Appleton and Higgins (1975) first described virus particles, isolated from infants with mild diarrhoea and vomiting, wh i c h were 29-30nm in diameter. These differed in size and morphology from rotavirus, enteric adenovirus and Norwalk virus, the recognised agents of this illness. The name astrovirus was first suggested by Madeley and Cosgrove (1975) to describe similar particles observed in the faeces of babies with gastroenteritis, approximately 10% of w h i c h exhibited a starshaped surface morphology under the electron microscope.
1.2a Astrovirus as a causative agent of gastroenteritis Since the initial observation of the virus, such agents have been found in the stools of infants and children with diarrhoea, frequently in hospital and paediatric wards (Kurtz
e t aJL. , 1977; Ashley ejt al_., 1978). In many cases the virus is excreted together with other enteric pathogens, especially rotavirus (Nazer ejt al. , 1982) making it difficult to assign an etiological role to the virus. Nazer e£ al. (1982) found the excretion of astrovirus to be associated only with clinical gastroenteritis although there have been reports of the virus isolated from symptom-free babies (Madeley and Cosgrove, 1975). The disease is most common in infants, a mean age of 6.5 months for children excreting astrovirus being observed by Nazer ejt a l . (1982). Antibodies to astrovirus had been acquired in 75% of the population by the age of 10 years (Kurtz a n d Lee, 1978). There have been cases of astrovirus-associated epidemics of gastroenteritis affecting adults as well as children (Konno e_t a_l., 1982; Kurtz et al. , 1977).
Kurtz et al. (1979) carried out a study inoculating human adult volunteers with a stool filtrate, shown to contain astrovirus particles, prepared from a child with mild gastroenteritis. One subject developed diarrhoea and shed large amounts of astrovirus in the faeces; the infection was shown to be serially transmissible to other volunteers. 81% (13/16) of subjects showed a fourfold or greater rise in astrovirus antibody titre, as detected by immunofluorescence.
children suffering from gastroenteritis showed the incidence of astrovirus excretion to be less common in summer than winter, although there was no distinctive peak comparable to that found for rotavirus (Nazer e_t a l ., 1982). The illness caused by the virus is generally m ild and brief, lasting approximately 48 hours (Konno e_t ajL., 1982)
The site of replication of astrovirus is an important consideration in terms of its pathogenicity and classification. Electron microscopy of biopsy material demonstrated the presence of astrovirus particles within epithelium in the lower part of the villus, suggesting the small intestine as the site of replication (Phillips e£ ¿1., 1982). The data obtained so far suggests that astroviruses cause a transmissible, mostly mild, gastrointestinal infection which, as previous observations of antibody levels would suggest (Kurtz and Lee, 1978), is of low pathogenicity in adults. The development of a radioimmune assay for the detection of IgM and IgG against astrovirus (Wilson and Cubitt, 1988) should help to determine the extent and nature of astrovirus infections.
1.2b Characteristics o f huaan astrovirus
The cultivation of human astrovirus in vitro was unsuccessful until Lee and Kurtz (1981) incorporated trypsin into the culture medium and achieved serial propagation in a continuous cell line of rhesus monkey kidney (LLCMK2) cells. Initial studies of astrovirus were confined to morphological and buoyant density analyses using material from stool isolates
and serological cross-reactions w i t h other viral agents (Caul and Appleton, 1982; Kurtz and Lee, 1984).
Morphology under the electron microscope
The u s e of electron microscopy for the analysis of virus particles, although important, is limited in its reliability regarding morphological features a n d potential classification. Caul and Appleton (1982) analysed viruses using the electron microscope but deliberately avoided the use of antibody to preserve the appearance of the particles and facilitate morphological distinction between them. Based on the theory that the u s e of antibody in immune electron m i c r oscopy may obscure s urface structure, alter the apparent size of the particle o r create artefactual shadowing.
Astrovirus particles are 28-30nm in diameter with a star shaped su r f a c e structure, distinct from that observed for caliciviruses (Fig 1.2). This distinctive morphological characteristic is seen on only a small proportion of the particles (Madeley and Cosgrove, 1975; Ashley e_t ¿ I., 1978; Caul and Appleton, 1982). Konno £ t al. (1982) found that the morphology of the particles was indistinct when uranyl acetate was used as the negative stain, whereas a clear star configuration was observed using phosphotungstic acid.
Such observations emphasise the fact that viral agents cannot be assigned to or disqualified from a particular taxonomic group on the basis of morphology alone.
