HAstV Infection Research Articles

The neonatal Fc receptor and DPP4 are human astrovirus receptors.

Targeting FcRn or DPP4 using available therapies effectively prevents human astrovirus infection in human enteroid cultures.

Structure and immunogenicity of the murine astrovirus capsid spike.

Human astroviruses (HAstVs) are small, non-enveloped icosahedral RNA viruses that are a significant cause of diarrhoea in young children. Despite their worldwide prevalence, HAstV pathogenesis studies and vaccine development remain challenging due to the lack of an animal model for HAstV infection. The recent development of a murine astrovirus (MuAstV) infection model in mice provides the opportunity to test proof-of-concept vaccines based on MuAstV antigens. To help establish a system in which an astrovirus capsid spike-based vaccine could be tested in vivo, we designed and produced a recombinant MuAstV capsid spike protein based on predicted secondary structure homology to HAstV spike proteins. The recombinant MuAstV spike can be expressed with high efficiency in Escherichia coli and retains antigenicity to polyclonal antibodies elicited by MuAstV infection. We determined the crystal structure of the MuAstV spike to 1.75 Å and assessed its structural conservation with HAstV capsid spike. Despite low sequence identity between the MuAstV and HAstV spikes and differences in their overall shapes, they share related structural folds. Additionally, we found that vaccination with MuAstV spike induced anti-MuAstV-spike antibodies, highlighting that the recombinant spike is immunogenic. These studies lay a foundation for future in vivo MuAstV challenge studies to test whether MuAstV spike can be the basis of an effective vaccine.

Molecular Epidemiology of Classic, MLB, and VA Astroviruses in Children with Acute Gastroenteritis, 2014-2021: Emergence of MLB3 Strain in Japan.

Human astroviruses (HAstVs) are important causative pathogens of acute gastroenteritis (AGE) in children worldwide. MLB and VA HAstVs, which are genetically distinct from the previously known classic HAstVs, have been detected since 2008. To investigate the role of HAstVs in AGE, we conducted molecular detection and characterization of HAstVs circulating in children with AGE in Japan from 2014 to 2021. Out of 2,841 stool samples, HAstVs were detected in 130 (4.6%). MLB1 was the predominant genotype detected (45.4%), followed by HAstV1 (39.2%), MLB2 (7.4%), VA2 (3.1%), HAstV3 (2.3%), HAstV4, HAstV5, and MLB3 (0.8% each). The results demonstrated that HAstV infection in pediatric patients in Japan was dominated by the two major genotypes MLB1 and HAstV1, with a small proportion of other genotypes. The overall infection rates of MLB and VA HAstVs were higher than those of classic HAstVs. The HAstV1 strains detected in this study belonged solely to lineage 1a. The rare MLB3 genotype was detected for the first time in Japan. All three HAstV3 strains belonged to lineage 3c based on the ORF2 nucleotide sequence and were shown to be recombinant strains. IMPORTANCE HAstVs are one of the pathogens of viral AGE and are considered the third most common viral agents of AGE after rotavirus and norovirus. HAstVs are also suspected to be the causative agents of encephalitis or meningitis in immunocompromised patients and elderly persons. However, little is known about the epidemiology of HAstVs in Japan, especially that of MLBs and VA HAstVs. This study demonstrated epidemiological features and molecular characterization of human astroviruses encompassing a 7-year study period in Japan. This study highlights the genetic diversity of HAstV circulating in pediatric patients with acute AGE in Japan.

Detection and characterization of human astrovirus and sapovirus in outpatients with acute gastroenteritis in Guangzhou, China

