Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities.

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 103 and 9.5 × 103 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis.

An emaciated subadult free-ranging California sea lion (Csl or Zalophus californianus) died following stranding with lesions similar to 11 other stranded animals characterized by chronic disseminated granulomatous inflammation with necrotizing steatitis and vasculitis, involving visceral adipose tissues in the thoracic and peritoneal cavities. Histologically, affected tissues had extensive accumulations of macrophages with perivascular lymphocytes, plasma cells, and fewer neutrophils. Using viral metagenomics on a mesenteric lymph node six mammalian viruses were identified consisting of novel parvovirus, polyomavirus, rotavirus, anellovirus, and previously described Csl adenovirus 1 and Csl bocavirus 4. The causal or contributory role of these viruses to the gross and histologic lesions of this sea lion remains to be determined.

Coinfection of parvovirus and astrovirus in gout-affected goslings.

Outbreaks of gosling gout have occurred in China since 2017 and caused a considerable economic impact on the poultry industry. While gosling astrovirus (GoAstV) is believed to be the main causal pathogen of gout, the full-blown disease of gout cannot be well reproduced by infecting the goslings with GoAstV, suggesting the possibility of other infectious agents being involved with the development of gosling gout. To assess other possible infectious agents, we collected tissues from gout-affected goslings in 12 goose farms in China, followed by PCR detection of GoAstV, goose reovirus (GRV), goose parvovirus (GPV), fowl adenovirus (FAdV), goose circovirus (GcoV), Tembusu virus (TMUV) and goose haemorrhagic polyomavirus (GHPV). Our data showed that all gout-affected goslings carried both of GoAstV and GPV determined by PCRs, and this was further confirmed by fluorescence multiplex immunohistochemical staining, and phylogenetic analysis of ORF2 gene of GoAstV and VP3 gene of GPV. In addition to the haemorrhage in the kidney, liver, spleen and lung of the gout-affected goslings, histological examinations showed also extensive infiltration of heterophil myelocytes in the kidney, liver, spleen, bursa of Fabricius, thymus, lungs and pancreas. Our findings strongly suggest that coinfection of GoAstV and GPV increases the severity of gout. While this is the first study to report GPV in gout-affected goslings, further studies including infection model are warranted to investigate the role of GPV and its coinfection with GoAstV in the development of gosling gout.

Tropism, pathology, and transmission of equine parvovirus-hepatitis.

Equine parvovirus-hepatitis (EqPV-H) has recently been associated with cases of Theiler's disease, a form of fulminant hepatic necrosis in horses. To assess whether EqPV-H is the cause of Theiler's disease, we first demonstrated hepatotropism by PCR on tissues from acutely infected horses. We then experimentally inoculated horses with EqPV-H and 8 of 10 horses developed hepatitis. One horse showed clinical signs of liver failure. The onset of hepatitis was temporally associated with seroconversion and a decline in viremia. Liver histology and in situ hybridization showed lymphocytic infiltrates and necrotic EqPV-H-infected hepatocytes. We next investigated potential modes of transmission. Iatrogenic transmission via allogeneic stem cell therapy for orthopedic injuries was previously suggested in a case series of Theiler's disease, and was demonstrated here for the first time. Vertical transmission and mechanical vectoring by horse fly bites could not be demonstrated in this study, potentially due to limited sample size. We found EqPV-H shedding in oral and nasal secretions, and in feces. Importantly, we could demonstrate EqPV-H transmission via oral inoculation with viremic serum. Together, our findings provide additional information that EqPV-H is the likely cause of Theiler's disease and that transmission of EqPV-H occurs via both iatrogenic and natural routes.

Cutavirus: A newly discovered parvovirus on the rise.

Cutavirus is a new member of the Parvoviridae family. It was first discovered in 2016 through unbiased metagenomics performed on fecal samples collected from patients with diarrhea, and also in skin biopsies collected from patients with cutaneous T-cell lymphoma (CTCL, also known as mycosis fungoides). We have systematically reviewed the literature to describe the discovery, genomic organization, prevalence, and geographic distribution of cutavirus.

High-Throughput Sequencing-Based Investigation of Viruses in Human Cancers by Multienrichment Approach.

BACKGROUND: Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data. METHODS: In this study, we applied a diverse selection of presequencing enrichment methods targeting all major viral groups, to characterize the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing, we generated 710 datasets constituting 57 billion sequencing reads. RESULTS: Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveal a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained 1 or more viruses; however, no link between specific viruses and cancer types were found. CONCLUSIONS: Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.

Retrospective investigation and molecular characteristics of the recombinant Muscovy duck parvovirus circulating in Muscovy duck flocks in China.

