Aichi Virus Research Articles

Detection and molecular characterization of enteric viruses in children with acute gastroenteritis in Northern Italy

Enteric viral infections are a major concern for public health, and viral acute gastroenteritis is the principal cause of pediatric morbidity and mortality worldwide, mostly in developing countries. The purpose of this study was to determine the prevalence of different enteric viruses detected in a pediatric population with acute gastroenteritis symptoms, and to characterize the strains detected.Stools were collected from children, aged from 2 months to 15 years old, admitted to one of the main hospitals of Northern Italy, between November 2015 and October 2016. Stools were tested for nine enteric viruses (adenovirus, rotavirus A, norovirus, astrovirus, sapovirus, enterovirus, parechovirus, bocavirus and aichivirus) by molecular methods. Furthermore, rotavirus, norovirus and adenovirus were deeply characterized by nucleotide sequencing and phylogenetic analysis.A total of 151 out of 510 (29.6%) stools analyzed resulted positive for at least one of the enteric virus investigated. The most common virus detected was rotavirus A (53/151, 35.1%), followed by norovirus (39/151, 25.8%), adenovirus (35/151, 23.1%), sapovirus (9/151, 6%), enterovirus (5/151, 3.3%), astrovirus (5/151, 3.3%), parechovirus (4/151, 2.6%) and bocavirus (1/151, 0.6%). Aichi virus was not detected in any sample. Co-infections were detected in 12 out of 510 faecal samples (2.3%).These data improved the knowledge of the enteric viruses circulating in children in Northern Italy. In fact, besides rotavirus, adenovirus and norovirus, several viruses circulated across the whole year in the pediatric population object of this study. The introduction of specific viral diagnosis in our clinical setting will improve patient care by reducing unnecessary use of antibiotics addressing the right etiologic diagnosis.

An overview of 20years of studies on the prevalence of human enteric viruses in shellfish from Galicia, Spain.

Galicia (NW Spain) has 1490km of coastline, and its particular topography, characterized by the presence of fiord-like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro-organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20years is presented, including not only the data for the well-known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas.

Virological Quality of Irrigation Water Sources and Pepper Mild Mottle Virus and Tobacco Mosaic Virus as Index of Pathogenic Virus Contamination Level.

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

Detection of Aichi virus genotype B in two lines of wastewater treatment processes.

Enteric viruses are released in important quantities into the environment where they can persist for a very long time. At very low doses, they can cause human gastroenteritis, and are responsible for a substantial number of waterborne diseases. The aims of this study were multiple: firstly, to study the circulation of Aichi viruses (AiV) in wastewater sampled at the scale of a pilot wastewater treatment plant; secondly, to evaluate the performance of two wastewater treatment procedures, as natural oxidizing lagoons and rotating Biodisks, concerning the AiV removal; and finally, to determine the different type of AiV genotype found during this study. Hence, the pilot wastewater treatment plant is principally irrigated by the wastewater of three neighbouring clinics. Wastewater samples were collected during 2011 from the two lines of biological treatment procedures. AiV detection in wastewater were achieved using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, and the identification of AiV genotype was realized by the direct sequencing of PCR products. The result revealed that AiV strains were identified in 50% (n=51) of the wastewater samples. A significant increase of the AiV detection frequency was registered from upstream to downstream of the five ponds constituting the natural oxidizing lagoon process, and at the exit of the rotating Biodisks procedure. All detected AiV strains showed the highest nucleotide sequence identity to genotype B that has been recently observed in patients in Asia. This finding represented the first Tunisian survey that revealed and mentioned the first detection of AiV genotype B in sewage and by the same argued for a noticeable resistance or survival of this type of virus in the two lines of treatment considered.

Ficus septica plant extracts for treating Dengue virus in vitro.

Dengue virus types 1-4 (DENV-1-4) are positive-strand RNA viruses with an envelope that belongs to the Flaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. In order to discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an enveloped virus such as DENV.

Linear antimicrobial peptides with activity against herpes simplex virus 1 and Aichi virus.

Viruses are the major cause of disease and mortality worldwide. Nowadays there are treatments based on antivirals or prophylaxis with vaccines. However, the rising number of reports of viral resistance to current antivirals and the emergence of new types of virus has concerned the scientific community. In this scenario, the search for alternative treatments has led scientists to the discovery of antimicrobial peptides (AMPs) derived from many different sources. Since some of them have shown antiviral activities, here we challenged 10 synthetic peptides from different animal and plant sources against, herpes simplex virus 1 (HSV-1), and Aichi virus. Among them, the highlight was Pa-MAP from the polar fish Pleuronectes americanus, which caused around 90% of inhibition of the HSV with a selectivity index of 5 and a virucidal mechanism of action. Moreover, LL-37 from human neutrophils showed 96% of inhibition against the Aichi virus, showing a selectivity index of 3.4. The other evaluated peptides did not show significant antiviral activity. In conclusion, the present study demonstrated that Pa-MAP seems to be a reliable candidate for a possible alternative drug to treat HSV-1 infections. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 108: 1-6, 2017.

