Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.

Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.

Occurrence of viral gastroenteritis in children below 5 years: A hospital-based study from Assam, India.

Viral gastroenteritis is an important cause of mortality and morbidity in children under 5 years of age. Many a time, these cases go unnoticed causing immense scarcity of data on viral diarrhoea. The study aimed to determine the occurrence of viral gastroenteritis among children below 5 years and the aetiological viral agents. Stool samples were collected from patients suffering from acute gastroenteritis. Real-time polymerase chain reaction was done for detection of rotavirus, adenovirus, norovirus, astrovirus and sapovirus. Viruses were detected in 55% of children. Adenovirus was found to be the most common virus (33.7%), followed by rotavirus infection (28.7%).

Construction of a reverse genetic system for porcine astrovirus.

In order to construct a full-length infectious cDNA clone of porcine astrovirus, three fragments covering the complete genome of PAstV1-GX1 strain were amplified by RT-PCR. All three PCR-amplified fragments were cloned into T-Vector pMD19 (Simple), and subsequently assembled into a full-length cDNA clone by subcloning. A silent nucleotide change creating a PstI site was engineered into the full-length cDNA clone to distinguish the rescued virus from the parental virus. Upon transfection of BHK-21 cells with the in vitro transcripts of both the original and constructed cDNAs, typical cytopathic effects were observed on PK-15 cells after serial passaging of the cell supernatant. The construction and recovery of the infectious cDNA clone of porcine astrovirus will provide a valuable experimental system to study the genome function and pathogenesis of astroviruses.

Novel and classical human astroviruses in stool and cerebrospinal fluid: comprehensive screening in a tertiary care hospital, Switzerland.

Classical human astroviruses (HAstV) are the third most common cause of non-bacterial acute gastroenteritis. Due to the lack of routine molecular assays, novel HAstV are underdiagnosed and the magnitude of their contribution to clinical disease remains unknown. To better understand their prevalence and the susceptible patient profile, we conducted a comprehensive screening of novel and classical HAstV in stool and cerebrospinal fluid (CSF) samples collected for clinical care in a tertiary care hospital using a specially designed rRT-PCR panel for the detection of novel (MLB1-3 and VA1-4) and classical HAstV. Of the 654 stool samples, 20 were positive for HAstV, and the novel (n=10; 3 MLB1, 4 MLB2; 3 VA2) and classical (n=10) serotypes were equally prevalent. None of the 105 CSF samples were positive. Investigating the patient profile, we found a higher prevalence (P=0.0002) of both novel and classical HAstV in pediatric stool samples (3.4% and 3%, respectively) compared with adult stool samples (0.5% and 0.7%, respectively). Furthermore, all novel and classical HAstV-positive pediatric subjects were ≤four years old, demonstrating similar susceptible populations. Forty-five percent of positive patients were immunocompromised (novel: 40%, classical: 50%). A comparison of novel and classical HAstV-positive cases showed a lower viral load for novel HAstV (P=0.0007) with significantly more upper respiratory symptoms (70% of subjects; P=0.02); this observation may suggest a unique pathogenic pathway. This study confirms the clinical and epidemiological relevance of novel HAstV and identifies a target population in which routine screening may yield clinically valuable information.

Case-Control Assessment of the Roles of Noroviruses, Human Bocaviruses 2, 3, and 4, and Novel Polyomaviruses and Astroviruses in Acute Childhood Diarrhea.

