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.

Immunity to calicivirus infection.

The evolution of our understanding of immunity to calicivirus infection, using Norwalk virus as the prototype, is discussed in three stages: (1) "ancient times (1972-1978), when human volunteer studies prevailed, (2) the "middle ages (1978-1990), which were characterized by the development and implementation of solid-phase immunoassays based on native viral antigens, and (3) "modern times (1990 to present), which began with the cloning of the genome of the noncultivatable 8FIIa strain of Norwalk virus and resulted in a readily available source of recombinant virus-like particles that have revolutionized the study of caliciviruses. Throughout these stages, it has been shown repeatedly that short-term immunity develops to homologous virus. However, the search for determinants of long-term immunity continues. These studies will likely be facilitated by the newest reagents-the noninfectious recombinant virus-like particles-used in the setting of human volunteer studies and large epidemiologic studies.

Non-invasive transgenic mouse genotyping using stool analysis.

Commonly applied genotyping of transgenic mice involves using tail or ear biopsies which may cause discomfort to the animal. We tested the possibility of polymerase chain reaction (PCR)-based mouse genotyping using stool specimens from three transgenic mouse lines that overexpress 10-18 transgene copies of human keratin polypeptide 18, as compared to genotyping using tail biopsies. Stool specimens were obtained with ease and provided easy detection of the human transgene product. The method was also able to detect endogenous mouse actin and keratin genes which presumably are present at two copies each. Nested PCR was not necessary for genotyping using stool-derived genomic material but did increase the relative magnitude of the signal obtained. The non-invasive genotyping method described herein offers a reproducible, sensitive and effective modality that could replace invasive tissue sampling procedures currently used to test thousands of genetically altered mice.

Comparison of the rotavirus gene 6 from different species by sequence analysis and localization of subgroup-specific epitopes using site-directed mutagenesis.

The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.

Host and viral factors affecting the decreased immunogenicity of Sabin type 3 vaccine after administration of trivalent oral polio vaccine to rural Mayan children.

Factors affecting immunogenicity of the first 2 doses of oral poliovirus vaccine (OPV) among unimmunized Mayan infants were prospectively evaluated. The relative impact of multiple variables, including mass or routine vaccination, concurrent enteric bacterial (salmonella, shigella, and campylobacter) and viral (adenovirus 40/41, astrovirus, nonpolio enteroviruses, and rotavirus) infections, interference among Sabin vaccine viruses, and preexisting poliovirus antibodies were studied. Sera were available from 181 infants after 2 OPV doses. Seroresponses were 86% to Sabin type 1, 97% to Sabin type 2, and 61% to Sabin type 3 vaccines. Mass versus routine vaccination and preexisting poliovirus antibodies did not affect immunogenicity. By multiple logistic regression analysis, fecal shedding of homologous Sabin strains was associated with increased seroresponses to all Sabin types, especially to Sabin type 3. Decreased OPV immunogenicity was primarily attributable to interference of Sabin type 3 by Sabin type 2. OPV formulations with higher doses of Sabin type 3 could improve immunogenicity among infants in developing countries.

Construction of a genome-length cDNA clone for human astrovirus serotype 1 and synthesis of infectious RNA transcripts.

We have constructed a genome-length cDNA clone for human astrovirus serotype 1. When a human colon cancer-derived cell line, CaCo-2, is transfected with RNA transcribed in vitro from this cDNA clone, infectious virus is produced at titers close to those observed after infection with intact astrovirus. A rodent cell line, BHK, which is largely refractory to astrovirus infection, was found to support efficient growth of the virus if transfected with viral RNA. The high transfection efficiency seen in the BHK cells allows studies of the viral replication in the transfected cells and thus should prove useful for the characterization of noninfectious astroviral mutants.

Studies of the astrovirus signal that induces (-1) ribosomal frameshifting.

