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Astroviruses (AstVs) are among the most important viruses causing diarrhea in human infants and many animals, posing a threat to public health safety and a burden on the economy. Five porcine AstV (PAstV) genotypes have been identified in various countries, including China. However, the epidemiology of PAstV in Yunnan province, China, remains unknown. In this study, 489 fecal samples from pigs in all 16 prefectures/cities of Yunnan were collected between April and August of 2020 for epidemiological investigation. The total infection rate of PAstV-2 or PAstV-5 was 39.9%, with suckling piglets having the highest infection rate (62.3%). The ORF2 genes of seven PAstV-2 and 10 PAstV-5 isolates were sequenced and phylogenetically analyzed. In addition to coinfections with PAstV-2 and PAstV-5, coinfections of PAstV with other diarrhea-inducing viruses (e.g., porcine bocavirus) were also discovered. A comparison of ORF2-encoded capsid protein sequences revealed that there were multiple insertions and deletions in the seven Yunnan PAstV-2 sequences, while point mutations, but no deletions or insertions, were found in the 10 Yunnan PAstV-5 sequences, which were very similar to the reference sequences. This is the first epidemiological investigation and genetic characterization of PAstV-2 and PAstV-5 in Yunnan province, China, demonstrating the current PAstV infection situation in Yunnan. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05311-8.

Porcine astrovirus type 3 (PoAstV3) is an emerging virus in the family Astroviridae that has been recently associated with polioencephalomyelitis/encephalitis. Herein, we describe the experimental oral and intravenous inoculation of an infectious central nervous system (CNS) tissue homogenate containing PoAstV3 to cesarean-derived, colostrum-deprived pigs, and the subsequent development of clinical signs, histologic lesions, specific humoral immune response, and detection of viral particles by electron microscopy (EM) and viral RNA by RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) and in situ hybridization (ISH). IgG against a portion of the PoAstV3 ORF2 capsid was first detected at 7 days post-inoculation (DPI) in 2 of 4 inoculated animals and in all inoculated animals by 14 DPI. At 21 and 28 DPI, 2 of 4 inoculated animals developed ataxia, tetraparesis, and/or lateral recumbency. All inoculated animals had histologic lesions in the CNS including perivascular lymphoplasmacytic cuffs, multifocal areas of gliosis with neuronal necrosis, satellitosis, and radiculoneuritis, and PoAstV3 RNA as detected by RT-qPCR within multiple anatomic regions of the CNS. Consistent viral structures were within the soma of a spinal cord neuron in the single pig examined by EM. Of note, PoAstV3 was not only detected by ISH in neurons of the cerebrum and spinal cord but also neurons of the dorsal root ganglion and nerve roots consistent with viral dissemination via axonal transport. This is the first study reproducing CNS disease with a porcine astrovirus strain consistent with natural infection, suggesting that pigs may serve as an animal model to study the pathogenesis of neurotropic astroviruses.

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes (120-GNNTFG-125, 485-RISDPTWFSA-494) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120-GNNTFG-125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years because of acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from astrovirus- and mock-infected cells showed that infection with HAstV Yuc8 enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Some of the extracellular virus was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extraintestinal presence of some astrovirus strains.ImportanceAstroviruses are an important cause of diarrhea in children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles seems to increase astrovirus infectivity in less susceptible cells, and renders virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

Human astroviruses (AstVs) are usually associated with acute gastroenteritis. In recent years, atypical animal-like AstVs have been identified, but their pathogenic role in humans has not been determined. Starting from 2010, there has been a growing evidence that AstVs may also be associated with encephalitis in human and animal hosts. Some human atypical AstV strains (VA1, MLB1/MLB2) display neurotropic potential, as they have been repeatedly identified in patients with AstV-related encephalitis, chiefly in immunosuppressed individuals. In this study, a VA1-like AstV was identified from a single stool sample from an outbreak of foodborne acute gastroenteritis occurred in Italy in 2018. On genome sequencing, the virus was related to the VA1-like strain UK1 (99.3% at the nucleotide level). Similar viruses were also found to circulate in paediatric patients hospitalized with AGE in the same time span, 2018, but at low prevalence (0.75%, 3/401). Gathering epidemiological data on atypical AstVs will be useful to assess the risks posed by atypical AstV infections, chiefly in medically fragile patients.

Domestic swine have been introduced by humans into a wide diversity of environments and have been bred in different production systems. This has resulted in an increased risk for the occurrence and spread of diseases. Although viromes of swine in intensive farms have been described, little is known about the virus communities in backyard production systems around the world. The aim of this study was to describe the viral diversity of 23 healthy domestic swine maintained in rural backyards in Morelos, Mexico, through collection and analysis of nasal and rectal samples. Next-generation sequencing was used to identify viruses that are present in swine. Through homology search and bioinformatic analysis of reads and their assemblies, we found that rural backyard swine have a high degree of viral diversity, different from those reported in intensive production systems or under experimental conditions. There was a higher frequency of bacteriophages and lower diversity of animal viruses than reported previously. In addition, sapoviruses, bocaparvoviruses, and mamastroviruses that had not been reported previously in our country were identified. These findings were correlated with the health status of animals, their social interactions, and the breeding/rearing environment (which differed from intensive systems), providing baseline information about viral communities in backyard swine.

Background To determine the prevalence of enteric infections in Aboriginal children aged 0–2 years using conventional and molecular diagnostic techniques and to explore associations between the presence of pathogens and child growth. Methods Cross-sectional analysis of Aboriginal children (n = 62) residing in a remote community in Northern Australia, conducted from July 24th - October 30th 2017. Stool samples were analysed for organisms by microscopy (directly in the field and following fixation and storage in sodium-acetate formalin), and by qualitative PCR for viruses, bacteria and parasites and serology for Strongyloides-specific IgG. Child growth (height and weight) was measured and z scores calculated according to WHO growth standards. Results Nearly 60% of children had evidence for at least one enteric pathogen in their stool (37/62). The highest burden of infection was with adenovirus/sapovirus (22.9%), followed by astrovirus (9.8%) and Cryptosporidium hominis/parvum (8.2%). Non-pathogenic organisms were detected in 22.5% of children. Ten percent of children had diarrhea at the time of stool collection. Infection with two or more pathogens was negatively associated with height for age z scores (− 1.34, 95% CI − 2.61 to − 0.07), as was carriage of the non-pathogen Blastocystis hominis (− 2.05, 95% CI - 3.55 to − 0.54). Conclusions Infants and toddlers living in this remote Northern Australian Aboriginal community had a high burden of enteric pathogens and non-pathogens. The association between carriage of pathogens/non-pathogens with impaired child growth in the critical first 1000 days of life has implications for healthy child growth and development and warrants further investigation. These findings have relevance for many other First Nations Communities that face many of the same challenges with regard to poverty, infections, and malnutrition.

Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk for contracting severe disease. However, no vaccines exist to combat human astrovirus infection. Evidence points to the importance of antibodies in protecting healthy adults from reinfection. To develop an effective subunit vaccine that broadly protects against diverse astrovirus serotypes, we must understand how neutralizing antibodies target the capsid surface at the molecular level. Here, we report the structures of the human astrovirus capsid spike domain bound to two neutralizing monoclonal antibodies. These antibodies bind two distinct conformational epitopes on the spike surface. We add to existing evidence that the human astrovirus capsid spike contains a receptor-binding domain and demonstrate that both antibodies neutralize human astrovirus by blocking virus attachment to host cells. We identify patches of conserved amino acids which overlap or border the antibody epitopes and may constitute a receptor-binding site. Our findings provide a basis for developing therapies to prevent and treat human astrovirus gastroenteritis. IMPORTANCE Human astroviruses infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies block astrovirus infection. Here, we determined the crystal structures of the astrovirus capsid protein in complex with two virus-neutralizing antibodies. We show that the antibodies bind to two distinct sites on the capsid spike domain, however, both antibodies block virus attachment to human cells. Importantly, our findings support the use of the human astrovirus capsid spike as an antigen in a subunit-based vaccine to prevent astrovirus disease.

Human astrovirus (HAstV) is recognized as one of the major causative agents of acute gastroenteritis in children worldwide. Data on the genetic diversity of HAstV in Nigeria are limited. The aim of this study was to determine the prevalence and molecular epidemiology of classical HAstV in children under 5 years of age with acute gastroenteritis in Ogun State, Nigeria. Fecal samples (331) as well as socio-demographic and clinical data were collected across the three senatorial districts of the state from February 2015 to April 2017. One hundred seventy-five samples were randomly selected and analyzed for the presence of HAstV using RT-PCR. PCR amplicons from positive samples were sequenced, and phylogenetic analysis was done to determine genotypes and lineages. The overall prevalence rate was 19.4% (34), with the highest occurrence observed in 2015 (41.4%). Viral coinfections were detected in 13 cases (38.2%). HAstV infection occurred throughout the year and in all age groups, mainly in the age group of 0-12 months. There was significant association between prevalence rate and collection year; however, no association was observed with gender, age, symptoms or risk factors. HAstV-5 was the predominant genotype (76.5%) circulating throughout the study period, followed by HAstV-1 (23.5%), which circulated only in the first 2 years of the study. Phylogenetic analysis showed that all HAstV-5 strains detected belonged to the 5a lineage, while HAstV-1 strains were grouped into lineage 1b. This study, to the best of our knowledge, is the first comprehensive report on molecular characterization of classical HAstV among children with gastroenteritis in the country, and this will serve as baseline information for implementing appropriate infection control practices.

Introduction Acute viral gastroenteritis is a major public health concern, especially among children younger than 5 years of age. The aim of this study was to determine the occurrence of human astrovirus infection in children with acute gastroenteritis. Methods Stool specimens were collected from 506 children under 5 years of age hospitalized with acute diarrhoea (289 male and 208 female), and human astrovirus was investigated by RT-PCR. Associations of socio-demographic, clinical, and behavioural conditions with infection were analysed. Results The overall prevalence of human astrovirus was found to be 10.3%. The mean age of positive cases was 12.41 ± 6.21 months and this was associated with infection (p = 0.013). Children >18 months of age were at three times the risk of infection when compared to those aged 0–6 months (odds ratio (OR) 3.19, 95% confidence interval (CI) 1.15–8.88; p = 0.026). Children living in houses with more than one room (OR 0.60, 95% CI 0.28–0.96; p = 0.036) and mothers using treated water (OR 0.47, 95% CI 0.25–0.86; p = 0.015) were associated with reduced infection. Conclusions In this study, infection with astrovirus was common in acute gastroenteritis cases among children younger than 5 years of age. Drinking treated water and living in non-crowded environments protected the children from infection.

Although human astroviruses (HAstVs) are important agents of gastroenteritis in young children, the studies aimed at characterizing their biology have been limited, in particular regarding their cell entry process. It has been shown that HAstV serotype 8 enters human cells by a classical clathrin-mediated endocytosis pathway, however, the cell receptor or other cell entry factors that may be relevant for an efficient viral infection are unknown. In this work we used a far-Western blotting approach to identify cellular proteins that interact with the recombinant capsid spike proteins of HAstV serotypes 1, 2, and 8, synthesized in Escherichia coli. We identified the 72 kDa protein disulfide isomerase A4 (PDIA4) as a binding partner for HAstV-1 and -8 spikes, but not for the HAstV-2 spike. In agreement with this observation, the PDI inhibitor 16F16 strongly blocked infection by HAstV serotypes 1 and 8, but not serotype 2. RNA interference of PDIA4 expression selectively blocked HAstV-8 infectivity. We also showed that the PDI activity does not affect virus binding or internalization but is required for uncoating of the viral genome.

Background Human 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. Methods For 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. Results Of 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). Conclusion HAstV and SaV are the common causes of AGE in Guangzhou, China.

Calf diarrhoea has been a major cause of economic losses in the global dairy industry. Many factors, including multiple pathogen infections, can directly or indirectly cause calf diarrhoea. This study compared the faecal virome between 15 healthy calves and 15 calves with diarrhoea. Significantly lower diversity of viruses was found in samples from animals with diarrhoea than those in the healthy ones, and this feature may also be related to the age of the calves. Viruses belonging to the families Astroviridae and Caliciviridae that may cause diarrhoea in dairy calves have been characterized, which revealed that reads of caliciviruses and astroviruses in diarrhoea calves were much higher than those in healthy calves. Five complete genomic sequences closely related to Smacoviridae have been identified, which may participate in the regulation of the gut virus community ecology of healthy hosts together with bacteriophages. This research provides a theoretical basis for further understanding of known or potential enteric pathogens related to calf diarrhoea.

