Divergent astrovirus associated with neurologic disease in cattle.

Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle.

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis E virus.

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T=3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~440Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. & Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA108, 12681-12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.

Association of the astrovirus structural protein VP90 with membranes plays a role in virus morphogenesis.

VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (mVP90), as well as in fractions enriched in cytosolic proteins (cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both mVP90 and cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with mVP90, but not with cVP90, suggesting that mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.

Development of antigen-capture enzyme-linked immunosorbent assay and RT-PCR for detection of turkey astroviruses.

Turkey astrovirus (TAstV) is an important agent of poult enteritis. The diagnosis of astroviruses has been dependent mainly on electron microscopy (EM) or immune EM (IEM). To develop other simple, rapid, and reliable diagnostic assays, two antigen-capture enzyme-linked immunosorbent assays (AC-ELISAs), polyclonal AC-ELISA and monoclonal AC-ELISA, were developed in this study. Monoplex and multiplex reverse transcription-polymerase chain reactions (RT-PCRs) were also developed using nondegenerate primer sets specific to the capsid region and degenerate primer pairs specific to the polymerase area of two TAstV. EM was included for comparison. Fecal or intestinal contents samples from naturally and experimentally infected poults with enteritis were examined using the developed assays. The polyclonal AC-ELISA had higher sensitivity and wider detection spectrum than the monoclonal AC-ELISA with group-specific monoclonal antibody (MAb), whereas the monoclonal AC-ELISA had very high specificity but lower sensitivity, which was estimated at 0.06 microg of viral proteins. Small round viruses (SRV) that could be astroviruses or other small viruses were detected in 34.4% of the samples examined by EM. The monoplex RT-PCR results amplified with primers SRV-1-3 and SRV-1-5 revealed that the positive rate of astroviruses was 45.3%, which was 10.9% higher than that of EM even if other SRVs were not excluded. Multiplex RT-PCR with SRV-1-3 and SRV-1-5 and AFCP-F1 and AFCP-R1 and the monoplex RT-PCR with degenerate primers verified that the positive rate of astroviruses was 59.4%, which was 25% higher than that of EM. Both RT-PCRs showed good specificity and wider detection spectrum compared with earlier published data.

Identification of gastroenteric viruses by electron microscopy using higher order spectral features.

BACKGROUND: Many paediatric illnesses are caused by viral agents, for example, acute gastroenteritis. Electron microscopy can provide images of viral particles and can be used to identify the agents. OBJECTIVES: The use of electron microscopy as a diagnostic tool is limited by the need for high level of expertise in interpreting these images and the time required. A semi-automated method is proposed in this paper. STUDY DESIGN: The method is based on bispectal features that capture contour and texture information while providing robustness to shift, rotation, changes in size and noise. The magnification or true size of the viral particles need not be known precisely, but if available can be used additionally for improved classification. Viral particles from one or more images are segmented and analyzed to verify whether they belong to a particular class (such as Adenovirus, Rotavirus, etc.) or not. Two experiments were conducted-depending on the populations from which virus particle images were collected for training and testing, respectively. In the first, disjoint subsets from a pooled population of virus particles obtained from several images were used. In the second, separate populations from separate images were used. The performance of the method on viruses of similar size was separately evaluated using Astrovirus, HAV and Poliovirus. A Gaussian Mixture Model was used for the probability density of the features. A threshold on the log-likelihood is varied to study false alarm and false rejection trade-off. Features from many particles and/or likelihoods from independent tests are averaged to yield better performance. RESULTS: An equal error rate (EER) of 2% is obtained for verification of Rotavirus (tested against three other viruses) when features from 15 viral particle images are averaged. It drops further to less than 0.2% when scores from two tests are averaged to make a decision. For verification of Astrovirus (tested against two others of the same size) the EER was less than 2% when 20 particles and two tests were used. CONCLUSION: Bispectral features and Gaussian mixture modelling of their probability density are shown to be effective in identifying viruses from electron microscope images. With the use of digital imaging in electron microscopes, this method can be fully automated.

The isolation and characterisation of astroviruses from chickens.

The isolation, cultivation and characterization of three chicken astroviruses (CAstV) isolates are described. They are antigenically related to each other but unrelated to avian nephritis virus (ANV) and duck hepatitis virus type 2 (DVH2) in neutralization, immunofluorescence and gel diffusion tests. CAstV, ANV and DVH2 all grew well in the LMH cell line, which was used for assay and serological tests. Serological surveys in 1982 and 2001 showed that antibody to CAstV virus was widespread in broiler and broiler breeder flocks and present in some turkey flocks. Infection of 1-day-old specific pathogen free chicks with one isolate in the laboratory resulted in mild diarrhoea and some distention of the small intestine. The virus could be isolated in high titres from all parts of the small intestine but rarely from other organs. Electron microscopic examination of purified particles of this agent revealed the presence of clusters of small round viruses with a diameter ranging from 25 to 30 nm. The amino acid sequence derived from the relatively conserved non-structural polyprotein region of this virus shows 62% identity with the corresponding region of turkey astrovirus 2, 58% identity with turkey astrovirus 1, 55% identity with avian nephritis virus and 33% identity with sheep astroviruses. Taken together, the results indicate that the agent is a new chicken astrovirus belonging to the family Astroviridae.