Buoyant density
Buoyant density estimations for human astrovirus in caesium chloride h a v e been made by several groups for virus preparations from diarrhoetic stools. The observed densities for the virus have bee n 1.39-1.40 g/ml (Konno ejt a l . , 1982) and 1.36-1.38 g/ml (Caul and Appleton, 1982). The differences in density may be related to the observation of multiple density peaks for hepatitis A virus (Lemon ej: a l . , 1985) and several other enteroviruses (Rowlands e ± a l. , 1975) within a single virus preparation and may be due to different capsid structures leading to variation in permeability to caesium chloride.
Similarly the variation in density estimations for astrovirus may a l s o be due to different methods used in preparing the virus from the faecal sample. It is possible that the virus may aggregate with faecal material and lipids so affecting its d e n s i t y in such gradients. This has been observed for hepatitis A virus grown in tissue culture (Heinricy £t al^., 1987).
Analysis of astrovirus grown in tissue culture will eliminate the possibility of contaminating faecal material and avoid harsh purification treatments which m a y affect the buoyant density, a n d provide a more reproducible value.
Five serotypes of human a s t r ovirus have been propagated in tissue culture and distinguished b y immunofluorescence (Kurtz and Lee, 1984) using antisera rais e d in rabbits (Lee and Kurtz, 1982). More recently a potential sixth serotype has been isolated and cultivated (T.W. Lee , personal communication). Type 1 has been shown to be the m o s t common strain (up to 77% of isolates) in the UK (Kurtz and Lee, 1984; Wilson and Cubitt, 1988). Immunological cross r e a ction studies have been carried out between the serotypes and th e i r antisera. Kurtz and Lee
(1984) showed little cross reaction using both
immunofluorescence tests and immunosorbent electron microscopy, except for a reduced one-way reaction between astrovirus serotype 3 and antiserum to type 1. These observations were supported by Herrmann e_t al. (1988) and Hudson e_t al_. (1989), using immunofluorescence and neutralisation assays respectively, showing the antisera to be predominantly type specific with only low titre o n e - w a y cross reactions occurring between some serotypes. Conversely, ELISA tests (Herrmann £t al., 1988) showed a high degree of cross reactivity between the serotypes as did a monoclonal a n t i b o d y to astrovirus type 2 antigen in both ELISA and immunofluorescence. These results have been taken to imply the exis t e n c e of a group antigen. It was suggested that degradation of the virions during adsorption in an ELISA may lead to the release of a group antigen and account for the d i f f erence in specificity seen between the two tests.
Serological cross reactions have been tested between astroviruses and other enteric viruses. Paired sera from patients infected with calicivirus, Norwalk vi r u s and Otofuke agent and antibodies to poliovirus types 1-3 and coxsackie B group showed no serological reaction with a s t r ovirus by immune electron microscopy (Konno e_t al_., 1982). An extensive ELISA test (Herrmann e_t a l . . 1988) using a group s p e cific monoclonal antibody to astrovirus and both cell c u l t ivated and stool derived enteric viruses (13 types) showed no relationship, indicating that astroviruses are serologically distinct from previously characterised viruses.
Recent studies on the Marin County agent, a virus associated with gastroenteritis (Oshiro e_t al,., 1981) and morphologically similar to astroviruses have suggested that it may be related to the human strain.
The Marin County agent has been adapted to tissue culture growth using trypsin-containing media (Herrmann ¿t a l .. 1987) and has been shown to react by immunofluorescence with the astrovirus specific monoclonal antibody deve l o p e d by Herrmann et a l . (1988). In addition, an adult volunteer infected with the Marin County agent developed an IgM and I g G response to astrovirus type 1, tests with the other sero t y p e s were not tried (Wilson and Cubitt, 1988). Immune e l e c t r o n microscopy and immunofluorescence studies (Herrmann e_t a l ., 1987) indicated a stronger serological relationship b e t w e e n the Marin County agent and astrovirus serotype 5, s u g g esting this agent should be classified as type 5 astrovirus.
Initial attempts to cultivate astrovirus in tissue c u l t u r e were unsuccessful. A limited infection was demonstrated (Lee and Kurtz, 1977) by detection of viral antigen in i n f e c t e d cells through immunofluorescence but without virus production. Lee and Kurtz (1981) incorporated trypsin into the cell m e d i u m on the basis that it has been shown to enhance the g r o w t h of reovirus in tissue culture (Spendlove e£ a l ., 1970). Astrovirus was serially passaged in primary human embryo k i d n e y (HEK) cells; rhesus monkey kidney (LLCMK2) cells and p r i m a r y baboon kidney (PBK) cells with trypsin (10 pg/ml) incorporated into the medium. Subsequent attempts to adapt astrovirus to a trypsin-free cell culture system were unsuccessful. Astroviruses were shown to infect cells in medium co n t a i n i n g a trypsin inhibitor, but no release of virus was detected w i t h o u t the presence of trypsin (Lee and Kurtz, 1981).