This mini-review describes observations of the 1990ies with culturing of gluten-specific and astrovirus-specific CD4+ T cells from duodenal biopsies from subjects who presumably had a long time between the exposure to gluten or astrovirus antigens and the sampling of the biopsy. In these studies, it was also observed that antigen-specific CD4+ T cells migrated out of the gut biopsies during overnight culture. The findings are suggestive of memory T cells in tissue which are resident, but which also can be mobilised on antigen stimulation. Of note, these findings were made years before the term tissue resident memory T cells was invoked. Since that time, many observations have accumulated on these gut T cells, particularly the gluten-specific T cells, and we have insight into the turnover of CD4+ T cells in the gut lamina propria. These data make it evident that human antigen-specific CD4+ T cells that can be cultured from gut biopsies indeed are bone fide tissue resident memory T cells.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus, the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Bovine astrovirus (BoAstV) belongs to genus Mamastravirus (MAstV). It can be detected in the faeces of both diarrhoeal and healthy calves. However, its prevalence, genetic diversity, and association with cattle diarrhoea are poorly understood. In this study, faecal samples of 87 diarrhoeal and 77 asymptomatic calves from 20 farms in 12 provinces were collected, and BoAstV was detected with reverse transcription-polymerase chain reaction (RT-PCR). The overall prevalence rate of this virus in diarrhoeal and asymptomatic calves was 55.17 % (95 % CI: 44.13, 65.85 %) and 36.36 % (95 % CI: 25.70, 48.12 %), respectively, indicating a correlation between BoAstV infection and calf diarrhoea (OR=2.15, P=0.024). BoAstV existed mainly in the form of co-infection (85.53 %) with one to five of nine viruses, and there was a strong positive correlation between BoAstV co-infection and calf diarrhoea (OR=2.83, P=0.004). Binary logistic regression analysis confirmed this correlation between BoAstV co-infection and calf diarrhoea (OR=2.41, P=0.038). The co-infection of BoAstV and bovine rotavirus (BRV) with or without other viruses accounted for 70.77 % of all the co-infection cases. The diarrhoea risk for the calves co-infected with BoAstV and BRV was 8.14-fold higher than that for the calves co-infected with BoAstV and other viruses (OR=8.14, P=0.001). Further, the co-infection of BoAstV/BRV/bovine kobuvirus (BKoV) might increase the risk of calf diarrhoea by 14.82-fold, compared with that of BoAstV and other viruses (OR=14.82, P n=3) and MAstV-33 (n=2) and two novel genotypes designated tentatively as MAstV-34 (n=1) and MAstV-35 (n=3). In addition, seven out of nine BoAstV strains showed possible inter-genotype recombination and cross-species recombination. Therefore, our results increase the knowledge about the prevalence and the genetic evolution of BoAstV and provide evidence for the association between BoAstV infection and calf diarrhoea.

Background In 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. Methods Fecal 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. Results Among 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. Conclusions Our 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.

Many enteric viruses are found in pig farms around the world and can cause death of animals or important production losses for breeders. Among the wide spectrum of enteric viral species, porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV) and porcine Astrovirus (PAstV) are frequently found in pig feces. In this study we investigated sixteen pig farms in Corsica, France, to evaluate the circulation of three enteric viruses (PKoV, PAstV-1 and PSV). In addition to the three viruses studied by RT–qPCR (908 pig feces samples), 26 stool samples were tested using the Next Generation Sequencing method (NGS). Our results showed viral RNA detection rates (i) of 62.0% [58.7–65.1] (n = 563/908) for PSV, (ii) of 44.8% [41.5–48.1] (n = 407/908) for PKoV and (iii) of 8.6% [6.8–10.6] (n = 78/908) for PAstV-1. Significant differences were observed for all three viruses according to age (P-value = 2.4e–13 for PAstV-1; 2.4e–12 for PKoV and 0.005 for PSV). The type of breeding was significantly associated with RNA detection only for PAstV-1 (P-value = 9.6e–6). Among the 26 samples tested with NGS method, consensus sequences corresponding to 10 different species of virus were obtained This study provides first insight on the presence of three common porcine enteric viruses in France. We also showed that they are frequently encountered in pigs born and bred in Corsica, which demonstrates endemic local circulation.ImportanceThis study provides important information in the comprehension of the epidemiology of different viruses circulating in swine farms. We have shown the great diversity of viruses that could be present in extensive farms. Moreover, to our knowledge, this is the first detection of these different viruses in France. So far, this study has to be considered as a first step in the study of enteric viruses in Corsican pig farms.

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses. Electronic supplementary material The online version of this article (10.1007/s00705-019-04394-8) contains supplementary material, which is available to authorized users.

In Quito, the microbiological contamination of surface water represents a public health problem, mainly due to the lack of sewage treatment from urban wastewater. Contaminated water contributes to the transmission of many enteric pathogens through direct consumption, agricultural and recreational use. Among the different pathogens present in urban discharges, viruses play an important role on disease, being causes of gastroenteritis, hepatitis, meningitis, respiratory infections, among others. This study analyzes the presence of viruses in highly impacted surface waters of urban rivers using next-generation sequencing techniques. Three representative locations of urban rivers, receiving the main discharges from Quito sewerage system, were selected. Water samples of 500 mL were concentrated by skimmed-milk flocculation method and the viral nucleic acid was extracted and processed for high throughput sequencing using Illumina MiSeq. The results yielded very relevant data of circulating viruses in the capital of Ecuador. A total of 29 viral families were obtained, of which 26 species were associated with infections in humans. Among the 26 species identified, several were related to gastroenteritis: Human Mastadenovirus F, Bufavirus, Sapporovirus, Norwalk virus and Mamastrovirus 1. Also detected were: Gammapapillomavirus associated with skin infections, Polyomavirus 1 related to cases of kidney damage, Parechovirus A described as cause of neonatal sepsis with neurological affectations and Hepatovirus A, the etiologic agent of Hepatitis A. Other emergent viruses identified, of which its pathogenicity remains to be fully clarified, were: Bocavirus, Circovirus, Aichi Virus and Cosavirus. The wide diversity of species detected through metagenomics gives us key information about the public health risks present in the urban rivers of Quito. In addition, this study describes for the first time the presence of important infectious agents not previously reported in Ecuador and with very little reports in Latin America.

This study aimed to investigate the molecular epidemiology and genetic diversity of astrovirus circulating in pediatric patients with acute gastroenteritis in Thailand. A total of 2034 fecal specimens collected between 2011 and 2016 from children hospitalized with acute gastroenteritis were analyzed for astrovirus infection using RT-PCR and nucleotide sequencing techniques. Overall, 54 samples were positive for astrovirus (2.6%). About half of the virus-positive samples (57.4%) were co-infection cases, mainly with rotavirus or norovirus. Genotyping revealed that three major clades, classic human astrovirus (HAstV) and newly emerging MLB and VA astroviruses co-circulated in the study population. At least seven different genotypes were identified and HAstV1 was the most predominant genotype detected (50%), followed by MLB1 (16.7%), HAstV4 (11.1%), HAstV5 and HAstV8 (7.4% each), MLB2 and VA2 (3.7% each). This study provided a useful information and contributed to molecular epidemiology of astroviruses including the novel MLB and VA astoviruses have been characterized and reported for the first time in Thailand.