Vaccinia virus recombinant expressing an 87-kilodalton polyprotein that is sufficient to form astrovirus-like particles.

Human astrovirus is an important cause of acute gastroenteritis. We have generated, for the first time, a vaccinia virus recombinant expressing the astrovirus 87-kDa structural polyprotein. The results demonstrate that this expression results in the formation of virus-like particles in the absence of other astrovirus proteins and genomic RNA. The purified trypsin-activated virus-like particles strongly resemble the complete astrovirus particles.

Astrovirus epidemiologically linked to pre-weaning diarrhoea in mink.

Diarrhoea and excessive secretion from the cervical apocrine glands in young, suckling mink kits is a well-known, but poorly defined, syndrome often referred to as "sticky", "greasy", or "wet" kits. We have performed a case-control study, at farm level as well as at mink kit level, in Denmark and Sweden to investigate whether enteric virus infections may be a risk factor in the development of pre-weaning diarrhoea. Tissue samples from the enteric tract of 180 sacrificed mink kits were analysed histologically. Faecal contents were examined by electron microscopy (EM). Astrovirus was detected in abundance and found to be a significant risk factor both at farm level (OR=21.60, p<0.001) and at mink kit level (OR=7.95, p<0.001). Other factors, i.e. low body weight, coccoid bacteria adherent to the enteric villi, and presence of calicivirus were also shown to increase the risk of pre-weaning diarrhoea, although with less impact than astrovirus.

Molecular biology of astroviruses: selected highlights.

Human astrovirus, the prototype of the Astroviridae family, is a non-enveloped positive-strand RNA virus with distinctive morphology. Initially named for a characteristic 5-6 point star evident on the surface of faecally shed viral particles by direct electron microscopy, a recent study using cryoelectron microscopy and image reconstruction indicates that viral particles consist of a smoothly rippled, solid capsid decorated with short spikes. Mechanisms underlying the assembly of these viral particles have not been fully elucidated. However, studies of two full-length cDNA clones of human astrovirus serotype 1 suggest that capsid residue Thr227 plays a critical role in the assembly of infectious viral progeny. The development of a full-length clone (pAVIC) from which infectious RNA can be transcribed has also facilitated studies of the viral 3C-like serine protease, encoded in ORF1a. These studies demonstrate that the full-length ORF1a product (101 kDa) is processed in vitro to an N-terminal 64 kDa fragment and a C-terminal 38 kDa fragment. Mutation of the predicted catalytic triad inhibits proteolysis. In other studies based on modifications of pAVIC, preliminary evidence supports the feasibility of developing a reporter cell line to facilitate astrovirus detection.

Characterisation of a South African human astrovirus as type 8 by antigenic and genetic analyses.

Human astroviruses (HAstV) can, on the basis of immunoassays using type-specific rabbit antisera, be classified into eight serotypes that correlate with genotypes. Very few isolates of HAstV type 8 have been described and there is a paucity of data available with regard to the antigenic and genetic relationships between HAstV type 8 (HAstV-8) and HAstV types 1 (HAstV-1) to 7 (HAstV-7). A wild-type HAstV from a South African paediatric patient with diarrhoea was analysed antigenically, by immune electron microscopy and enzyme immunoassay, and genetically in selected regions of the ORF1a, ORF1b and ORF2 and characterised as a HAstV-8. This HAstV-8 strain exhibited greatest homology with HAstV-4 in the 5' end of the capsid gene and ORF1a and 1b, and greatest homology with HAstV-5 in the 3' end of the capsid region. This study confirms, by both antigenic and genetic analyses, that HAstV-8 represents a distinct antigenic and genotype and is the first report of a HAstV-8 from a hospitalised paediatric patient with diarrhoea in southern Africa.

Proteolytic processing of the astrovirus capsid.

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35S]Smethionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10(5)-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.

Application of electronmicroscopy, enzyme immunoassay, and RT-PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit.

During 1997, an extensive outbreak of astrovirus occurred in a unit where paediatric patients were being treated for leukaemias and inherited immune deficiency disorders. Prolonged shedding of virus for many months following infection was demonstrated in three patients who had undergone bone marrow transplantation. Comparison of reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA), and electronmicroscopy (EM) to monitor the outbreak showed that many subclinical infections, mainly in children aged > 3 years could only be detected by RT-PCR. Use of RT-PCR revealed that several patients were infected earlier and shed virus for longer than by using EM or EIA. The virus responsible for the outbreak was identified as HAstV-1 and was shown to have a sequence that differed from a strain obtained in 1988.