Quantification and Genotyping of Aichi Virus 1 in Water Samples in the Kathmandu Valley, Nepal.

Aichi virus 1 genomes were detected by quantitative PCR in groundwater from shallow dug (10/22) and tube wells (1/15), river water (14/14), and sewage (1/1), with the maximum concentration of 4.0×109 copies/l. Nucleotide sequencing analysis demonstrated the prevalence of genotype B in the virus positive samples.

Aichivirus in Children with Diarrhea in Northern Italy

Objective: Since its discovery, Aichivirus (AiV) A has been detected, with an incidence of 0.9–4.1%, primarily when studying outbreaks of diarrhea in children or young adults. In this paper, we report the first detection of AiV in Piedmont, Italy, in pediatric patients. Methods: A total of 159 fecal specimens (from 96 males and 63 females) previously screened for rotaviruses, adenoviruses, noroviruses, human parechoviruses, saliviruses, and sapoviruses were collected from infants and children with acute gastroenteritis. Results: The most commonly detected virus was norovirus GII (33.80%), fol lowed by rotavirus (21.30%), astrovirus (18.87%), boca virus (13.92%), sapovirus (10.90%), parechovirus (8%), norovirus GI (6.70%), adenovirus (1%), and salivirus (0.52%). Real-time polymerase chain reaction detected AiV A in 1 (0.62%) case subjects. AiV A was detected in monoinfection only in January. Conclusions: Our results indicate that AiV may be associated with a limited number of diarrhea cases in pediatric patients.

Low prevalence of Aichi virus in molluscan shellfish samples from Galicia (NW Spain).

The aim of this study was to detect and quantify Aichi virus (AiV) in shellfish from three estuaries in Galicia, the main producer of molluscs in Europe. A total of 249 shellfish samples were analysed using a reverse transcription-quantitative PCR procedure. AiV was detected in 15 of 249 (6·02%) samples. Ría de Ares-Betanzos showed the highest prevalence (11·1%), followed by Ría do Burgo (3·7%) and Ría de Vigo, (2·56%). AiV quantifications ranged from nonquantifiable (under the limit of quantification of the method) to 6·9×103 RNAc per g DT, with a mean value of 1·9×102 RNAc per g DT. Results obtained indicated that the prevalence of this enteric virus in the studied area is considerably lower than those of other enteric viruses, such as Norovirus, Sapovirus, HAV or HEV. This is the first study that detects the presence of AiV in shellfish from authorized harvesting areas in Spain. Further studies with clinical samples are needed to determine the potential risk of AiV for human health in Galicia.

The Molecular Basis of Aichi Virus 3A Protein Activation of Phosphatidylinositol 4 Kinase IIIβ, PI4KB, through ACBD3

Phosphatidylinositol 4-kinase III beta (PI4KIIIβ) is an essential enzyme in mediating membrane transport, and plays key roles in facilitating viral infection. Many pathogenic positive-sense single-stranded RNA viruses activate PI4KIIIβ to generate phosphatidylinositol 4-phosphate (PI4P)-enriched organelles for viral replication. The molecular basis for PI4KIIIβ activation during viral infection has remained largely unclear. We describe the biochemical reconstitution and characterization of the complex of PI4KIIIβ with the Golgi protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3) and Aichi virus 3A protein on membranes. We find that 3A directly activates PI4KIIIβ, and this activation is sensitized by ACBD3. The interfaces between PI4KIIIβ-ACBD3 and ACBD3-3A were mapped with hydrogen-deuterium exchange mass spectrometry (HDX-MS). Determination of the crystal structure of the ACBD3 GOLDdomain revealed a unique N terminus that mediates the interaction with 3A. Rationally designed complex-disrupting mutations in both ACBD3 and PI4KIIIβ completely abrogated the sensitization of 3A activation by ACBD3.

Analysis of Aichi virus and Saffold virus association with pediatric acute gastroenteritis

BackgroundAichi virus (AiV) and Saffold virus (SAFV) have been reported in children with acute gastroenteritis and respiratory disease worldwide; however, their causative role in acute gastroenteritis remains ambiguous. ObjectivesTo assess the clinical association of AiV and SAFV with acute gastroenteritis in the pediatric population. Study designA case-control study involving 461 paired stool samples from pediatric cases with diarrhea and healthy controls was conducted in China. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to screen AiV and SAFV. ResultsIn the 461 paired samples, AiV and SAFV were more prevalent among asymptomatic children than children with acute gastroenteritis (0.87% vs. 0.43% and 2.8% vs. 1.5%, respectively), with no significant differences between groups (p=0.142 and p=0.478, respectively). Cox regression model analysis revealed no correlation between AiV (odds ratio, OR=2.24; 95% confidence interval, CI, 0.76–6.54) or SAFV infection (OR=1.36; 95% CI, 0.86–2.15) and diarrhea. High viral loads were found in both AiV- and SAFV-positive groups, with no significant difference in viral load between the groups (p=0.507 and p=0.677, respectively). No other known enteric pathogens were found in the AiV-positive samples but common in SAFV-positive cases. Phylogenetic analysis revealed that all 6 AiV subjects clustered with genotype B. All 7 SAFV-positive cases and 8 of 13 SAFV-positive controls were genotyped successfully; the genotypes identified included SAFV-1, SAFV-2 SAFV-3, and SAFV-6. ConclusionOur study revealed no association of these viruses in acute gastroenteritis in children. These viruses may have the ability to replicate in humans; however, the infections are usually asymptomatic.