We estimated the prevalence of astrovirus, sapovirus, and norovirus among patients enrolled in research protocols and receiving medical care at the Clinical Center of the National Institutes of Health, Bethesda, MD, a clinical research hospital with a large immunocompromised patient population. We identified patients whose fecal specimens were submitted to the Clinical Center for testing on the Biofire FilmArray Gastrointestinal Panel from September 15, 2015 through November 30, 2016. Among 442 patients with fecal specimens submitted for multiplex testing, 11% had norovirus identified, 2% had astrovirus, and 2% had sapovirus. Like norovirus, astrovirus was detected in multiple sequential samples from a single patient, consistent with chronic infection or the occurrence of multiple reinfections. Coinfection with non-viral gastrointestinal pathogens was detected in 31% of patients with positive results for norovirus, astrovirus, or sapovirus. Norovirus remains common in this immunocompromised patient population, and both sapovirus and astrovirus are present.

Human astrovirus was one of the most common causative agent of acute gastroenteritis in children with infection rate estimated to range from 2% to 9% worldwide. This study was aimed to investigate the 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 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 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 was 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 diseases spectrum 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. This article is protected by copyright. All rights reserved.

Between 2016 and 2018, the prevalence of porcine kobuvirus (PKoV) and porcine astrovirus (PAstV) in Korean wild boars (n = 845) was 28.0% and 10.7%, respectively. Coinfection by both viruses was detected in 5.1% of boars. Phylogenetic analysis revealed that 134 PKoV isolates belonged to diverse lineages within the species Aichivirus C; however, one strain (WKoV16CN-8627) clustered with bovine kobuvirus (Aichivirus B). Forty-seven PAstVs belonged to lineage PAstV4, and only one strain (WAst17JN-10931) was a novel addition to lineage PAstV2. The two viruses were more prevalent in boars weighing ≤ 60 kg than in boars weighing > 61 kg.

An outbreak of cat vomiting was observed in an animal shelter. Testing for known enteric feline pathogens did not identify a causative agent. Viral metagenomics on four mini pools of feces from cases and controls housed in the same area revealed the presence of feline astrovirus in all pools. Also found with fewer reads in one pool each were rotavirus I, carnivore bocaparvovirus 3, norovirus (NoV) GVI, and a novel dependovirus. The genome of the highly prevalent astrovirus was sequenced and classified into mamastrovirus species two, also known as feline astrovirus. Real-time RT-PCR on longitudinally acquired fecal samples from 11 sick cases showed 10 (91%) to be shedding astrovirus for as long as 19 days. Affected cats were sick for an average of 9.8 days, with a median of 2.5 days (range = 1–31 days). Unaffected control cats housed in the same areas during the outbreak showed five out of nine (56%) to also be shedding astrovirus. Feline fecal samples collected from the same animal shelter ~1 year before (n = 8) and after (n = 10) showed none to be shedding astrovirus, indicating that this virus was temporarily associated with the vomiting outbreak and is not part of the commensal virome for cats in this shelter. Together with the absence of highly prevalent known pathogens, our results support a role for feline astrovirus infection, as well as significant asymptomatic shedding, in an outbreak of contagious feline vomiting.

Porcine astrovirus (PAstV) is distributed worldwide and has been reported to cause diarrhea in pigs. PAstV belongs to the family Astroviridae and genus Mamastrovirus. PAstVs are divided into five diverse genotypes (PAstV1-PAstV5) on the basis of phylogenetic analysis of a part of the RNA-dependent RNA polymerase (RdRp) gene and the capsid gene. However, knowledge regarding the clinical significance and molecular characteristics of PAstV in Haryana, India, is limited. In this study, we investigated the presence of PAstV by RT-PCR of the partial RdRp gene in 110 rectal swabs collected from diarrheic pigs in different parts of Haryana, India. Of these, 35 samples (31.8%) tested positive for PAstV, with the highest positivity observed among weaning piglets 3 to 9 weeks of age (47.7%, 21/44), followed by fattening pigs 9 to 24 weeks of age (28.5%, 8/28). Phylogenetic analysis of the partial RdRp gene revealed circulation of four different genotypes (PAstV1, PAstV2, PAstV4, and PAstV5) in Haryana, with PAstV1 being the predominant genotype. To the best of our knowledge, this is the first report of the presence of PAstV1 and PAstV5 in the pig population of India. The PAstV sequences revealed high genetic variability and genetic heterogeneity in a relatively confined area.

Background: Astroviruses (AstVs) are common pathogens of a wide range of animal hosts, including mammals and avians, causing gastrointestinal diseases, mainly gastroenteritis and diarrhea. They prompt a significant health problem in newborns and young children and economic losses in the poultry sector and mink farms. Recent studies revealed a growing number of bat species carrying astroviruses with a noticeable prevalence and diversity. Here, we demonstrate the first detection of bat astroviruses (BtAstVs) circulating in the population of insectivorous bats in the territory of Poland. Results: Genetically diverse BtAstVs (n = 18) were found with a varying degree of bat species specificity in five out of 15 bat species in Poland previously recognized as BtAstV hosts. Astroviral RNA was found in 12 out of 98 (12.2%, 95% CI 7.1&ndash, 20.2) bat intestines, six bat kidneys (6.1%, 95% CI 2.8&ndash, 12.7) and two bat livers (2.0%, 95% CI 0.4&ndash, 7.1). Deep sequencing of the astroviral RNA-dependent RNA polymerase (RdRp) region revealed co-infections in five single bat individuals with highly distinct astrovirus strains. Conclusions: The detection of highly distinct bat astroviruses in Polish bats favors virus recombination and the generation of novel divergent AstVs and creates a potential risk of virus transmission to domestic animals and humans in the country. These findings provide a new insight into molecular epidemiology, prevalence of astroviruses in European bat populations and the risk of interspecies transmission to other animals including humans.