The recombinant Muscovy duck parvovirus (rMDPV) has been recently characterized and identified in China. However, whether other additional rMDPV field isolates exist, and whether these strains possess common molecular characteristics, remain to be explored. In this retrospective study, two new rMDPV isolates, namely, JH06 and JH10, were identified through genome sequencing and recombination analysis. JH06, JH10, and four previously characterized rMDPV strains (SAAS-SHNH, ZW, FJM3, and PT97) underwent the same recombination events in a 1.1-kb region in their VP3 genes and displayed highly consistent beginning and ending breakpoints. JH06, JH10, SAAS-SHNH, ZW, and FJM3, but not PT97, underwent recombination in their P9 promoter regions. In both recombination events, the classical MDPV strain YY acted as the major parent, whereas the virulent strain DY16 and the vaccine strain SYG61v of goose parvovirus (GPV) served as the minor parents. The sequence alignments of inverted terminal repeats (ITRs) revealed that rMDPV strains shared higher identities (96.0%-97.2%) with classical MDPV strains than with GPV and contained typical one-nucleotide-pair deletions in the palindromic stems of their ITRs. This work elucidated the common molecular characteristics and differences of six rMDPV strains. The results of this work will facilitate the preparation of an efficacious vaccine for the protection of Muscovy ducks against rMDPV infection.

Kawasaki disease triggered by parvovirus infection: an atypical case report of two siblings.

BACKGROUND: There are reports of the familial occurrence of Kawasaki disease but only a few reports described Kawasaki disease in siblings. However, the familial cases were not simultaneous. In these patients the idea of infective agents as trigger must be considered. CASE PRESENTATION: We describe two siblings with atypical presentations of Kawasaki disease; the sister was first diagnosed as having parvovirus infection with anemia and the brother was diagnosed as having myocarditis. The first patient was a 9-month-old Caucasian girl with fever, conjunctivitis, rash, and pharyngitis, and later she had cervical adenopathy, diarrhea and vomiting, leukocytosis, and anemia, which were explained by positive immunoglobulin M against parvovirus. However, coronary artery lesions with aneurysms were documented at day 26 after fever onset. An infusion of intravenous immunoglobulin and high doses of steroids were not efficacious to resolve the coronary lesions. She was treated with anakinra, despite a laboratory test not showing inflammation, with prompt and progressive improvement of coronary lesions. Her 7-year-old Caucasian brother presented vomiting and fever at the same time as she was unwell, which spontaneously resolved after 4 days. Four days later, he again presented with fever with abdominal pain, associated with tachypnea, stasis at the pulmonary bases, tachycardia, gallop rhythm, hypotension, secondary anuria, and hepatomegaly. An echocardiogram revealed a severe hypokinesia, with a severe reduction of the ejection fraction (20%). He had an increase of immunoglobulin M anti-parvovirus, tested for the index case of his sister, confirming the suspicion of viral myocarditis. He received dopamine, dobutamine, furosemide plus steroids, with a progressive increase of the ejection fraction to 50%. However, evaluating his sister's history, the brother showed a myocardial dysfunction secondary to Kawasaki shock syndrome. CONCLUSIONS: We report on familial Kawasaki disease in two siblings which had the same infectious trigger (a documented parvovirus infection). The brother was diagnosed as having post-viral myocarditis. However, in view of the two different and simultaneous evolutions, the girl showed Kawasaki disease with late coronary artery lesions and aneurysms, whereas the brother showed Kawasaki shock syndrome with myocardial dysfunction. We stress the effectiveness of anakinra in non-responder Kawasaki disease and the efficacy on coronary aneurysms.

Detection of adenovirus, papillomavirus and parvovirus in Brazilian bats of the species Artibeus lituratus and Sturnira lilium.

Bats play a significant role in maintaining their ecosystems through pollination, dispersal of seeds, and control of insect populations, but they are also known to host many microorganisms and have been described as natural reservoirs for viruses with zoonotic potential. The diversity of viruses in these animals remains largely unknown, however, because studies are limited by species, location, virus target, or sample type. Therefore, the aim of this study was to detect fragments of viral genomes in bat samples. We performed high-throughput sequencing analysis and specific PCR and RT-PCR on pools of anal and oropharyngeal swabs from Artibeus lituratus and Sturnira lilium collected in southern Brazil. As a result, a member of the family Adenoviridae related to human adenovirus C was detected in anal swabs from S. lilium. In addition, we detected a papillomavirus in an anal swab from A. lituratus. Our analyses also allowed the detection of adenoviruses and parvoviruses in oropharyngeal swabs collected from A. lituratus. These results increase our knowledge about viral diversity and illustrate the importance of conducting virus surveillance in bats.