BACKGROUND: The etiology of acute childhood diarrhea often eludes identification. We used a case-control study-stool archive to determine if nucleic acid tests for established and newly identified viruses diminish our previously published 32% rate of microbiologically unexplained episodes. METHODS: Using polymerase chain reaction, we sought to detect noroviruses GI and GII, classic and novel astroviruses, and human bocaviruses (HBoVs) 2, 3, and 4 among 178 case and 178 matched control stool samples and St. Louis and Malawi polyomaviruses among a subset of 98 case and control stool samples. We calculated adjusted odds ratios and 95% confidence intervals using conditional logistic regression. RESULTS: Noroviruses were more common in cases (GI, 2.2%; GII, 16.9%) than in controls (GI, 0%; GII, 4.5%) (adjusted odds ratio, 5.2 [95% confidence interval, 2.5-11.3]). Astroviruses and HBoVs 2, 3, and 4 were overrepresented among the cases, although this difference was not statistically significant. Malawi polyomavirus was not associated with case status, and St. Louis polyomavirus was identified in only 1 subject (a control). When identified in cases, HBoVs 2, 3, and 4 were frequently (77%) found in conjunction with a bona fide diarrheagenic pathogen. Thirty-five (20%) case and 3 (2%) control stool samples contained more than 1 organism of interest. Overall, a bona fide or plausible pathogen was identified in 79% of the case stool samples. Preceding antibiotic use was more common among cases (adjusted odds ratio, 4.5 [95% confidence interval, 2.3-8.5]). CONCLUSION: Noroviruses were found to cause one-third of the diarrhea cases that previously had no identified etiology. Future work should attempt to ascertain etiologic agents in the approximately one-fifth of cases without a plausible microbial cause, understand the significance of multiple agents in stools, and guide interpretation of nonculture diagnostics.

Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients.

BACKGROUND: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. METHODS: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. RESULTS: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. CONCLUSIONS: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Estudo retrospectivo dos astrovirus humanos associados a casos de gastrenterite aguda em crianças residentes em tres regioes do Brasil: nordeste, sudeste e sul no período de 1994 a 2011 / Retrospective study of human astrovirus associated with cases of acute gastroenteritis in children living in three regions of Brazil: northeast, southeast and south in the period 1994-2011

Os astrovírus humanos (HAstVs) pertencem a família Astroviridae e são associados agastrenterite aguda (GA) em crianças menores de cinco anos, tanto nos paísesdesenvolvidos como naqueles em desenvolvimento, o que os tornam de interesse nocampo da Saúde Pública. A família Astroviridae é dividida em dois gêneros:Avastrovirus e Mamastrovirus. No gênero Mamastrovirus, encontram-se os astrovirusassociados à infecção em mamíferos, tanto humanos como animais. Até 2008, osastrovirus associados a doenças em humanos eram restritos a oito genótipos,conhecidos como HAstV 1-8. A partir de então novos HAstVs foram sendo descritos,associados a doenças em humanos, como os HAstVs MLB1-3 e os HAstVs VA1-4.Opresente estudo consiste no estudo epidemiológicos retrospectivos (1994 a 2011) paradetecção e caracterização molecular de HAstV em amostras de fezes provenientes decrianças com menos de cinco anos de idade com GA, em diferentes regiões do Brasil:Nordeste, Sudeste e Sul. Incluem-se neste trabalho três estudos: 1) Estudo dosHAstV em casos esporádicos de GA ocorridos em crianças menores de cinco anos deidade, em três regiões brasileiras (Nordeste, Sudeste e Sul), durante o período de2005 a 2011, incluindo a pesquisa dos novos HAstV; 2) Estudo dos HAstV em criançascom GA, atendidas na creche Bertha Lutz, FIOCRUZ-RJ, durante o período de janeirode 1994 a dezembro de 2008; 3)...