Historical background and classification of caliciviruses and astroviruses.

Infections caused by caliciviruses, i.e., vesicular exanthema virus of swine were recognised as a major cause of economic loss in the 1930s. However, it was not until the application of electronmicroscopy in the 1970s that caliciviruses and astroviruses were recognised and proven to be a cause of diarrhoea and vomiting. The following review briefly describes the steps which have led to the development of diagnostic tests and enabled the characterization of several members of the Caliciviridae and Astroviridae. In the past five years this has culminated in the sequencing of their genomes and the expression of viral proteins. This in turn has led to the development of improved diagnostic tests e.g., RT-PCR and enzyme immunoassays, and may pave the way towards producing effective vaccines in the future.

The changing epidemiology of astrovirus-associated gastroenteritis: a review.

Our understanding of the epidemiology of astrovirus-associated gastroenteritis has changed markedly with each improvement in detection method. In early surveys based on electronmicroscopy (EM), astroviruses appeared to be a rare cause of gastroenteritis, being found in fewer than 1% of children with diarrhea, usually in small outbreaks of disease and primarily during the winter season. The development and use of monoclonal antibodies and enzyme immunoassays (EIA) to detect astroviruses led to reports of a higher prevalence (2.5%-9%) of astrovirus infection among patients hospitalized with diarrhea. Astroviruses appeared second only to rotaviruses as a cause of hospitalization for childhood viral gastroenteritis. Studies based on EIA detection of astroviruses indicate that astroviruses are common causes of diarrhea in children worldwide, and that most children are infected during their first two years of life. The elderly and the immunocompromised represent high-risk groups as well. The observations that newborns monitored prospectively rarely have repeat disease and that the rate of detection decreases with increasing age suggest that immunity to astroviruses, as immunity to rotaviruses, may develop early in life. The cloning and sequencing of astroviruses have led to more sensitive assays to detect the viruses by reverse transcription, polymerase chain reaction (RT-PCR). Application of RT-PCR for detection of astroviruses in children in day-care centers showed a marked increase in the detected prevalence of astrovirus-associated diarrhea, the rate of asymptomatic infection, and the duration of shedding of virus among those infected, when compared with studies that used other methods. As with rotaviruses, neither the mode of transmission nor the reservoir of astrovirus infection has been identified. Both immune and molecular-based assays to detect astrovirus serotypes indicate that serotype 1 is most common worldwide, although the predominant serotypes may vary by region and time. In the absence of obvious strategies to prevent astrovirus-associated diarrhea, vaccines might be considered if further studies establish that the disease burden would render such a vaccine cost-effective.

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Astrovirus infection in hatchling turkeys: histologic, morphometric, and ultrastructural findings.

In three separate experiments, 2- or 5-day-old commercial turkey poults were inoculated orally with astrovirus and examined for clinical signs and for gross and microscopic lesions over a period of 14 days. By day 2 postinoculation (PI), inoculated poults had developed diarrhea, generalized loss of intestinal tone, and dilated ceca that contained light-yellow fluid feces and gas; these changes persisted through day 10 PI. Mild crypt hyperplasia was the only change discernible by light microscopy, and it was first noted in the proximal jejunum on day 1 PI, in the distal jejunum and ileum on day 3 PI, and in the duodenum on day 5 PI. A significant (P < 0.05) increase in crypt depth and area was documented by image analysis on day 3 PI. Ultrastructural evaluation revealed intracytoplasmic aggregates of astrovirus in enterocytes on the sides and base of villi in the ileum and distal jejunum on day 3 PI. Based on the findings, it was concluded that astrovirus caused lesions and replicated in both upper and lower segments of the small intestine in turkey poults.

Serotyping of human astrovirus strains by immunogold staining electron microscopy.

Human astrovirus strains were propagated in CaCo-2 cell cultures, and virus multiplication was demonstrated by immunosorbent electron microscopy (ISEM). Serotyping of the virus strains was carried out in cell culture fluids or directly in faecal extracts by an indirect immunogold staining (IGS) electron microscopy technique, using specific rabbit antisera against astrovirus types 1-6 as primary antibodies and goat anti-rabbit IgG gold conjugate as secondary antibody. Thirty-seven astrovirus strains were examined, of which 26 grew in the cell cultures in several passages. IGS of the cell-derived viruses showed that 16, 3, 3, and 4 of the strains were types 1, 2, 3, and 4 respectively. Types 5 and 6 were not demonstrated. Eleven strains did not grow in cell cultures, and attempts to serotype these strains by IGS directly in the faecal extracts were unsuccessful, except for one strain which was found to be type 1. The results indicate that IGS may be a specific and suitable method for serotyping astroviruses grown in cell cultures.