Rapid and sensitive detection of goose parvovirus and duck-origin novel goose parvovirus by recombinase polymerase amplification combined with a vertical flow visualization strip.

Goose parvovirus (GPV) is one of the most serious viral pathogens in goslings. Recently, a new pathogen to the Chinese mainland-duck-origin novel goose parvovirus (N-GPV)-was found to be 90.8-94.6% identical to the nucleotide sequence of GPV, and typically causes growth disorders and high infection rates in meat ducks. The spread of both of these viruses hinders the healthy development of the waterfowl breeding industry. In this study, recombinase polymerase amplification (RPA) was combined with a vertical flow (VF) visualization strip to develop a universal assay for the rapid detection of GPV and N-GPV. A set of specific primers and probes were designed to target the VP3 gene. Detection was possible at a constant temperature of 37 °C within 5-10 min. The assay successfully detected GPV and N-GPV with high-specificity and did not exhibit cross-reactivity with other waterfowl viruses and bacteria. The analytical sensitivity of the GPV-RPA-VF assay was 2 × 102 copies of GPV plasmid. Validation of the GPV-RPA-VF assay-using 60 samples from the field--confirmed 100% similarity between the results of GPV-RPA-VF and conventional qPCR. The results indicate that the GPV-RPA-VF assay was accurate, sensitive, and specific. This assay can be performed with minimal equipment and training to rapidly detect GPV and N-GPV during the early phase of an outbreak, especially when timely veterinary diagnoses are needed in the field and in rural areas.

Proceedings of the 2017 Viral Clearance Symposium, Session 4: Submission Strategies.

Appropriate performance of virus validation studies and testing of unprocessed bulk harvests for retrovirus particle count are procedures in the demonstration of an acceptable level of viral safety for cell-derived biotechnology products. Product-specific validation studies on virus reduction with two model viruses [usually murine leukemia virus (MuLV) and a parvovirus] performed in duplicate runs are standard for clinical trial applications. For the retroviral safety margin, a 6 log reduction is normally expected. Retroviral particle counts are measured traditionally by transmission electron microscopy (TEM) and are commonly performed at contract laboratories. These procedures are quite time-consuming and can be associated with significant costs. In particular, the time factor is a hurdle for companies that want to quickly bring their new products to the clinic. In this session, several strategies on how to lower time, cost, and workload in the evaluation of viral safety for early clinical trial applications, while still ensuring sufficient level of viral safety of the product, were presented. In addition, virus reduction strategies for molecules that do not have the standard antibody structure are presented. Also presented in this session is the feasibility of the use of retrovirus-like particle (RVLP) in the prevalidation of virus removal and the use of quantitative polymerase chain reaction (qPCR) as an alternative to infectivity assays in virus validation studies as well as its use as an alternative to quantitative TEM analysis for determining RVLP count in the bulk harvest of a perfusion bioreactor.LAY ABSTRACT: In this session, several strategies on how to lower time, cost, and workload in the evaluation of viral safety for early clinical trial applications of cell-derived biotechnology products, while still ensuring sufficient level of viral safety of the product, were presented. In addition, virus reduction strategies for molecules that do not have the standard antibody structure are presented. Also presented in this session is the feasibility of the use of retrovirus-like particle (RVLP) in the prevalidation of virus removal and the use of quantitative polymerase chain reaction (qPCR) as an alternative to infectivity assays in virus validation studies as well as its use as an alternative to quantitative TEM analysis for determining RVLP count in the bulk harvest of a perfusion bioreactor.

Effect of selected gastrointestinal parasites and viral agents on fecal S100A12 concentrations in puppies as a potential comparative model.

BACKGROUND: Previous data suggest that fecal S100A12 has clinical utility as a biomarker of chronic gastrointestinal inflammation (idiopathic inflammatory bowel disease) in both people and dogs, but the effect of gastrointestinal pathogens on fecal S100A12 concentrations is largely unknown. The role of S100A12 in parasite and viral infections is also difficult to study in traditional animal models due to the lack of S100A12 expression in rodents. Thus, the aim of this study was to evaluate fecal S100A12 concentrations in a cohort of puppies with intestinal parasites (Cystoisospora spp., Toxocara canis, Giardia sp.) and viral agents that are frequently encountered and known to cause gastrointestinal signs in dogs (coronavirus, parvovirus) as a comparative model. METHODS: Spot fecal samples were collected from 307 puppies [median age (range): 7 (4-13) weeks; 29 different breeds] in French breeding kennels, and fecal scores (semiquantitative system; scores 1-13) were assigned. Fecal samples were tested for Cystoisospora spp. (C. canis and C. ohioensis), Toxocara canis, Giardia sp., as well as canine coronavirus (CCV) and parvovirus (CPV). S100A12 concentrations were measured in all fecal samples using an in-house radioimmunoassay. Statistical analyses were performed using non-parametric 2-group or multiple-group comparisons, non-parametric correlation analysis, association testing between nominal variables, and construction of a multivariate mixed model. RESULTS: Fecal S100A12 concentrations ranged from < 24-14,363 ng/g. Univariate analysis only showed increased fecal S100A12 concentrations in dogs shedding Cystoisospora spp. (P = 0.0384) and in dogs infected with parvovirus (P = 0.0277), whereas dogs infected with coronavirus had decreased fecal S100A12 concentrations (P = 0.0345). However, shedding of any single enteropathogen did not affect fecal S100A12 concentrations in multivariate analysis (all P > 0.05) in this study. Only fecal score and breed size had an effect on fecal S100A12 concentrations in multivariate analysis (P < 0.0001). CONCLUSIONS: An infection with any single enteropathogen tested in this study is unlikely to alter fecal S100A12 concentrations, and these preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation.