Human astroviruses use a (-1) ribosomal frameshift mechanism to regulate expression of the viral RNA-dependent RNA polymerase gene. To evaluate the efficiency of the astrovirus frameshift signal in cell culture, different regions of the frameshift signal were cloned into the rhesus rotavirus VP4 gene and expressed in an infection-transfection transient expression cell-culture system. The various astrovirus-VP4 constructs were transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7-3). All constructs contain a T7 promoter, a picornavirus internal ribosome entry site, and a T7 terminator. Frameshifted and non-frameshifted proteins were distinguished by immunoprecipitation with monoclonal antibodies specific for either the VP4 amino- or carboxy-terminus. Frameshifting efficiency was calculated as the ratio of radioactive counts in the frameshifted protein to the total counts in both the frameshifted and nonframeshifted proteins as determined by Phosphorimager analysis. We found the efficiency of astrovirus frameshifting in intact cells to be 25-28%, significantly greater than the 5-7% efficiency reported previously in a cell-free uncoupled translation system. Since the transfected plasmid is expressed in the cytoplasm in the infection-transfection system, the frameshifting efficiency determined by this assay may be a more accurate reflection of the level of frameshifting for this RNA virus in which transcription and translation are likely coupled in the cytoplasm of infected cells. This hypothesis is supported by the observation that the level of astrovirus frameshifting is increased three-fold when evaluated in a cell-free coupled transcription-translation system. These studies also confirm in intact cells what was previously determined in cell-free studies: the shifty heptamer is an absolute requirement for astrovirus ribosomal frameshifting, but deletion of sequences downstream of the stem-loop that are potentially involved in pseudoknot formation does not affect the efficiency of frameshifting.

Astrovirus ribosomal frameshifting in an infection-transfection transient expression system.

Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection-transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal-specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell-free translation system. Coupling of transcription and translation in a cell-free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting.

Monoclonal antibodies for detection of Norwalk virus antigen in stools.

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.

An astrovirus frameshift signal induces ribosomal frameshifting in vitro.

Expression of the astrovirus RNA-dependent RNA polymerase has been hypothesized to be regulated by (-1) ribosomal frameshifting. Sequence analysis of the 70 nucleotide region between open reading frames 1a and 1b indicates the presence of a shifty heptamer consensus sequence and downstream sequences that may be needed for ribosomal frameshifting. We constructed four astrovirus cassettes that spanned this region and inserted each into the rhesus rotavirus VP4 gene. The constructs were expressed in an in vitro system, and products were immunoprecipitated by rotavirus amino and carboxy terminal-specific monoclonal antibodies. Ribosomal frameshifting, at an efficiency of 6-7%, was demonstrated in all constructs containing the shifty heptamer and stem-loop. Deletion of the downstream sequence potentially involved in pseudoknot formation did not affect frameshifting efficiency. However, deletion of the shifty heptamer resulted in no detectable frameshift activity.

Etiology and outcome of diarrhea after marrow transplantation: a prospective study.

BACKGROUND/AIMS: Acute diarrhea after marrow transplant is usually ascribed to acute graft-vs.-host disease (GVHD) or infection, with a reported 40%-50% incidence of infection. The aim of this study was to determine the incidence of acute diarrhea after transplantation, its causes, and its outcome. METHODS: Two hundred ninety-six patients were followed up; patients with diarrhea were studied using standard evaluation of stool plus immunoelectron microscopy; assays for astrovirus, picobirnavirus, and Norwalk virus; and gene-probe methods for toxin-producing Escherichia coli. In 38 patients with diarrhea, intestinal biopsy specimens and duodenal fluid were also analyzed. RESULTS: One hundred fifty acute diarrheal episodes developed in 126 patients (an incidence of 43%). Intestinal infection was found in 20 of 150 episodes: viruses (astrovirus, adenovirus, cytomegalovirus, and rotavirus) in 12 patients, nosocomially acquired bacteria (Clostridium difficile and Aeromonas) in 7 patients, and mixed infection in 1 patient. Acute GVHD was responsible for 72 of 150 episodes (48%). Clinical signs and symptoms of infection and GVHD were similar. In 58 of 150 episodes (39%), no clear etiology could be found for self-limited diarrhea. CONCLUSIONS: Intestinal infection accounted for 13% and acute GVHD for 48% of diarrheal episodes. The most common infecting organisms were astrovirus, C. difficile, and adenovirus. Most cases of diarrhea after marrow transplant are not caused by infection.

Rapid and sensitive method for detection of hepatitis C virus RNA by using silica particles.

