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Background Viruses are major etiological agents of childhood gastroenteritis. In recent years, several molecular platforms for the detection of viral enteric pathogens have become available. Objective/study design We evaluated the performance of three multiplex platforms including Biofire’s Gastrointestinal Panel (FilmArray), Luminex xTAG ® Gastrointestinal Pathogen Panel (GPP), and the TaqMan Array Card (TAC) for the detection of five gastroenteritis viruses using a coded panel of 300 archived stool samples. Results The FilmArray detected a virus in 199 (96.1%) and the TAC in 172 (83.1%) of the 207 samples (187 samples positive for a single virus and 20 samples positive for more than one virus) whereas the GPP detected a virus in 100 (78.7%) of the 127 (97 positive for one virus and three positive for more than one virus) samples. Overall the clinical accuracy was highest for the FilmArray (98%) followed by TAC (97.2%) and GPP (96.9%). The sensitivity of the FilmArray, GPP and TAC platforms was highest for rotavirus (100%, 95.8%, and 89.6%, respectively) and lowest for adenovirus type 40/41 (97.4%, 57.9% and 68.4%). The specificity of the three platforms ranged from 95.6% (rotavirus) to 99.6% (norovirus/sapovirus) for the FilmArray, 99.6% (norovirus) to 100% (rotavirus/adenovirus) for GPP, and 98.9% (astrovirus) to 100% (rotavirus/sapovirus) for TAC. Conclusion The FilmArray demonstrated the best analytical performance followed by TAC. In recent years, the availability of multi-enteric molecular testing platforms has increased significantly and our data highlight the strengths and weaknesses of these platforms.

Background The family Caliciviridae consists of a genetically diverse group of RNA viruses that infect a wide range of host species including noroviruses and sapoviruses which cause acute gastroenteritis in humans. Typing of these viruses relies on sequence-based approaches, and therefore there is a need for rapid and accurate web-based typing tools. Objective To develop and evaluate a web-based tool for rapid and accurate genotyping of noroviruses and sapoviruses. Methods The Human Calicivirus Typing (HuCaT) tool uses a set of curated reference sequences that are compared to query sequences using a k-mer (DNA substring) based algorithm. Outputs include alignments and phylogenetic trees of the 12 top matching reference sequences for each query. Results The HuCaT tool was validated with a set of 1310 norovirus and 239 sapovirus sequences covering all known human norovirus and sapovirus genotypes. HuCaT tool assigned genotypes to all queries with 100 % accuracy and was much faster (17 s) than BLAST (150 s) or phylogenetic analyses approaches. Conclusions The web-based HuCaT tool supports rapid and accurate genotyping of human noroviruses and sapoviruses.

Highlights • We validated genogroup-specific one-step conventional RT-PCR assays (PC assays) for sequence-based dual typing of GI and GII norovirus strains. • The PC assays use a combination of oligonucleotide primers that target a genomic region spanning the 3’-end of ORF1 and 5’end of ORF2 of GI and GII noroviruses. • The PC assays are sensitive (5 to 50 copies/rx) and detect all currently identified norovirus P-types and capsid genotypes from different geographic regions. • The PC assays have been successfully implemented by CaliciNet USA and CaliciNet China., Background Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. Objective Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. Results The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010–2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. Conclusion The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.