Astroviruses are common pathogens of the human gastrointestinal tract, but they have been recently identified from cases of fatal meningoencephalitis. Astrovirus VA1 is the most frequently detected astrovirus genotype from cases of human encephalitis, but the prevalence of neutralizing antibodies to VA1 in human sera is unknown. We developed a focus reduction neutralization assay (FRNT) for VA1 and measured the seroprevalence of neutralizing antibodies from two cohorts of adult and pediatric serum samples: (i) an age-stratified cohort from St. Louis, MO, collected from 2007 to 2008 and (ii) a cohort from the Peruvian Amazonian River Basin collected in the late 1990s. In the St. Louis cohort, the lowest seropositivity rate was in children 1 year of age (6.9%), rising to 63.3% by ages 9 to 12, and 76.3% of adults ≥20 years were positive. The Peruvian Amazon cohort showed similar seropositivity rates across all ages, with individuals under age 20 having a rate of 75%, while 78.2% of adults ≥20 years were seropositive. In addition, we also identified the presence neutralizing antibodies to VA1 from commercial lots of intravenous immunoglobulin (IVIG). Our results demonstrate that a majority of humans are exposed to VA1 by adulthood, with the majority of infections occurring between 2 and 9 years of age. In addition, our results indicate that VA1 has been circulating in two geographically and socioeconomically divergent study cohorts over the past 20 years. Nonetheless, a significant proportion of the human population lacks neutralizing immunity and remains at risk for acute infection. IMPORTANCE Astroviruses are human pathogens with emerging disease associations, including the recent recognition of their capacity to cause meningoencephalitis. Astrovirus VA1 is the most commonly identified astrovirus genotype from cases of human encephalitis, but it is unknown what percentage of the human population has neutralizing antibodies to VA1. We found that 76.3 to 78.2% of adult humans ≥20 years of age in two geographically and socioeconomically distinct cohorts are seropositive for VA1, with the majority of infections occurring between 2 and 9 years of age. These results demonstrate that VA1 has been circulating in human populations over the past 2 decades and that most humans develop neutralizing antibodies against this virus by adulthood. However, a subset of humans lack evidence of neutralizing antibodies and are at risk for diseases caused by VA1, including encephalitis.

International audience; Enteroviruses (Picornaviridae) and astroviruses (Astroviridae) cause various diseases in humans and animals, including in non-human primates (NHPs). Some enteroviruses and astroviruses detected in NHPs are genetically related to those infecting humans, indicating the occurrence of interspecies transmissions. In this study, we screened 200 fecal samples of 56 free-ranging mandrills (Mandrillus sphinx) by nested reverse transcription-PCR with primers targeting the VP1 and RdRp genes, to evaluate the diversity of enterovirus and astrovirus infection, respectively, and the associated zoonotic risk. Overall, ten samples from six mandrills were enterovirus-positive (5%), and three samples from three mandrills were astrovirus-positive (1.5%). This is the first evidence of astrovirus infection in mandrills. Phylogenetic analyses based on the VP1 sequences revealed that all ten enterovirus sequences were part of the species Enterovirus J, suggesting low zoonotic risk. Phylogenetic analysis of the three astrovirus sequences showed that they all belonged to the Mamastrovirus genus. Two astrovirus sequences were highly divergent from all human astrovirus sequences (63.4-73% nucleotide identity), while one sequence (AstV-5) suggested cross-species transmission from humans to mandrills. Additional studies are needed to better characterize the identified astroviruses and to confirm whether mandrills are host of astroviruses than can be transmitted to humans.

Astroviruses, isolated from numerous avian and mammalian species including humans, are commonly associated with enteritis and encephalitis. Two astroviruses have previously been identified in cats, and while definitive evidence is lacking, an association with enteritis is suggested. Using metagenomic next-generation sequencing of viral nucleic acids from faecal samples, we identified two novel feline astroviruses termed Feline astrovirus 3 and 4. These viruses were isolated from healthy shelter-housed kittens (Feline astrovirus 3, 6448 bp) and from a kitten with diarrhoea that was co-infected with Feline parvovirus (Feline astrovirus 4, 6549 bp). Both novel astroviruses shared a genome arrangement of three open reading frames (ORFs) comparable to that of other astroviruses. Phylogenetic analysis of the concatenated ORFs, ORF1a, ORF1b and capsid protein revealed that both viruses were phylogenetically distinct from other feline astroviruses, although their precise evolutionary history could not be accurately determined due to a lack of resolution at key nodes. Large-scale molecular surveillance studies of healthy and diseased cats are needed to determine the pathogenicity of feline astroviruses as single virus infections or in co-infections with other enteric viruses.

Astroviruses (AstVs) are widely distributed and are associated with gastroenteritis in human and animals. The knowledge of the genetic diversity and epidemiology of AstVs in Africa is limited. This study aimed to characterize astroviruses in asymptomatic smallholder piglets in Kenya and Uganda. Twenty-four samples were randomly selected from a total of 446 piglets aged below 6 months that were initially collected for rotavirus study and sequenced for whole genome analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of seven strains with complete (or near complete) AstV genome revealed variable nucleotide and amino acid sequence identities with known porcine astrovirus (PoAstV) strains. The U083 and K321 strains had nucleotide sequence identities ranging from 66.4 to 75.4% with the known PoAstV2 strains, U460 strain had nucleotide sequence identities of 57.0 to 65.1% regarding the known PoAstV3, and K062, K366, K451, and K456 strains had nucleotide sequence identities of 63.5 to 80% with the known PoAstV4 strains. The low sequence identities (&lt, 90%) indicate that novel genotypes of PoAstVs are circulating in the study area. Recombination analysis using whole genomes revealed evidence of multiple recombination events in PoAstV4, suggesting that recombination might have contributed to the observed genetic diversity. Linear antigen epitope prediction and a comparative analysis of capsid protein of our field strains identified potential candidate epitopes that could help in the design of immuno-diagnostic tools and a subunit vaccine. These findings provide new insights into the molecular epidemiology of porcine astroviruses in East Africa.

Astroviruses (AstVs) are emerging, potentially zoonotic pathogens, associated with enteric and neurological signs in various animal species. In pigs, five lineages have been identified, porcine AstVs (PoAstVs) 1-5. We now report PoAstV5-associated atrophic enteritis in a colostrum-deprived (CD) pig and a similar disease in field cases of enteritis. Four newborn CD piglets were housed under biosafety level 2 conditions and monitored for the presence of PoAstV RNA in rectal swabs and tissues. PoAstV4 RNA was detected in low numbers (0.9-5.3 log10 genomic copies/rectal swab) in rectal swabs from all of these pigs while all animals were negative for PoAstV1, PoAstV2, PoAstV3 and PoAstV5. At 19 and 20 days of age, high levels of PoAstV5 RNA (7.4-7.5 log10 genomic copies/rectal swab) were detected in one pig, which had developed enteritis. At necropsy 1 day after the first evidence of PoAstV5 shedding, this pig was strongly positive for PoAstV5 RNA in most tissues with strong immunolabelling of PoAstV5 in enterocytes. Gene sequencing confirmed PoAstV5 infection. A retrospective investigation of PoAstVs 3, 4 and 5 in archival tissues from field cases of post-weaning enteritis identified high PoAstV4 or PoAstV5 antigen levels in intestinal tissues. These pigs were often concurrently infected with porcine rotavirus. Our findings suggest that PoAstV5 may contribute to the pathogenesis of enteritis in young pigs.