Recently Hudson e£ al.. (1989) have described the development of a plaque assay system for astrovirus sero t y p e s 1, 2 and 5, incorporating trypsin and diethylaminoethyl-dextran (DEAE-dextran) into the agar overlay as previously d e s c r i b e d for influenza virus (Appleyard and Maber, 1974) and rota v i r u s (Smith et a l . , 1979).
1.2d Protease enhancement of virus growth
Proteases have been shown to enhance the growth in tissue culture of reoviruses (Spendlove e£ al. , 1970), rotavirus (Almeida e_t _al., 1978) and influenza virus (Klenk e_t a l ., 1975). The mechanism of enhancement is not the same for all viruses. Possible mechanisms include: effects on the cell or virion surface, influencing virus attachment or penetration; digestion of viral inhibitors; dispersion of viral aggregates or an effect expressed during replication. The precise mechanisms are obscure for some viruses but from the data obtained for reo-, rota- and influenza viruses it appears that the increased growth is due to an effect on the particle rather than on the cell.
In the case of reovirus the removal of the capsid by enzyme action leads to the activation of a functional viral RNA polymerase (Spendlove et a l . , 1975).
The enhancement of influenza virus growth has been shown to be independent of the host cell type, but achieved through the cleavage of the haemagglutinin (HA) protein (Klenk e£ a l ., 1975; Lazarowitz et al., 1975). The yield from cells infected with virions with their HA already in the cleaved form, as a result of their propagation in chick embryo cells or in the presence of plasminogen, could not be improved by protease treatment. Lazarowitz and Choppin (1975) showed that cleavage of the HA into HAj and HA 2 subunits occurs at a trypsin specific site on the molecule suggesting a distinct reaction is required. The proteolytic cleavage of HA is not essential for
adsorption but is necessary for fusion of the viral envelope with the cellular membrane during penetration of the particle into the cell (Huang et al., 1980).
Almeida e£ al_. (1978) observed that adsorption of rotavirus in the presence of trypsin before maintenance in trypsin-free medium leads to only slight enhancement, suggesting that the enzyme is required during the whole course of replication. Time course experiments (Graham and Estes, 1980) have shown that the effect of the trypsin is greatest when added at the initiation of infection and absent when added 3-5 hours after inoculation. This implies that trypsin acts extracellularly on the virus, being required for longer than the 60 minute adsorption period but not for the entire replication cycle. Graham and Estes (1980) observed that the addition of trypsin 10 hours post-infection again resulted in increased titres as at this stage newly synthesised particles are being released from cells and the addition of trypsin allows further rounds of infection. Trypsin does not seem to affect the cellular attachment of rotavirus (Clarke e_t a l . , 1981) but seems to induce the ability to uncoat and release RNA in infected cells, similar to that seen for influenza virus.
It has been suggested that a specific structural polypeptide in simian rotavirus SA11, the VP3 protein, is cleaved to yield two subunits VP5 and VP8, when the virus is treated with trypsin (Espejo e t ¿1., 1981; Estes e£ a l ., 1981). This concept of cleavage of a large structural polypeptide could be extended as a general mechanism of enhancement for all
rotaviruses allowing more efficient penetration and replication (Espejo e_t a l . , 1981).
From these observations trypsin seems to mostly enhance virus growth by a cleavage event in the viral capsid enabling p e netration of the virus into the cell, more efficient unco a t i n g of the nucleic acid and multiple rounds of replication. Lee and Kurtz (1981) found that astrovirus grown in the presence of trypsin could infect cells but did not lead to the release of virus particles, suggesting trypsin m a y be invol v e d in events other than at the initial stages of infection. However, cleavage of a capsid protein may occur from the action of residual trypsin in the culture m e dium a l l owing the infection of fresh cells in trypsin-free m e d i u m but no multiple rounds of replication. The systems used for analysis of astrovirus, namely immunofluorescence and immune e l e ctron microscopy, may not be sensitive enough to allow d e t e c t i o n of low titres of virus. A similar mechanism may exist as that described for influenza and rotaviruses, whereby a specific polypeptide is cleaved to facilitate penetration, wit h o u t which the virus is still able to adsorb to the cells.