Background Despite calicivirus food-borne outbreaks posing major public health concern worldwide, little information is at present available about the impact of caliciviruses mixed infection in an oyster-associated outbreak in China. Objectives To investigate the clinical and epidemiologic characteristics of an oyster-associated calicivirus outbreak initiated by a food festival in Shanghai, China, in April 2014. Study design Molecular epidemiological studies based on nucleotide sequencing and phylogenetic analysis of calicivirus strains from patients. Results A total of 65 of the 78 (83%) cases from this outbreak were associated with raw oyster consumption. Forty-six calicivirus strains were identified from 25 stool specimens with norovirus (NoV) GII.4 Sydney_2012, GII.13, GI.2, GI.5 and sapovirus (SaV) GI.2 being predominant genotypes and with a prevalence of triple-, double- and single-infection being 20%, 48% and 28%, respectively. Meanwhile, 13 putative NoV recombinants were indicated by the phylogenetic inconsistency between capsid and polymerase genotype, mainly including GII.Pe/GII.4 Sydney_2012. Molecular epidemiological investigation showed possible multiple route transmission in the field. The clinical and epidemiologic characteristics of the mixed point-source calicivirus outbreak also conformed to Kaplan’s criteria. Conclusions This is the first reported oyster-associated calicivirus outbreak with a high prevalence of mixed infection during a food festival described in China. Our investigation underscores the importance of early surveillance and comprehensive etiologic identification of mixed point-source outbreaks and the need for reliable standards of monitoring oysters to prevent and control calicivirus food-borne outbreaks in China.

Background Human sapovirus (SaV) causes sporadic and endemic acute gastroenteritis worldwide. However, little is known about the relationship between the mode of transmission and genetic characteristics of SaV. Objective To investigate the molecular characteristics of SaV-associated acute gastroenteritis among sporadic cases, foodborne, and nonfoodborne outbreaks. Study design We performed a systematic review of publications and genetic analysis of SaV in fecal specimens from 98 outpatients with acute gastroenteritis, 32 stool samples from 8 foodborne outbreaks, and 63 stool samples from 23 nonfoodborne outbreaks in Miyagi Prefecture, Japan from 1993 and between 2004 and 2020. Reverse transcription polymerase chain reaction (RT-PCR) was employed for the detection of SaV, and the partial capsid gene was sequenced for genotyping and phylogenetic analysis. Results The overall detection rate of SaV in sporadic cases, foodborne, and nonfoodborne outbreaks was 5.8, 1.7, and 4.3%, respectively. Genotypic analysis revealed GI.1 to be the predominant genotype in sporadic cases (31.5%) and nonfoodborne outbreaks (52.1%), whereas it was not detected in foodborne outbreaks. Some outbreaks occurred following sporadic cases with the same genotype. Conclusions The distribution of SaV genotypes was different between foodborne outbreaks and other settings. The effective SaV infection control may differ depending on the genomic characteristics.

Background Human sapovirus (SaV) is a causative agent of acute gastroenteritis. Recently, SaV detection has been increasing worldwide due to the emerging SaV genotype I.2. However, SaV infection has not been reported in the Philippines. Objectives To evaluate the prevalence and genetic diversity of SaV in hospitalized children aged less than 5 years with acute gastroenteritis. Study design Stool samples were collected from children with acute gastroenteritis at three hospitals in the Philippines from June 2012 to August 2013. SaV was detected by reverse transcription real-time PCR, and the polymerase and capsid gene sequences were analyzed. Full genome sequencing and recombination analysis were performed on possible recombinant viruses. Results SaV was detected in 7.0% of the tested stool samples (29/417). In 10 SaV-positive cases, other viruses were also detected, including rotavirus ( n =6), norovirus ( n =2), and human astrovirus ( n =2). Four known SaV genotypes (GI.1 [7], GI.2 [2], GII.1 [12], and GV [2]) and one novel recombinant ( n =3) were identified by polymerase and capsid gene sequence analysis. Full genome sequencing revealed that the 5ʹ nontranslated region (NTR) and nonstructural protein region of the novel recombinant were closely related to the GII.1 Bristol/98/UK variant, whereas the structural protein region and 3ʹ NTR were closely related to the GII.4 Kumamoto6/Mar2003/JPN variant. Discussion and conclusions SaV was regularly detected in hospitalized children due to acute gastroenteritis during the study period. A novel recombinant, SaV GII.1/GII.4, was identified in three cases at two different study sites.