BackgroundHuman astrovirus (HAstV) and sapovirus (SaV) are common pathogens that can cause acute gastroenteritis (AGE). However, very few studies have reported the molecular epidemiology and clinical information on HAstV and SaV in China. This study aims to determine the molecular epidemiology and clinical features of HAstV and SaV in patients with AGE in Guangzhou, China.MethodsFor this study, 656 patients with AGE were enrolled. Their stool samples were screened for 15 enteropathogens using Luminex xTAG® Gastrointestinal Pathogen Panel. HAstV and SaV were detected through an in-house multiplex reverse transcriptase polymerase chain reaction followed by phylogenetic analysis. We described and compared clinical features of AGE in patients with HAstV and SaV.ResultsOf the 656 stool samples, 63.72% (418/656) were found to be positive, with 550 enteropathogens (296 bacteria and 254 viruses). HAstV and SaV were detected in 20 (3.0%) and 12 (1.8%) samples, respectively. Four genotypes (genotypes 1, 2, 3, and 8) of HAstV and three genotypes (GI.1, GI.2 and GIV) of SaV were identified. Coinfection was observed in ten HAstV-positive and two SaV-positive samples. HAstV was more likely to occur in winter, while SaV in early spring. The median age of the patients with single HAstV infection was higher than that of the patients with other viruses (rotavirus, norovirus, and enteric adenovirus; P = 0.0476) and unknown etiology (P = 0.006). Coinfection with HAstV or SaV were not associated with disease severity (P > 0.05).ConclusionHAstV and SaV are the common causes of AGE in Guangzhou, China.

Molecular and epidemiological characterization of human adenovirus and classic human astrovirus in children with acute diarrhea in Shanghai, 2017\u20132018

BackgroundIn addition to rotavirus and norovirus, human adenovirus (HAdV) and classic human astrovirus (classic HAstV) are important pathogens of acute diarrhea in infants and young children. Here, we present the molecular epidemiology of HAdV and classic HAstV in children with acute diarrhea in Shanghai.MethodsFecal specimens were collected from 804 outpatient infants and young children diagnosed with acute diarrhea in Shanghai from January 2017 to December 2018. All of the samples were screened for the presence of HAdV and classic HAstV. HAdV and classic HAstV were detected using traditional PCR and reverse-transcription PCR, respectively. All of the HAdV and classic HAstV positive samples were genotyped by phylogenetic analysis.ResultsAmong the 804 fecal samples, 8.58% (69/804) of samples were infected with either HAdV or classic HAstV, and five were co-infected with two diarrhea viruses. The overall detection rates of HAdV and classic HAstV were 3.47% (28/804) and 5.22% (42/804), respectively. Four subgroups (A, B, C, and F) and seven genotypes (HAdV-C1, −C2, −B3, −C5, −A31, −F40, and -F41) of HAdV were detected. Subgroup F had the highest constituent ratio at 64.29% (18/28), followed by non-enteric HAdV of subgroup C (21.43%, 6/28) and subgroup B 10.71% (3/28). HAdV-F41 (60.71%, 17/28) was the dominant genotype, followed by HAdV-C2 (14.29%, 4/28) and HAdV-B3 (10.71%, 3/28). Two genotypes of classic HAstV (HAstV-1 and HAstV-5) were identified in 42 samples during the study period; HAstV-1 (95.24%, 40/42) was the predominant genotype, and the other two strains were genotyped as HAstV-5. No significant differences were found between boys and girls in the detection rates of HAdV (P = 0.604) and classic HAstV (P = 0.275). Over half of the HAdV infections (82.14%, 23/28) and classic HAstV infections (66.67%, 28/42) occurred in children less than 36 months. Seasonal preferences of HAdV and classic HAstV infections were summer and winter, respectively. In this study, the common clinical symptoms of children with acute diarrhea were diarrhea, vomiting, fever and abdominal pain.ConclusionsOur findings indicate that HAdV and classic HAstV play important roles in the pathogenesis of acute diarrhea in children in Shanghai. Systematic and long-term surveillance of HAdV and classic HAstV are needed to monitor their prevalence in children and prevent major outbreak.

#51: Neurotropic Astrovirus-VA1: Identifying the distinctions from classical genotypes