Human astrovirus (HAstVs), belong to Astroviridae family, and are associatedwith acute gastroenteritis (GA) in children under five years-old, both indeveloped and in developing countries, which makes them of interest in thePublic Health field. The Astroviridae family is divided into two genera:Avastrovirus and Mamastrovirus. Mamastrovirus are the astrovirusesassociated to infection in mammals, both humans and animals. By 2008, theastrovirus associated with human disease were restricted to eight genotypes,known as HAstV 1-8. Since then, new HAstVs have been described, associatedwith human disease, such as HAstVs MLB1-3 and HAstVs VA1-4. The presentstudy is the retrospective epidemiological study (1994 to 2011) for the detectionand molecular characterization of HAstV in stool samples from children underfive years old presenting GA, in different regions of Brazil: Northeast, Southeastand South. Three studies are presented: 1) Study of HAstV in sporadic cases ofGA occurred in children under five years old in three Brazilian regions(Northeast, Southeast and South) from 2005 to 2011, including the descriptionof a new HAstV; 2) Study of HAstV in children with GA, attending the day careBertha Lutz, FIOCRUZ-RJ from January 1994 to December 2008 and 3) Studyof HAstV in children under two years old presenting GA and hospitalized inNiteroi, Rio de Janeiro from April to September 2003. The detection of HAstVwas performed using different protocols for detection and molecularcharacterization such as: Reverse–transcriptase polymerase chain reaction,(RT- PCR), Single step RT -PCR (OneStep RT-PCR) and RealTime RT- PCR.The HAstV detected were characterized by partial sequencing of ORF2 regionof the viral genome. The study 1 demonstrated the HAstV detection frequencyin 7.1 % of samples, and described the first ASTV MLB1 in Brazil. Themolecular characterization identified the circulation genotypes HAstV -1, 2, 3, 4,5, 6 and 8...

Increase in the detection rate of viral and parasitic enteric pathogens among Egyptian children with acute diarrhea.

INTRODUCTION: Acute diarrhea continues to be a major cause of morbidity and mortality in children from developing countries. Determination of the frequency of diarrhea in an area, along with the proportion of disease caused by specific enteric agents of different origins, is considered the first step in controlling diarrheal diseases. METHODOLOGY: From 2005 to 2007, a hospital-based surveillance was conducted in two locations in Egypt to determine the causes of acute diarrhea in children younger than 5-years seeking treatment. Five additional enteric viral and parasitic pathogens were tested using commercially-available enzyme immunoassays (EIA) to re-evaluate the prevalence of diarrheal pathogens in undiagnosed cases. RESULTS: Adenovirus, astrovirus, norovirus and G. lamblia were detected as the sole pathogen in 2% (n=34), 3% (n=56), 9% (n=191) and 7% (n=146) of the cases, respectively. E. histolytica was never detected as the sole pathogen. The percentage of diarrheal cases with a known cause increased significantly, from 48% (n=1,006) to 74% (n=1,568) (P < 0.0001). CONCLUSION: In our study, the incorporation of immunoassays yielded useful data in identifying pathogens in previously pathogen-negative diarrhea cases.

Structural and biochemical analysis of human pathogenic astrovirus serine protease at 2.0 A resolution.

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.

C-terminal nsP1a protein of human astrovirus colocalizes with the endoplasmic reticulum and viral RNA.

Computational and biological approaches were undertaken to characterize the role of the human astrovirus nonstructural protein nsP1a/4, located at the C-terminal fragment of nsP1a. Computer analysis reveals sequence similarities to other nonstructural viral proteins involved in RNA replication and/or transcription and allows the identification of a glutamine- and proline-rich region, the prediction of many phosphorylation and O-glycosylation sites, and the occurrence of a KKXX-like endoplasmic reticulum retention signal. Immunoprecipitation analysis with an antibody against a synthetic peptide of the nsP1a/4 sequence detected polyprotein precursors of 160, 75, and 38 to 40 kDa as well as five smaller proteins in the range of 21 to 27 kDa. Immunofluorescence labeling showed that the nsP1a/4 protein is accumulated at the perinuclear region, in association with the endoplasmic reticulum and the viral RNA. These results suggest the involvement of nsP1a/4 protein in the RNA replication process in endoplasmic reticulum-derived intracellular membranes.

Caspases mediate processing of the capsid precursor and cell release of human astroviruses.