Astroviruses (AstVs) cause disease in a wide variety of species. Porcine AstVs are highly genetically diverse and conventionally assigned to five genetic lineages (PoAstV1-5). Due to the increasing evidence that porcine astrovirus type 3 (PoAstV3) is a cause of encephalomyelitis in swine and to elucidate important ecologic characteristics, the infection dynamics and environmental distribution of PoAstV3 were investigated in a herd with PoAstV3-associated neurologic disease. Over a 22 week period, the frequency of PoAstV3 fecal shedding varied by pig and age. The peak detection by RT-qPCR of PoAstV3 on fecal swabs (95%, 61 of 64) occurred at 3 weeks of age. The lowest frequency of detection was at 21 weeks of age (4%, 2 of 47), however, the frequency increased to 41% (19 of 46) at the final sampling time point (25 weeks of age). Viremia was rare (0.9%: 4 of 433). Detection in oral fluid was consistent with 75% to 100% of samples positive at each time point. Pens and feeders also had a high rate of detection with a majority of samples positive at a majority of sampling time points. Based on the data presented, PoAstV3 can be consistently detected in the environment with a majority of pigs being infected and a subset intermittently shedding the virus in feces out to 25 weeks of age. These findings suggest the importance of as-yet unidentified risk factors associated with the development of PoAstV3-associated polioencephalomyelitis.

Human astroviruses (HAstVs) were first identified in 1975 and can be classified into three clades: classic HAstVs (HAstV 1-8), MLB (MLB1-3) and VA (VA1-5), with MLB and VA were newly identified. Recombination and a high mutation rate make HAstV as one of the rapidly evolving infectious agents. This study reported a novel identified recombinant human astrovirus (Y/1-CHN) and its long existence in two immunocompromised patients with diarrhea following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The identified Yu/1-CHN genome contains 6801 base pairs encoding three open reading frames, with ORF1a best hit to the HAstV1 (Pune strain, 97 % nucleotide identity), while ORF1b and ORF2 best hit to HAstV-5 (DL30 strain, 99 % nucleotide identity). Possible recombination breakpoint was predicted to be located in the boundary of ORF1a and ORF1b. Different quasispecies were found in the host, and the dN/dS ratios of the S and P domains were determined to be 1.189 and 1.444, respectively, suggesting a positive selection existed. Fecal samples collected in different clinical phases from the two patients were all positive for Yu/1-CHN, suggesting a long existence of the virus in the host. It was indicated that immunocompromised patients may a reservoir for astrovirus, their excreta should be monitored even after discharge from hospital.

Sapovirus (SaV) and astrovirus (AstV) increasingly are recognized as cause of acute viral gastroenteritis (AGE). We evaluated the real-time RT-PCR assays RIDA®GENE SaV and viral stool panel II (RGN RT-PCR) for detection of SaV, AstV, adenovirus (AdV) F40/41 and rotavirus (RoV) in clinical stool samples (n = 69). Results were compared with reference singleplex RT-PCRs. The sensitivity for SaV, AstV and RoV are 100%, the specificity ranges from 98.1% to 100%. In 10 out of 11 AdV (all types) samples, the RGN RT-PCR for AdV F40/41 displayed negative results. Retrospectively, 196 stool specimens from adult patients previously tested negative for norovirus (NoV) were analyzed. In about 10% of NoV-negative stool samples, AdV (n = 9), RoV (n = 6), AstV (n = 3) or SaV (n = 3) were found. The RGN RT-PCR assays are useful for detection of enteric viruses other than NoV. This study emphasizes the need for further testing of NoV-negative stool samples in patients with AGE.

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%).

ObjectiveTo estimate the number of deaths from foodborne disease in the UK from 11 key pathogens.DesignFour different models were developed using data from a range of sources. These included enhanced surveillance, outbreaks, death certificates and hospital episode statistics data. For each model, median estimates were produced with 95% credible intervals (CrI). The results from the different models were compared.ResultsThe estimates for foodborne deaths for each pathogen from the different models were consistent, with CrIs largely overlapping. Based on the preferred model for each pathogen, foodborne norovirus is estimated to cause 56 deaths per year (95% CrI 32 to 92), foodborne Salmonella 33 deaths (95% CrI 7 to 159), foodborne Listeria monocytogenes 26 deaths (95% CrI 24 to 28), foodborne Clostridium perfringens 25 deaths (95% CrI 1 to 163) and foodborne Campylobacter 21 deaths (95% CrI 8 to 47). The considerable overlap in the CrIs means it is not possible to make any firm conclusions on ranking. Most of these deaths occur in those aged over 75 years. Foodborne deaths from Shigella, Cryptosporidium, Giardia, adenovirus, astrovirus and rotavirus are all rare.ConclusionsWe estimate that there are 180 deaths per year in the UK (95% CrI 113 to 359) caused by foodborne disease based on these 11 pathogens. While this is a small fraction of the estimated 2.4 million cases of foodborne illness per year it still illustrates the potential severity of these illnesses demonstrating the importance in continuing efforts to reduce these infections.

Neonatal diarrhea in piglets may cause major losses in affected pig herds. The present study used random high-throughput RNA sequencing (metagenomic next generation sequencing, mNGS) to investigate the virome of sows from a farm with persistent neonatal piglet diarrhea in comparison to two control farms without diarrhea problems. A variety of known swine gastrointestinal viruses was detected in the control farms as well as in the problem farm (Mamastrovirus, Enterovirus, Picobirnavirus, Posavirus 1, Kobuvirus, Proprismacovirus). A substantial increase in normalized viral read counts was observed in the affected farm compared to the control farms. The increase was attributable to a single viral species in each of the sampled sows (porcine astrovirus 4 and Posavirus 1). The complete genomes of a porcine astrovirus 4 and two co-infecting Posavirus 1 were de novo assembled and characterized. The 6734 nt single-stranded RNA genome of porcine astrovirus 4 (PoAstV-4) strain Belgium/2019 contains three overlapping open reading frames (nonstructural protein 1ab, nonstructural protein 1a, capsid protein). Posavirus 1 strains Belgium/01/2019 and Belgium/02/2019 have a 9814 nt single-stranded positive-sense RNA genome encoding a single open reading frame (polyprotein precursor) containing the five expected Picornavirales-conserved protein domains. The study highlights the potential of mNGS workflows to study unexplained neonatal diarrhea in piglets and contributes to the scarce availability of both PoAstV-4 and Posavirus-1 whole genome sequences from Western Europe.