Abstract Background Human astroviruses (HAstV) are a leading cause of acute gastroenteritis in children, particularly those under the age of 2 or with immunosuppressive conditions. Indeed, our studies suggest that children with hematological malignancies are at high risk of infection. However, it has become increasingly clear that HAstV infections can also be associated with respiratory and even central nervous system (CNS) diseases. In the last decade, there have been at least 12 cases of astrovirus-induced CNS disease resulting in encephalitis and meningitis, including 2 St Jude patients. The CNS-associated infections are overwhelmingly fatal and are primarily caused by a novel astrovirus genotype, VA1, that is genetically more similar to animal astroviruses that also induce CNS-associated infections. The recent ability to propagate the VA1 strain in cell lines affords us the opportunity to better understand VA1 infection and how it compares to the better-studied HAstV1 strain. Methods Viral kinetics were determined by infection of human intestinal adenocarcinoma Caco-2 cells with either HAstV1 or VA1 and monitored for 24 hours post-infection (hpi). Infected cells were identified via immunofluorescent microscopy using antibodies against double-stranded RNA and viral capsid. Epithelial permeability of astrovirus-infected Caco-2 cells was measured using transepithelial electrical resistance (TER) and monitored for 24hpi. Nitazoxanide efficacy was identified by immunofluorescent analysis as described previously. Results Our findings demonstrate that HAstV1 and VA1 replicate in intestinal epithelial cells without inducing cell death or a pro-inflammatory cytokine response. However, these two strains have distinct replication kinetics. VA1 appears to have an ~8 hour lag in the expression of dsRNA and capsid protein compared to HAstV1 and does not require exogenous proteases to process the viral outer coat (capsid) protein, Additionally, we demonstrated that the increase in epithelial barrier permeability associated with HAstV1-infection is not found in VA1-infected cells, which is intriguing and may explain why HAstV1 is more likely to cause diarrhea. Of clinical importance, we have revealed a similar susceptibility of VA1 to the antiviral drug, nitazoxanide, which we have recently demonstrated its effectiveness inhibiting classical HAstV strains. Conclusion Overall, our studies highlight that VA1 pathogenesis is distinct from HAstV1, which could explain the differences in vivo. Future studies will be necessary to investigate viral replication and pathogenesis in distinct neuronal cells and in vivo.

Molecular and clinical epidemiological features of human astrovirus infections in children with acute gastroenteritis in Shandong province, China.

Human astrovirus (HAstV) isone of the most common causative agents of acute gastroenteritis in children with an infection rate estimated to range from 2% to 9% worldwide. This study was aimed atinvestigatingthe molecular and clinical epidemiological features of human astrovirus infections in children under 5 years old with acute gastroenteritis in Shandong province, China from July 2017 to June 2018. In total, 376 fecal samples and the corresponding clinical information were collected and analyzed. HAstV infections were detected in all age groups with an overall positive rate of 8.51%. In addition to acute diarrhea, the main clinical manifestations were fever, abdominal pain, vomiting, and dehydration, in which fever was the most common complication. Infections could be seen throughout the year with a peak in the colder season. Four genotypes were detected in which HAstV-1 was the most prevalent genotype with a prevalence of 78.12%, followed by HAstV-5 (9.38%), MLB-1 (9.38%), and MLB-2 (3.12%). HAstV-1 strains were classified as lineage 1a, 1b, and 1d, in which lineage 1a strains were the most prevalent followed by lineage 1b and lineage 1d strains. All HAstV-5 strains were classified as lineage 5b and no other lineages were detected. The results showed that HAstV infection was an important cause of acute gastroenteritis among children under 5 years old in Shandong province. Given that their diseasespectrum had been broadened, HAstVs should be paid more attention, not only as a causative agent of acute gastroenteritis but also as a potential pathogen of unexpected diseases.

Epidemiology and Genetic Characterization of Classical Human Astrovirus Infection in Shanghai, 2015-2016.

ObjectiveHuman astroviruses (HAstVs) are single-stranded RNA viruses of the Astroviridae family. Infection of classic HAstVs is one of the most common causes of acute viral gastroenteritis (infectious viral diarrhea). There is a lack of data on the prevalence and genetic characterization of classic HAstVs in acute viral gastroenteritis in the whole population. This study aimed to investigate the epidemiological trend, genotypes, viral co-infections, and viral loads of classic HAstVs in Shanghai, China, from January 2015 to December 2016.MethodsA total of 6,051 non-redundant stool samples were collected in outpatients with acute diarrhea in Shanghai from January 2015 to December 2016. One-step real-time RT-PCR was used for screening viral diarrhea, including rotavirus A, rotavirus B, rotavirus C, norovirus genotype I and II, classic human astroviruses, and sapovirus. Real-time PCR was used for screening human enteric adenoviruses. Conventional RT-PCR was used for the amplification of viral fragments for genotyping. PCR products were sequenced and used for the construction of phylogenetic trees.ResultsThe detection rate of classic HAstVs was 1.55% (94/6,051). The prevalence of HAstV infection displayed a typical winter/spring (December to March) seasonality and was highest in the 5–14 age group. Eighty-six samples were genotyped, which revealed HAstV-1 as the most prevalent genotype, followed by HAstV-5, HAstV-4, HAstV- 2, HAstV-8, and HAstV-3. There was a dramatic rise in the prevalence of HAstV-4 from December 2015 to March 2016, and the viral loads of HAstV-4 were significantly higher than those of other genotypes. Among the mixed infection samples, noroviruses were found to be the most frequently co-infected enteric viruses with HAstV.ConclusionMultiple genotypes of classic HAstVs circulated in Shanghai from January 2015 to December 2016. For the first time, HAstV-3\\4\\5\\8 were detected in Shanghai.