In this work we have shown that astrovirus infection induces apoptosis of Caco-2 cells, since fragmentation of cellular DNA, cleavage of cellular proteins which are substrate of activated caspases, and a change in the mitochondrial transmembrane potential occur upon virus infection. The human astrovirus Yuc8 polyprotein capsid precursor VP90 is initially processed to yield VP70, and we have shown that this processing is trypsin independent and occurs intracellularly through four cleavages at its carboxy-terminal region. We further showed that VP90-VP70 processing is mediated by caspases, since it was blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk), and it was promoted by the apoptosis inducer TNF-related apoptosis-inducing ligand (TRAIL). Although the cell-associated virus produced in the presence of these compounds was not affected, the release of infectious virus to the cell supernatant was drastically reduced in the presence of z-VAD-fmk and increased by TRAIL, indicating that VP90-VP70 cleavage is important for the virus particles to be released from the cell. This is the first report that describes the induction and utilization of caspase activity by a virus to promote processing of the capsid precursor and dissemination of the viral particles.

Astrovirus-induced synthesis of nitric oxide contributes to virus control during infection.

Astrovirus is one of the major causes of infant and childhood diarrhea worldwide. Our understanding of astrovirus pathogenesis trails behind our knowledge of its molecular and epidemiologic properties. Using a recently developed small-animal model, we investigated the mechanisms by which astrovirus induces diarrhea and the role of both the adaptive and innate immune responses to turkey astrovirus type-2 (TAstV-2) infection. Astrovirus-infected animals were analyzed for changes in total lymphocyte populations, alterations in CD4(+)/CD8(+) ratios, production of virus-specific antibodies (Abs), and macrophage activation. There were no changes in the numbers of circulating or splenic lymphocytes or in CD4(+)/CD8(+) ratios compared to controls. Additionally, there was only a modest production of virus-specific Abs. However, adherent spleen cells from infected animals produced more nitric oxide (NO) in response to ex vivo stimulation with lipopolysaccharide. In vitro analysis demonstrated that TAstV-2 induced macrophage production of inducible nitric oxide synthase. Studies using NO donors and inhibitors in vivo demonstrated, for the first time, that NO inhibited astrovirus replication. These studies suggest that NO is important in limiting astrovirus replication and are the first, to our knowledge, to describe the potential role of innate immunity in astrovirus infection.

Viruses causing gastroenteritis.

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viruses are recognized as important causes of this disease, particularly in children. Since the Norwalk virus was identified as a cause of gastroenteritis, the number of viral agents associated with diarrheal disease in humans has steadily increased. Rotavirus is the most common cause of severe diarrhea in children under 5 years of age. Astrovirus, calicivirus and enteric adenovirus are also important etiologic agents of acute gastroenteritis. Other viruses, such as toroviruses, coronaviruses, picobirnaviruses and pestiviruses, are increasingly being identified as causative agents of diarrhea. In recent years, the availability of diagnostic tests, mainly immunoassays or molecular biology techniques, has increased our understanding of this group of viruses. The future development of a safe and highly effective vaccine against rotavirus could prevent, at least, cases of severe diarrhea and reduce mortality from this disease.

Proteolytic processing of a serotype 8 human astrovirus ORF2 polyprotein.

Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.

Induction of functional defects in macrophages by a poult enteritis and mortality syndrome-associated turkey astrovirus.