Novel human astroviruses (HAstV) were discovered 10 years ago and have been associated with fatal cases of central nervous system infections. Their role in gastroenteritis is controversial, as they have been identified in symptomatic and asymptomatic subjects. The aim of the study was to investigate novel HAstV in a gastroenteritis case-control study including a pediatric population in Spain over a one-year period. We included stool samples from patients with gastroenteritis and negative results for viruses screened by routine diagnostics, and stool samples of control subjects who sought for a routine medical consultation. All samples were screened by real-time RT-PCR assays for novel HAstV. An additional screening for rotavirus, norovirus GI, GII, sapovirus, classic HAstV and adenovirus was also performed for the control group. Overall, 23/363 stool samples from case patients (6.3%) and 8/199 stool samples from control patients (4%) were positive for ≥1 novel HAstV. MLB1 was predominant (64.5% of positives). Seasonality was observed for the case group (p = 0.015), but not the control group (p = 0.95). No difference was observed in the prevalence of novel HAstV between the case and control groups (OR 1.78, 95% CI 0.68–5.45; p = 0.30). Nevertheless, MLB genome copy numbers/ml of fecal suspension was significantly higher in the control group than in the case group (p = 0.008). In our study, we identified a lack of association between novel HAstV and gastroenteritis in the studied population, which could indicate a potential role of reservoir for children, especially given the higher viral load observed in the asymptomatic group for some of them.

Background Recent recognition of invasive astrovirus infections, including encephalitis and viremia in humans, have highlighted the need for effective anti-astrovirus therapeutics. However, there is a paucity of data regarding the in vitro activity of broad-spectrum RNA antivirals against astroviruses, including ribavirin and favipiravir. Objectives We quantified the EC50 values for ribavirin and favipiravir against two human astrovirus strains, astrovirus VA1 (VA1) and human astrovirus 4 (HAstV4). Study Design Caco-2 cells were infected with VA1 or HAstV4 in the presence of ribavirin or favipiravir (dose range 0.1–1000 μM), and the cells were maintained in media containing the drugs for 72 h. Viral RNA was extracted and quantified by qRT-PCR. As a surrogate for cytotoxicity, cellular adenosine triphosphate (ATP) from each drug treatment was also measured. Results VA1 replication was inhibited 10-100-fold by both ribavirin (EC50 = 154 μM) and favipiravir (EC50 = 246 μM). In contrast, ribavirin inhibited HAstV4 replication (EC50 = 268 μM) but favipiravir only reduced replication by 44% at the highest dose. Mild reductions in ATP (17–31%) was only observed at the highest concentration of ribavirin (1000 μM) and no significant decrease in ATP was detected for any concentration of favipiravir. Conclusions Ribavirin inhibited both human astrovirus species and favipiravir was only active against VA1. In the future, the in vivo efficacy of these drugs could be tested with development of an animal model of human astrovirus infection.

Background Porcine astroviruses (PAstVs) are common in pigs worldwide. There are five distinct lineages with each lineage representing a different ancestral origin. Recently, multiple reports have demonstrated the evidence of extra-intestinal infection of PAstVs, but little is known about viremia. Results In this study, a total of 532 fecal samples and 120 serum samples from healthy pigs were collected and tested from 2013 to 2015 in Guangxi province, China; of these 300/532 (56.4%) and 7/120 (5.8%) of fecal samples tested positive for PAstVs, respectively. Our study revealed that there was wide genetic diversity and high prevalence of the virus in the pig population. All five of the known PAstVs genotypes (1–5) prevailed in the pig population of Guangxi province and were distributed in all age groups of pigs, from suckling piglets to sows, with PAstV2 (47.7%), PAstV1 (26.2%) and PAstV5 (21.5%) seen predominantly. Phylogenetic analysis of partial ORF1b and partial capsid sequences from fecal and serum samples revealed that they were divided into the five lineages. Among these genotypes, based on partial ORF2 genes sequencing 23 strains were grouped as PAstV1, including 6 serum-derived strains, and were regarded as the causative agents of viremia in pigs. Conclusions Due to the information regarding the types of PAstV in blood is limit. This is the first report for the presence of PAstV1 in blood and PAstV3 in the feces of nursery pigs of China. This study provides a reference for understanding the prevalence and genetic evolution of PAstVs in pigs in Guangxi province, China. It also provides a new perspective for understanding of the extra-intestinal infection of PAstVs in pigs.

The Sichuan takin inhabits the bamboo forests in the Eastern Himalayas and is considered as a national treasure of China with the highest legal protection and conservation status considered as vulnerable according to The IUCN Red List of Threatened Species. In this study, fecal samples of 71 Sichuan takins were pooled and deep sequenced. Among the 103,553 viral sequences, 21,961 were assigned to mammalian viruses. De novo assembly revealed genomes of an enterovirus and an astrovirus and contigs of circoviruses and genogroup I picobirnaviruses. Complete genome sequencing and phylogenetic analysis showed that Sichuan takin enterovirus is a novel serotype/genotype of the species Enterovirus G, with evidence of recombination. Sichuan takin astrovirus is a new subtype of bovine astrovirus, probably belonging to a new genogroup in the genus Mamastrovirus. Further studies will reveal whether these viruses can also be found in Mishmi takin and Shaanxi takin and their pathogenic potentials.

Background Human astroviruses have increasingly been identified and are important agents of diarrheal disease, especially in infants and young children. This article presents the real-time polymerase chain reaction TaqMan assay for the detection and quantification of human astrovirus for clinical fecal samples collected from hospitalized children with acute gastroenteritis in Piedmont (northern Italy) from December 2014 to November 2015. Methods A total of 159 fecal specimens from hospitalized children with acute gastroenteritis, previously screened for rotavirus, adenovirus, norovirus, human parechovirus, salivirus and sapovirus, were tested for human astrovirus. Results The most commonly detected virus was norovirus GII (33.8%), followed by rotavirus (21.3%), sapovirus (10.9%), human parechovirus (8%), norovirus GI (6.7%), adenovirus (1%) and salivirus (0.52%). A total of 30 of 159 (18.87%) episodes of acute gastroenteritis were associated with human astrovirus genomic detection. Conclusions Our data showed that the detection rate of astrovirus in diarrheal children (18.87%) was higher than observed in other countries, where they were reported in diarrheal children in 10.3%-0.8% of patients and a mean incidence worldwide of 11%. Our data showed that the detection rate of astrovirus in pediatric gastroenteritis was greater than previously reported in Italy.