Background Human caliciviruses ( Norovirus and Sapovirus ) are important acute gastroenteritis agents. The Norovirus (NoV) disease is usually self-limited; however, prolonged viral excretion and complications have been reported, mainly in immunosuppressed individuals. Objectives In this prospective study, we have monitored allogeneic stem cell transplant (ASCT) patients for human calicivirus infection. Study design Ten ASCT patients were monitored for NoV and sapoviruses (SaV) infection, for a period of five months to a maximum of one year. Prolonged NoV excretion and long term viral RNA in the blood were assessed by multiplex RT-PCR targeting region C of the viral capsid. Secretor status of the patients was determined by enzyme immunoassay using Ulex Europaeus agglutinin. Partial genomic sequencing and phylogenetic analysis were performed to characterize the viral genotypes. Results NoV was detected in six out of ten patients (60%). Prolonged viral excretion in feces (mean of 61.6 days) and long term presence of NoV RNA in the sera (mean of 33.6 days) of the patients were observed. SaV was not detected in any of the samples. All patients had diarrhea, vomiting and fever during NoV positivity. All NoV-positive samples were characterized as GI.3 NoV. Three Nov-infected patients presented with acute intestinal graft versus host disease. Conclusions This study brings important information on NoV course of infection in ASCT patients. It also provides evidence for long term viral RNA in the blood highlighting the importance of the inclusion of NoV screening in the routine testing performed before transplantation and during follow-up of these patients.

Sapovirus has been accepted as a major cause of acute gastroenteritis worldwide. It can affect all age groups, ranging from young adults to the elderly, while little is known about the epidemiological patterns and genetic characteristics of sapovirus infections in China.To investigate the prevalence of sapovirus infections among adult outpatients suffering from acute gastroenteritis in Shanghai, China.From April 2011 to March 2013, fecal specimens from 1125 adult outpatients (≥16 years of age) with acute gastroenteritis were collected. Reverse transcription polymerase chain reaction (RT-PCR) was employed for detection of sapovirus, and 5' end of capsid gene were sequenced for genotyping and phylogenetic analysis.The overall occurrence of sapovirus infection in adult outpatients was 3.73% (42 in 1125) through the two-year surveillance period, and sapovirus diarrhea is more common in spring and winter. The highest sapovirus positive rate was observed in adults of ≥56 years old, and statistically significant relationship was observed when compared with other age groups (p0.05). Only three genotypes were detected, whereas GI.2 was proved to be the predominant strain, occupying 78.57% (33 in 42) of all strains, followed by GIV, GI.1 and GII.3.Sapovirus was commonly found in adults with acute gastroenteritis in Shanghai, China, while no specific seasonal variation of sapovirus diarrhea could be distinguished. GI.2 strains established themselves in a short time span as the predominant genotype in Shanghai, China.

This deposit is composed by a publication in which the IGC's authors have had the role of collaboration (it's a collaboration publication). This type of deposit in ARCA is in restrictedAccess (it can't be in open access to the public), and can only be accessed by two ways: either by requesting a legal copy from the author (the email contact present in this deposit) or by visiting the following link: https://www.sciencedirect.com/science/article/pii/S1386653215001535?via%3Dihub#sec0040 This work can also be accessed by visiting the following link: http://repositorio.insa.pt/handle/10400.18/3513 This deposit is composed by the main article, and it hasn't any supplementary materials associated. This publication hasn't any creative commons license associated. Background Gastroenteritis is one of the most common infectious diseases in the military populations and can diminish operational effectiveness and impede force readiness. Objectives The present study investigates the cause and the source of an acute gastroenteritis outbreak that occurred during a military exercise of the Portuguese Army, in February 2013. Study Design A retrospective investigation was performed and stool samples, food items and water were screened for common foodborne bacteria and viruses, namely Norovirus GI, Norovirus GII, Astrovirus, Rotavirus, Adenovirus and Sapovirus. Results From the total of 160 soldiers that participated in the military exercise 20 developed gastroenteritis (attack rate of 12.5%). Symptoms were predominantly vomiting (n = 17, 85%) and diarrhoea (n = 9, 45%). The first cases occurred 24–48 h after drinking water from the creek, the plausible origin of the outbreak. The epidemic peak was registered 2 days after and the last cases 6 days after, upon returning to base. No pathogenic bacteria were found in stools however virological analysis revealed the presence of multiple enteropathogenic viruses, namely Norovirus GI (GI.3), Norovirus GII (GII.4 New Orleans 2009), Astrovirus and Sapovirus, as single or co-infections. Food and water samples were not tested for the presence of viruses due to exhaustion of samples on bacteriological analysis. Conclusions To the best of our knowledge this is the first report of a viral gastroenteritis outbreak among military personnel in the Portuguese Army. There are no funders and sponsors indicated explicitly in the document. info:eu-repo/semantics/publishedVersion