The interaction of a poult enteritis and mortality syndrome (PEMS)-turkey astrovirus-Ohio State University (TAst-OSU) with the mononuclear phagocytic system cells, namely macrophages, was examined after in vitro and in vivo exposure. In vitro exposures were performed by incubating adherent turkey macrophages with various volumes of 10(6) 50% embryo infective dose (EID50)/ml TAst-OSU stock, whereas for in vivo challenge, poults were given a 200 microl inoculum of 10(6) EID50/ml TAst-OSU stock at 7 days of age. Results show that TAst-OSU in vitro exposure reduced macrophage viability relative to controls (P < 0.05) and decreased phagocytosis (P < 0.05) and intracytoplasmic killing of Escherichia coli (P < 0.05) after a 42-48-hr exposure. Poults challenged with TAst-OSU in vivo recruited almost 50% fewer Sephadex-elicited inflammatory cells in the abdominal cavity (P < 0.05) as compared with the sham controls. Similar to in vitro exposure, macrophages isolated from in vivo TAst-OSU-challenged poults exhibited reduced percentage of phagocytic macrophages (P < 0.05) as well as fewer intracytoplasmic E. coli per phagocytic macrophage (P < 0.05). TAst-OSU-challenged poults had a greater number of viable E. coli in their spleens (P < 0.05) after an intravenous E. coli challenge as compared with the non-TAst-OSU-challenged control poults. Macrophage-mediated cytokines and metabolites were also examined during this study. Both in vitro and in vivo TAst-OSU challenge resulted in reduced interleukin (IL)-1 and IL-6 activity. On the contrary, nitrite levels in macrophage culture supernatant fraction of TAst-OSU-challenged macrophages were significantly higher (P < or = 0.05). The findings of these studies indicated that TAst-OSU challenge induced defects in macrophage effector functions, implying that PEMS-turkey astrovirus can potentially impair the immune response of turkeys, thereby leading to enhanced susceptibility of turkeys to secondary, perhaps even fatal, bacterial infections.

Capsid protein composition of reference strains and wild isolates of human astroviruses.

Astroviruses are small RNA viruses that are frequently associated with gastroenteritis in humans and animals. Despite much work on the genetic analysis of astrovirus strains, little progress has been made in the characterization of the proteins composing mature virions. We have analyzed the capsid protein composition of the reference strains and several wild isolates of human astroviruses using high-resolution polyacrylamide gradient gels. For reference strains of the seven serotypes analyzed, a consistent pattern of three infection-specific proteins--designated P1, P2, and P3 -was generally observed. The strains could be divided into two groups, based upon the reactivity of these proteins in immune precipitation assays that used homologous rabbit serum. One group included reference types 1 4 for which all three proteins were precipitated by homologous rabbit sera; for the other group, types 5 7, only proteins P2 and P3 were precipitated. When wild isolates from around the world were compared to the reference strains, a correlation between genetic type and the pattern of protein sizes and immune reactivity was observed for strains of the common types (1-4). Strains of types 2 and 4 consistently exhibited P3 proteins larger than those of types 1 and 3. Unusual patterns of proteins or immune reactivity were detected in strains of types 5-7, indicating that there may be incomplete processing of the capsid precursor during growth in cell culture.

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.

Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney.

A cytopathic astrovirus was isolated from pigs with acute diarrhea in an established cell line that was derived from porcine embryonic kidneys with the aid of trypsin. The virus showed a distinct cytopathic effect characterized by an enlargement of cells and the appearance of fine granules in the cytoplasm. Porcine astrovirus was shown to have an RNA genome, as determined by the effect of 5-iodo-2'-deoxyuridine on its replication, and five polypeptides with molecular masses of 13,000, 30,000, 31,000, 36,000, and 39,000 daltons; and it was shown to be stable to lipid solvents and heating at 50 degrees C for 30 min but somewhat labile to acid (pH 3.0). The buoyant density of the isolate determined in CsCl was 1.35 g/ml. Seroconversion to the virus was evident in the paired serum specimens obtained from pigs with diarrhea that were housed at the farm where the disease occurred. The neutralization test on serum specimens collected randomly from 128 adult pigs of eight herds revealed that 50 of the serum specimens were positive for antibody to porcine astrovirus, although there was considerable variation in the prevalence among herds, ranging from 0 to 83%. Hysterectomy-produced, colostrum-deprived, 4-day-old pigs developed mild diarrhea after oral exposure to porcine astrovirus propagated in the cell culture; and the virus was isolated again from diarrheal stool specimens.