Background Until today, classic human astroviruses have not been associated with central nervous system infections in immunocompetent patients. Case presentation A 16-month-old Caucasian girl presented with repetitive generalized seizures with a 4-day history of watery diarrhea, which had already gradually improved. Initially, the prolonged seizures ceased after systemic midazolam treatment and were thought to be fever associated. However, her mental status remained altered, and after seizure recurrence, she was transferred to our pediatric intensive care unit. Seizure control was achieved by a combination of high-dose levetiracetam and phenobarbital, but she remained unconscious. An electroencephalogram at this time revealed generalized high voltage theta activity. All laboratory analyses, including extended blood and cerebrospinal fluid analyses, and a brain magnetic resonance imaging were normal. On day 4, the child gradually became conscious, but was very agitated and not able to walk. Since an electroencephalogram at this time still revealed generalized high voltage theta activity, although she had not received sedative medications for 72 hours, she was diagnosed as having encephalopathy. At that time, results of diagnostic testing of the stool sample were positive for classic astrovirus infection, and we decided to analyze the initially obtained cerebrospinal fluid for astrovirus as well. Cerebrospinal fluid was also found positive for human astrovirus. Sequencing analysis revealed a classic astrovirus genotype 1 with exactly the same nucleotide sequence as in the feces. Clinically, the child gradually improved and was discharged on day 9. Conclusions Whereas the new human astrovirus subtypes have been recently associated with central nervous system infection, this is the first case of encephalitis in an immunocompetent child due to classic human astrovirus. Considering that classic human astroviruses are the third most common etiological agents of viral gastroenteritis in children, we believe that human astroviruses as causative agents for central nervous system infections should be considered more often, especially in children and infants with preceding gastroenteritis.

Although classical human astroviruses (HAstV) are known to be a leading cause of viral gastroenteritis, the pathogenesis and clinical manifestations of novel HAstV remain largely unknown. There is mounting evidence that, in contrast to classical astroviruses, novel HAstV exhibit tropism for the upper respiratory tract. This one-year period prevalence screened all available clinical nasopharyngeal swab samples collected from pediatric patients aged ≤5 years for novel and classical HAstV using real-time reverse transcription polymerase chain reaction. A total of 205 samples were tested; two novel HAstV cases were detected for a prevalence of 1.3%, with viral loads suggesting active upper respiratory tract replication. No classical HAstV was detected.

Classical human astroviruses (HAstV) are agents of nonbacterial acute gastroenteritis (AGE), being predominant among children. There are only a few studies reporting HAstV loads in samples from patients with AGE, data are even scarcer regarding asymptomatic patients. The aim of this study was to evaluate the occurrence and estimate the viral load of HAstV and to perform molecular characterization of positive samples obtained from children, up to 6 years old, with and without AGE. One fecal sample was obtained from each of the 250 children enrolled in the study, from May 2014 to April 2015. Real-time reverse transcription-polymerase chain reaction (RT-qPCR TaqMan) was performed, followed by a conventional RT-PCR directed to ORF2, region C, of the positive samples. Then, these amplicons were sequenced and a phylogenetic analysis was performed to determine the HAstV-1 lineages. A global positivity index of 3.2% (8 of 250) was observed for HAstV with a similar frequency (50%) in both symptomatic and asymptomatic group. Viral loads ranged from 2.8 × 105 to 1.6 × 1011 genome copy/mL Four samples were characterized as HAstV-1, lineage 1a and two as HAstV-4, lineage 4c. Our findings show similar HAstV positivity rates for children with and without AGE, providing evidence of HAstV-1a and HAstV-4c lineage cocirculation in the Central West region of Brazil. Data contributes to the molecular epidemiology of these agents in the region.

Background Viral gastroenteritides are among the causes of higher morbidity and mortality in the childhood period, especially in infants. Although viral-induced diarrheal diseases are important problems in Erzurum, there have been no studies on the molecular prevalence of viral gastroenteritis agents in this region's children. The aim of the prospective study is to determine the molecular prevalence of the most commonly seen viral etiologic agents and their coinfection rates in children under 5 years of age with gastroenteritis in Erzurum, Turkey. Methods Stool samples from 375 children between 0 and 5 years of age who suffered from acute diarrhea were investigated for the presence of Rotavirus, Norovirus, Astrovirus and Adenovirus by reverse transcriptase polymerase chain reaction, followed by conventional PCR techniques. The presence of Rotavirus, Norovirus, Astrovirus and Adenovirus in the specimens was detected by amplification of the VP6, RdRp, ORF-1b and Hexon regions, respectively. Stool samples were also investigated non-viral enteropathogens by conventional techniques. Results At least one viral pathogen was detected in 59.2% of the stool samples. Rotavirus was the most frequently observed agent (32.3%), followed by Norovirus (20.3%), Adenovirus (9.6%) and Astrovirus (5.6%). All specimens were negative for bacterial pathogens. Twenty seven (7.2%) specimens were positive for intestinal helminths and protozoan. A total of 39 coinfection (10.4%) including 38 dual and 1 triple were detected. The most frequent coinfections were observed between Norovirus plus Rotavirus and Norovirus plus Adenovirus. Conclusions Single infections or coinfections of the enteropathogenic viruses occur at a significant rate in Erzurum's children. This study draws attention to the necessity of taking account of multiple viral infections in studies on combined vaccines and the treatment of gastroenteritis.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions., Author summary Human astroviruses (HAstV) are understudied positive-strand RNA viruses that typically cause gastroenteritis mostly in children and the elderly, but more recent studies also implicate them in neurological disease in immunocompromised patients. To better understand these viruses, a physiologically relevant cell culture model that supports growth of all clades of HAstV would be highly beneficial. Herein, we demonstrated robust infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts from different patients and intestinal regions, making HIE a valuable model to study HAstV biology. Using this system, we identify for the first time that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes. Analysis of the antiviral host response to infection demonstrated that HIE respond to infection with a type I and III interferon response. This response reduced HAstV replication and when blocked resulted in increased infection. Establishment of the HIE system for HAstV research lays the foundation for future basic and translational discoveries.