Background Noroviruses (NoVs) are important enteric pathogens that cause gastroenteritis worldwide. The first documented NoV outbreaks in South Africa (SA) were described in 1993. The current NoV prevalence and circulating genotypes are unknown. SA lacks NoV outbreak reporting systems and therefore the number and impact of NoV infections is underestimated. Objectives This study aimed to determine the prevalence and genetic diversity of NoV infections in hospitalised paediatric patients with gastroenteritis in SA during 2008. Study design Stool specimens referred for virological analysis from hospitalised children ≤13 years, with gastroenteritis, were screened for rotavirus, human adenovirus and human astrovirus by enzyme immunoassay and for NoV genogroup I (GI), II (GII) and sapovirus by real-time RT-PCR. NoV strains were genotyped, and variants identified, based on sequence and phylogenetic analyses of the 5′ end or the full length of the capsid gene, respectively. Results Rotavirus was the most prevalent virus detected in 24.2% (61/252) of specimens, followed by NoV in 14.3% (35/245) and adenovirus, astrovirus and sapovirus in 9.6%, 6.7% and 4% of specimens, respectively. NoVs were only detected in children ≤2 years. The GII NoVs (89%) predominated and eight types were identified with GII.4 (43%) detected most frequently. The emerging 2008 GII.4 variant represented 80% of the GII.4 strains. Conclusions A diverse range of NoV genotypes were identified in hospitalised children with gastroenteritis. The 2008 GII.4 variant was the most frequently detected strain in the study. This is the first report of NoV GII.4 viruses in SA.

Background Sapoviruses are known to cause gastroenteritis mainly in young children. Objectives To establish a collection of sapoviruses and to gain knowledge about the genetic diversity and epidemiology of sapoviruses circulating in children in Denmark. Study design During a 24-month period in 2005–2007 samples from 1104 children, aged 0–3 years, submitted for acute gastroenteritis diagnostics, were analysed for sapoviruses by real-time PCR. Genotyping of positive findings was carried out by sequencing part of the capsid gene, and in several cases also part of the polymerase gene. Results Sapoviruses were detected in stool samples from 97 children (9%), with the highest prevalence in the 7–18 months age group. Viruses from three genogroups and seven genotypes were found. Genotype I.1 was demonstrated in half of the positive samples and was observed throughout the study period. The less common types seemed to appear during shorter periods, often in succession of each other. Positive samples were detected throughout the study period. The only months, in both years studied, with high proportions of positive samples were September, November and February. Conclusions Sapoviruses were commonly found in children with gastroenteritis in Denmark. No clear seasonal pattern could be seen. Genotype I.1 was clearly the most common genotype found, but several other genotypes circulated during shorter periods.