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.

Molecular Epidemiology of Human Astrovirus in Stool Samples From Patients With Acute Gastroenteritis in Korea, 2013?2017

BackgroundHuman astroviruses (HAstVs) are one of the major causes of acute gastroenteritis. Classic HAstVs can be classified into eight genotypes. We investigated the positive rate of HAstVs and the distribution of HAstV genotypes in strains isolated from patients with acute gastroenteritis in Hwaseong, Korea, in 2013–2017.MethodsBetween November 2013 and December 2017, 3,519 stool samples were collected from patients with symptoms of acute gastroenteritis and tested for HAstV using multiplex PCR. For HAstV-positive samples, the ORF2 gene, which encodes a capsid protein, was genotyped by reverse-transcription PCR and sequencing. Phylogenetic analysis was performed to determine whether the sequences of the HAstVs differed by year.ResultsThe positive rate of HAstV was 1.9% (67 samples). HAstV Type 1 was the most prevalent genotype (82.4%), and Types 4, 5, and 8 were also detected. Infection occurred year-round, with no distinct seasonal variation. Infection occurred at nearly all ages (55 days–81 years; median: 3 years), and the positive rate was substantially higher in children younger than five years. Phylogenetic analysis revealed three distinct clades of HAstV Type 1 according to the collection time.ConclusionsOur results provide recent epidemiological data on HAstVs in Korea between 2013 and 2017. The finding of three distinct clades of HAstV Type 1 according to collection time suggests genetic evolution of HAstVs. These findings can enhance our knowledge on HAstV infection and viral evolution.

Epidemiology of human astroviruses among children younger than 5 years: Prospective hospital-based sentinel surveillance in South Africa, 2009-2014.

The epidemiology of human astroviruses (HAstVs) in hospitalised patients less than 5 years of age from selected sites in South Africa was investigated. Diarrheagenic stool specimens collected from April 2009 to May 2014 were screened retrospectively for selected viruses, bacteria and parasites. Patient data were analysed to identify epidemiologic factors most frequently detected with HAstV infections. The following case-comparisons were investigated; HAstV-positive and HAstV-negative children, human immunodeficiency virus (HIV)-infected and HIV-uninfected (HAstV-positive) children and HIV-exposed and unexposed (HAstV-positive HIV-uninfected) children. Astrovirus was identified in 7.0% (234/3340) of cases and most frequently in ages 7 to 12 months (9.2%; 90/975) compared with 5.8% to 6.6% in other 6-month age groups. No seasonal trends were observed. More HAstVs were detected in children from homes that used outdoor water sources (7.6%) compared to indoor sources [5.7%; adjusted odds ratio (aOR), 1.5; 95% CI, 1.1-2.1; P = 0.009]. Astroviruses were detected in 8.4% (67/799) of HIV-uninfected patients that were exposed to HIV compared with 5.9% (74/1257) of HIV-unexposed patients ( P = 0.032). Astroviruses were most prevalent in children aged 7 to 12 months and were detected throughout the study period. The study was limited as only hospitalised patients were investigated and no comparisons were made to diarrhoea-free control groups. Future HAstV surveillance should include community-based studies and children presenting at outpatient facilities.