Comparative genetic analysis and pathological characteristics of goose parvovirus isolated in Heilongjiang, China.

BACKGROUND: Goose parvovirus (GPV) causes acute enteritis, hepatitis, myocarditis and high morbidity and mortality in geese and ducks. GPV H strain was isolated from a Heilongjiang goose farm where the geese were showing signs of hemorrhage in the brain, liver, and intestinal tract. In this study, we explored the genetic diversity among waterfowl parvovirus isolates and the pathological characteristics of GPV H in Shaoxing ducklings. METHODS: The complete capsid protein (VP) and non-structural (NS) sequences of the isolated H strain were sequenced, and phylogenetic trees of VP and NS were constructed in MEGA version 5.05 using the neighbor-joining method. Three-day-old Shaoxing ducklings were inoculated with GPV and were euthanized at 1, 2, 4, 6, and 8 days post-inoculation (PI), and their organs were removed and collected. The organs of 6-day PI ducklings were fixed in formalin, embedded in paraffin, sectioned for histology, stained with HE and analyzed for pathological lesions. The distribution of the GPV H strain in the tissues of the inoculated ducklings was detected using the polymerase chain reaction (PCR) method. RESULTS: Genetic analysis of the NS and VP genes indicated that the H strain was closely related to strains circulating in China during 1999-2014, and the nucleic acid identity of those strains was 98%-99%. Classical symptoms were observed in the inoculated ducklings. GPV remained in many tissues and replicated in a majority of the tissues, leading to histopathological lesions in four tissues. CONCLUSIONS: We first reported the distribution and histopathological lesions of a Chinese strain of GPV in infected shaoxing ducklings. This H strain was moderate pathogenic for Shaoxing ducklings.

Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene.

Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1) showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, VP2, in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized.

Identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in Mexico

ABSTRACT This is the first report on circulating canine rotavirus in Mexico. Fifty samples from dogs with gastroenteritis were analyzed used polymerase chain reaction and reverse transcription polymerase chain reaction in order to identify parvovirus and rotavirus, respectively; 7% of dogs were infected with rotavirus exclusively, while 14% were co-infected with both rotavirus and parvovirus; clinical signs in co-infected dogs were more severe.

Differentiation of minute virus of mice and mouse parvovirus by high resolution melting curve analysis.

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/µL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV.

ENDEMIC INFECTION OF STRANDED SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) WITH NOVEL PARVOVIRUS, POLYOMAVIRUS, AND ADENOVIRUS.

Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened subspecies under federal law and a fully protected species under California law, US. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter polyomavirus 1 and the complete coding sequence of sea otter parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SSO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

Cosavirus, Salivirus and Bufavirus in Diarrheal Tunisian Infants.

Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), two picornaviruses, and Bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Tunisian infants. From October 2010 through March 2012, stool samples were collected from 203 children <5 years-old suffering from AGE and attending the Children's Hospital in Monastir, Tunisia. All samples were screened for CosV, SalV and BuV as well as for norovirus (NoV) and group A rotavirus (RVA) by molecular biology. Positive samples for the three screened viruses were also tested for astrovirus, sapovirus, adenovirus, and Aichi virus, then genotyped when technically feasible. During the study period, 11 (5.4%) samples were positive for one of the three investigated viruses: 2 (1.0%) CosV-A10, 7 (3.5%) SalV-A1 and 2 (1.0%) BuV-1, whereas 71 (35.0%) children were infected with NoV and 50 (24.6%) with RVA. No mixed infections involving the three viruses were found, but multiple infections with up to 4 classic enteric viruses were found in all cases. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain since all infected children also presented infections with several enteric viruses, suggesting here potential water-borne transmission. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.