No: 1752 Presentation at ESCV 2015: Oral 38 Salivirus Type 1 and Type 2 in patients with acute gastroenteritis, Germany S. Aldabbagh1,∗, I. Eckerle1, A. Muller2, E.L. Delwart3, A.M. Eis-Hubinger1 1 Institute of Virology, University of Bonn Medical Centre, Germany 2 Department of Neonatology and Pediatric Intensive Care, Childrens Hospital, University of Bonn Medical Centre, Germany 3 Blood Systems Research Institute, Department of Laboratory Medicine, University of California, USA Background: Salivirus (SaV-A) is a novel member of the family Picornaviridae and the only species within the novel picornaviral genus Salivirus. The virus was found in up to 9% of fecal samples from patients, mostly children, with gastroenteritis, with highest prevalences in Asian countries. Thus, the virus was suspected to cause acute gastroenteritis. Recently, a second type of SaV-A (SaVA2) was identified in a sewage sample from Thailand. Because no information is available on SaV-A infection in Western Europe, we analysed stool samples from patients with symptoms suspicious of acute viral gastroenteritis for SaV-A and evaluated the clinical course of SaV-A-positive individuals. Methods: A total of 3019 fecal samples from 1941 hospitalised patients with symptoms of acute gastroenteritis were analysed. Samples were collected during 2012–2013 at the University Hospital Bonn and were routinely screened by (RT-)PCR for human norovirus, sapovirus, astrovirus, rotavirus, and adenovirus. A SaVA real-time RT-PCR was designed targeting a conserved nucleotide sequence in the 5′-untranslated region. Positive results were verified by sequencing the viral capsid protein 1 gene allowing also typing of the virus andwhole genome sequencing. Medical records of SaV-A-infected patients were reviewed for clinical features and laboratory data. Results: SaV-A was detected in five unrelated patients, with viral concentrations ranging between 7.1×106 and 7.2×108 copies/g feces, in the absence of common gastroenteritis viruses. The viruses from four patients were classified as SaV-A1 while in one patient SaV-A2 was present. In none of the five patients SaV-A infection was considered as the major cause of the gastroenteritis symptoms. Conclusion: SaV-A infection can be detected in Germany,Western Europe, albeit at low levels. The detection of SaV-A2 in Europe suggests global spread of SaV-A2. Presence of SaV-A, even at high concentration, in a stool sample provides no conclusive evidence that SaV is the major cause of the patient’s gastroenteritis symptoms. http://dx.doi.org/10.1016/j.jcv.2015.07.048 Abstract No: 1526 Presentation at ESCV 2015: Oral 39 Disseminated adenovirus infection following allogeneic haemopoetic stem cell transplantation – A report on the use of brincidofovir and 10 year retrospective review of an adult transplant cohort I. Ramsay1,∗, C. Attwood2, D. Irish1, D. Lowe3, P.D. Griffiths1, Kyriacou Charalampia2 1 Department of Virology, Royal Free Hospital, London UK 2 Department of Haematology, Royal Free Hospital, London UK 3 Department of Immunology, Royal Free Hospital

s / Journal of Clinical Virology 70 (2015) S1–S126 S39 the samples were tested either by electron microscopy (EM) or norovirus PCR depending on the patient demographics. Since June 2014, all samples regardless of age have been tested for norovirus, rotavirus, sapovirus, astrovirus and enteric adenovirus by real-time PCR. We present the results of the first years testing using the PCR method across all age groups. In addition to the front line PCR a rotavirus genotyping service will also be introduced at the NVRL. Following the recent successful introduction of national vaccination programmes in many European countries, vaccination is likely to be included in the national infant immunisation schedule in Ireland in the near future. The genotyping data will determine the current circulating strains and will also monitor the genetic evolution post implementation of vaccination. Methods: An in-house real time PCR was developed and validated to detect the five main viral causes of gastroenteritis. A reliance on automation has led to a lean streamlined processwhich can test up to approximately 100 samples per day. The genotyping method was a WHO recommended method involving PCR of the VP7 and VP4 regions and gel analysis to determine the G and P genotype. This testing method will be supplemented by Sanger sequencing. Results: The PCR data collated over one year will be presented; thiswill show the seasonal trends of the five viruses across the year and also the prevalence in different age groups. Preliminary data of the circulating strains of rotavirus will also be presented. Conclusion: The introduction of the molecular detection of the main viruses causing gastroenteritis has resulted in a significant increase in the detection of viruses detected when compared with EM. As expected the predominant virus was norovirus in both adults and children. The next prevalent virus was rotavirus which mainly affects children but was also detected in adults to a lesser extent. Sapovirus and astrovirusweremorewidespread than anticipated and finally the enteric adenovirus was the least common virus. http://dx.doi.org/10.1016/j.jcv.2015.07.094