Astrovirus infection in hospitalized children: Molecular, clinical and epidemiological features

BackgroundAstrovirus (HAstV) is a common viral pathogen that causes gastroenteritis worldwide. It is classified into eight classical human types (HAstV-1/8) and seven other less prevalent types, described as HAstV VA1, VA2, VA3, VA4, MLB-1, MLB-2 and MLB-3. During outbreaks, the elderly and children are the most affected, and the spread of the virus is associated with person-to-person contact, food ingestion and contaminated water. ObjectivesThe aim of the present study was to investigate the prevalence of infection and genetic diversity of HAstV strains. Samples were collected from children with acute gastroenteritis admitted to a large pediatric hospital during a surveillance period of three years (2008–2011) in Belém city, Pará State, Amazon Region, Northern Brazil. Study designScreening and genotyping tests were conducted using RT-PCR to detect the classical and non-classical HAstV types using specific primers. A semi-nested RT-PCR protocol was developed to improve viral detection in samples with a low viral load. ResultsThe overall positivity observed in this study was 3.9% (19/483). The age distribution showed a high prevalence of positive cases in children under one year old (5.3%). We found vomiting associated with 75% of the positive cases, fever with 82.3%, and dehydration with 76.9%. Most patients with positive cases demonstrated two to five days of diarrhea, two to three episodes of vomiting during hospitalization, and three bowel movements per day. Co-infection with HAstV and norovirus was observed in three cases (15.8%), and no pattern of seasonality or any relationship between the HAstV positivity rate and climate variables was observed. Eighteen positive samples (94.7%–18/19) were genotyped based on the ORF 2 region, and the greatest prevalence was of HAstV-1a (66.6%–12/18), followed by HAstV-2 (22.2%–4/18, comprising two type-2b and two type-2c genotypes), HAstV-3c (5.6%–1/18) and HAstV-4c (5.6%–1/18). No non-classical types were detected in the clinical samples analyzed. ConclusionsThe present study showed that although HAstV infections occur at low frequency, they are involved in severe pediatric cases of acute gastroenteritis presenting with a high diversity of strains, including the lineages 3c and 4c, which were never before detected in Brazil.

Epidemiology and genetic diversity of classic human astrovirus among hospitalized children with acute gastroenteritis in Uruguay.

Classic Human Astrovirus (Classic HAstV) are one of the most important causes of pediatric acute gastroenteritis (AGE), after rotaviruses and arguably caliciviruses. The aim of this study was to determine the molecular epidemiology of Classic HAstV from 175 clinical samples, being 153 stools and 22 vomits, collected from pediatric hospitalized patients with AGE in Salto city, Uruguay, from January 2011 to December 2012. Classic HAstV were detected and genotyped by using a qualitative Retro Transcription-Polimerase Chain Reaction (RT-PCR) directed to the Open Reading Frame-2 (ORF2) region C. Amplicons were sequenced and phylogenetic analyses were carried out in order to determine genotypes and lineages. Classic HAstV were detected in 18 out of 175 analyzed samples (10.3%) and 14 of them (78.0%) were successfully sequenced being 6 (42.8%) classified as HAstV-1 (1a lineage), 4 (28.6%) as HAstV-2 (2c lineage), and 4 (28.6%) as HAstV-3 (3c lineage). A higher detection of Classic HAstV infections was observed in autumn for both years of surveillance, and the majority of the positive cases were observed in 2011. The group of children between 2 and 5 years old presented the higher percentage of infections. To our knowledge, the present study represents the first report of astrovirus from acute gastroenteritis cases in Uruguay, evidencing its role as a relevant etiologic agent in severe cases of this disease.

Temporal variation in the distribution of type-1 human astrovirus lineages in a settled population over 14years.

Human astroviruses (HAstVs) are important enteric pathogens that are genetically and antigenically heterogeneous and can be classified into eight sero/genotypes (HAstV-1 to -8) and different lineages within each HAstV type. This study describes the genetic diversity of HAstVs circulating in southern Italy over 14 years. Molecular analysis of HAstV-1 strains showed that three different lineages (1a, 1b and 1d) of the predominant genotype were circulating during the study period. The study of an archival collection of HAstV strains offers a unique opportunity to evaluate the patterns of variation of HAstV infections over the years and to correlate the observed epidemiological changes to the genetic variability of HAstVs.

Characterization of Human Astrovirus Cell Entry

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.