We describe a rapid, sensitive, and economic method for detection of hepatitis C virus (HCV) RNA. This method uses silica particles for purification of nucleic acid and then a modified reverse transcription-PCR that minimizes the risk of contamination and reduces the amount of reagents used. We found purification by silica particles to be at least as sensitive and in certain circumstances more sensitive than that by traditional phenol-chloroform extraction. This improved sensitivity may be due to more efficient recovery of HCV RNA by silica particles. HCV RNA appears to bind to silica particles in a saturable fashion, and the addition of extraneous nucleic acids (salmon sperm DNA or tRNA) decreases the binding in a dose-related fashion. The reverse transcription-PCR is performed by using a modified single tube method which further simplifies and reduces the cost of this assay. Finally, this method may be applied to clinical specimens such as liver tissue.

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

Cloning and characterization of human astrovirus immunoreactive epitopes.

We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.

Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes.

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.

Isolation and characterization of a novel reassortant between avian Ty-1 and simian RRV rotaviruses.

A reassortant, TyRh, was isolated after coinfection of MA104 cells with avian Ty-1 and simian RRV rotaviruses. Hybridization and serological studies showed that the reassortant's 4th gene, which encodes Vp4, was derived from the simian RRV rotavirus parent, whereas the remaining 10 genes were derived from the avian Ty-1 rotavirus parent.

Astroviruses and caliciviruses: emerging enteric pathogens.

Acute, infectious gastroenteritis is an extremely common disease that contributes significantly to morbidity and mortality worldwide. In the United States, it is the second most frequent illness encountered in families. While this illness generally runs a self-limited course, it may be temporarily incapacitating and impact substantially on numbers of days lost from work or school. At present, 30-40% of infectious gastroenteritis cases in the United States are attributable to viral agents, while 20-30% are due to bacteria and parasites. These estimates are almost certainly low, since the cause of gastroenteritis is not discernible in approximately 40% of the cases, and gastroenteritis may be caused by viruses or other pathogens that cannot be identified at this time. Rotavirus and enteric adenovirus are two of the most prevalent and well-studied of the viral agents and have been reviewed extensively elsewhere. This review focuses on two broad groups of small round structured viruses (SRSV), astroviruses and caliciviruses (classic, Norwalk, and Norwalk-like). Although recognized in association with acute, nonbacterial gastroenteritis since the early 1970s, the study of these viruses has been hampered by the relatively low levels of viral shedding in feces, difficulty in propagating the virus in cell or organ culture, and the lack of widely available, well-standardized reagents for their detection. In spite of these obstacles, much has been learned about these viruses using standard virologic (electron microscopy, biophysical characterization, immunoassays) and epidemiologic methods. More recently, substantial progress has been made in studying astroviruses and caliciviruses at the molecular level. Molecular techniques are now being used as diagnostic aids to characterize the epidemiology of these agents in greater detail.

The isolation and characterization of a Norwalk virus-specific cDNA.

Norwalk virus, an important cause of epidemic, acute, nonbacterial gastroenteritis in adults and children, has eluded adaptation to tissue culture, the development of an animal model, and molecular cloning. In this study, a portion of the Norwalk viral genome encoding an immunoreactive region was cloned from very small quantities of infected stool using sequence-independent single primer amplification. Six overlapping complementary DNA (cDNA) clones were isolated by immunologic screening. The expressed recombinant protein from a representative clone reacted with six of seven high titer. Norwalk-specific, postinfection sera but not with corresponding preinfection sera. Nucleic acid sequence for all clones defined a single open reading frame contiguous with the lambda gt11-expressed beta-galactosidase protein. Only oligonucleotide probes specific for the positive strand (defined by the open reading frame) hybridized to an RNaseA-sensitive, DNaseI-resistant nucleic acid sequence extracted from Norwalk-infected stool. Furthermore, RNA extracted from serial postinfection, but not preinfection, stools from three of five volunteers hybridized to a Norwalk virus cDNA probe. Clone-specific oligonucleotide probes hybridized with cesium chloride gradient fractions containing purified Norwalk virion. In conclusion, an antigenic, protein-coding region of the Norwalk virus genome has been identified. This epitope has potential utility in future sero- and molecular epidemiologic studies of Norwalk viral gastroenteritis.