastidious viruses will grow readily in cell culture. however, it may be possible in the foreseeable to find culture systems for many of these viruses.

4. Virus Antigen Detection - These tests are specific tests using polyclonal and monoclonal antibodies. Most of these tests are home made tests by individual laboratories although commercial tests are becoming available. ELISA and Latex Agglutination are the most commonly used tests.

5. PCR - PCR has become the method of choice for diagnosing norovirus infections, particularly in outbreak situations.

Rotaviruses

A. Properties

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.

B. Epidemiology

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

1. Point mutations - RNA polymerase has a high error rate. The error rate is 10^-4 for each site. These errors accumulate very quickly.
2. Reassortments - natural reassortments occur, rather like influenza A.

3. Introduction of animal rotavirus into the human population, the group B rotaviruses, responsible for causing disease in China, is found in household animals. This is confirmed by serological and PAGE studies.

4. Gene rearrangements - children with immunodeficiencies (SCID) are prone to develop chronic infection. The electropherotypes which emerge 1 year later is very different to that of the original strain. The rearranged genes may consist of a translated region and an untranslated duplicated region. Therefore these viruses have additional nucleotides in their sequence. Atypical PAGE profiles are common in group A. In one case, a novel protein has been identified in one such isolate. The biological properties of this virus is also different. Viruses with genome rearrangements have also been isolated from asymtomatically infected immunocompetent children and animals. Isolates with rearrangements in segments 5, 6, 8, 10, and 11 have been characterized, with the greatest number having rearrangements in segment 11. Viruses containing rearranged segments are generally not defective, and the rearranged segments can reassort and replace normal RNA segments structurally and functionally.
      

C. Pathogenicity

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 WHO’s 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. Most children (about 9 out of 10) who get the vaccine will be protected from severe rotavirus illness. While about 7 out of 10 children will be protected from rotavirus illness.

Adenoviruses

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.

Astroviruses

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.

Noroviruses