Full sequence analysis and characterization of a human astrovirus type 1 isolate from South Korea

Human astroviruses are recognized as an important cause of infantile gastroenteritis around the world. In South Korea, sporadic cases of HAstV infection have been reported since 2002. However, hitherto, there have been no studies reporting the whole genome sequence of an HAstV isolate from South Korea. Hence, we sequenced and analyzed the entire genome of an HAstV-1 strain (lhar) that was isolated in Seoul, South Korea. The whole-genome sequence analysis revealed 3 open reading frames comprising the whole genome: ORF1a (2,763 bp), ORF1b (1,548 bp), and ORF2 (2,364 bp). The lhar strain showed amino acid identities with 8 other reference strains of 87.6-98.7%, 94.2-98.8%, and 62.6-99.0% in the ORF1a, ORF1b, and ORF2 regions, respectively. The amino acid sequence of the capsid region encoded by ORF2 was compared with a total of 19 HAstV-1 strains and 8 HAstVs reference strains isolated in various countries. This revealed 1 amino acid substitution, at aa412 (Pro → Arg) in ORF2. This study, the first to report the full-length sequence of an HAstV isolated in South Korea, is meaningful in that it can be used as a full-length HAstV sequence standard for future comparison studies. It may also prove useful to the field of public health field by facilitating the diagnosis and the prediction of new emerging variants.

A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of human astrovirus

A one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) method targeting the 5' end of the capsid gene for rapid and quantitative detection of human astrovirus serotype 1 (HAstV 1) was developed. The assay is highly sensitive and comparable to real-time RT-PCR (rRT-PCR), with a detection limit of ∼100 RNA copies per assay. The specificity of the method was validated by the absence of any cross-reaction with RNA samples of HAstV 2-8 and other gastroenteritis viruses, followed by nucleotide sequencing of the amplified product. Fecal specimens (n=120) obtained from children under five years of age with gastroenteritis were tested by rRT-LAMP, rRT-PCR and transmission electron microscopy (TEM). Six (5%) of these samples were determined to be positive by both rRT-LAMP and rRT-PCR assay, and these two nucleic acid amplification methods resulted in a 200% increase in detection rates for HAstV infection compared with TEM alone. Furthermore, the rRT-LAMP assay is much more rapid than rRT-PCR and generates results in less than 20min for positive samples. The quantitation of viral load in stool specimens was determined from the standard curve plot of time-of-positivity versus initial RNA concentration. Most viral loads were determined to be within the range of 10(5)-10(8) copies. The results highlight the significance of the rapid rRT-LAMP method as a diagnostic and routine screening tool for the analysis of stool samples in hospital laboratories.

Prevalence and characterization of astroviruses in Argentinean children with acute gastroenteritis.

Among viral agents causing gastroenteritis, human astroviruses (HAstVs) take second or third place, after rotaviruses and caliciviruses, as the most frequent cause of illness. The aims of this study were to determine the prevalence of HAstV infection and to characterize the circulating HAstV strains in children with diarrhea under 3 years of age treated between 1995 and 1998 at out- or in-patient facilities of the children's hospital in Mendoza, Argentina. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay (EIA) were used to detect HAstVs in stool specimens. Positive specimens were tested further by EIA and/or sequenced to type detected HAstV strains. HAstVs were detected in 40 (3.7%) of 1,070 samples that were rotavirus and calicivirus-negative: 14 (3.5%) of 402 from outpatients and 26 (3.9%) of 668 from inpatients. HAstV infection tended to be more severe in children during their first year of life: 18 (4.7%) of 383 HAstV-positive children 0-11 months old were hospitalized versus 8 (2.8%) of 285 children 1 year of age or older (P = 0.29). Type 1 (HAstV-1) was the most common type (41%), followed by HAstV-4 (25%), HAstV-2 (13%), HAstV-3 (13%), and HAstV-5 (8%). In this first epidemiological study of HAstV infection in this region, we confirmed HAstV to be a cause of severe gastroenteritis in children, more often among children younger than 12 months of age. HastV-4 caused 25% of HastV infections in Mendoza, although it has been detected commonly elsewhere. Distinct genetic lineages were apparent but their epidemiological significance remains to be demonstrated.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.