Structure of Aichi Virus 1 and Its Empty Particle: Clues to Kobuvirus Genome Release Mechanism.

ABSTRACTAichi virus 1 (AiV-1) is a human pathogen from the Kobuvirus genus of the Picornaviridae family. Worldwide, 80 to 95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. We present here the structure of the AiV-1 virion determined to a resolution of 2.1 Å using X-ray crystallography. The surface loop puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue BC loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 Å using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at least in vitro, and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release.IMPORTANCE Aichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80 to 95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from the Picornaviridae family. We also determined that AiV-1 capsids form compact shells even after genome release. Therefore, AiV-1 genome release requires large localized and probably reversible reorganization of the capsid.

Structural biology: A picornavirus unlike the others.

Technological breakthroughs in cryo-electron microscopy are allowing the capture of virus structures, leading to advances in the field of viral microbiology almost daily. An innovative structure–function study on Aichi virus reveals that novel entry mechanisms, yet undiscovered, may exist for non-enveloped viruses.

Survival of hepatitis A virus and Aichi virus in cranberry-based juices at refrigeration (4 °C)

Viral foodborne illness continues to be a health-concern globally, with numerous fruit and juice outbreaks of Hepatitis A virus (HAV) reported worldwide. Aichi virus (AiV) is an emerging pathogen with limited epidemiological data. Both, HAV and AiV are resistant to low pH and can survive under adverse environmental conditions leading to transmission ease. The objective of this study was to evaluate the survival of HAV and AiV in commercially-available cranberry-based juices (Cranberry juice cocktail, CJ and a 100% juice with cranberry, MJ) over 21 days at refrigeration (4 °C). Equal volumes of juice was mixed with each virus individually (final titer of 6 log PFU/mL) and stored at refrigeration over 21 days. At each time interval, the inoculated juices were serially diluted in cell culture media and infectious virus survival was determined by standard plaque assays. Each experiment was carried out in duplicate and replicated thrice. Reductions of 0.72 ± 0.06 (after day 1) to 2.3 ± 0.18 log PFU/mL (after day 21) and 0.63 ± 0.02 (after day 1) to 1.84 ± 0.14 log PFU/mL (after day 21) were obtained for AiV with MJ and CJ, respectively. Reductions ranging from 0.67 ± 0.03 (after day 1) to 1.09 ± 0.1 log PFU/mL (after day 21) and 0.93 ± 0.27 (after day1) to 1.49 ± 0.18 log PFU/mL (after day 21) were obtained for HAV at refrigeration in MJ and CJ, respectively. HAV showed greater survival than AiV in these juices over refrigerated storage. These results provide survival data of HAV and AiV in cranberry-based juices that can be used in risk-modeling and risk assessment studies.

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.

Structure of human Aichi virus and implications for receptor binding

Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries1,2. The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life3,4. There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

Virus Reduction during Advanced Bardenpho and Conventional Wastewater Treatment Processes.

The present study investigated wastewater treatment for the removal of 11 different virus types (pepper mild mottle virus; Aichi virus; genogroup I, II, and IV noroviruses; enterovirus; sapovirus; group-A rotavirus; adenovirus; and JC and BK polyomaviruses) by two wastewater treatment facilities utilizing advanced Bardenpho technology and compared the results with conventional treatment processes. To our knowledge, this is the first study comparing full-scale treatment processes that all received sewage influent from the same region. The incidence of viruses in wastewater was assessed with respect to absolute abundance, occurrence, and reduction in monthly samples collected throughout a 12 month period in southern Arizona. Samples were concentrated via an electronegative filter method and quantified using TaqMan-based quantitative polymerase chain reaction (qPCR). Results suggest that Plant D, utilizing an advanced Bardenpho process as secondary treatment, effectively reduced pathogenic viruses better than facilities using conventional processes. However, the absence of cell-culture assays did not allow an accurate assessment of infective viruses. On the basis of these data, the Aichi virus is suggested as a conservative viral marker for adequate wastewater treatment, as it most often showed the best correlation coefficients to viral pathogens, was always detected at higher concentrations, and may overestimate the potential virus risk.

Detection and characterization of Aichi virus 1 in pediatric patients with diarrhea in Thailand.

Kobuvirus is a newly discovered virus that belongs to the Kobuvirus genus in Picornaviridae family, which comprised of three species including Aichivirus A, Aichivirus B, and Aichivirus C. The kobuvirus isolated from human has been classified as Aichi virus 1 and belongs to Aichivirus A species. The present study aimed to assess the epidemiology and to perform molecular characterization of Aichi virus 1 in children admitted to hospitals with acute gastroenteritis in Chiang Mai, Thailand. A total of 923 fecal specimens collected from January, 2011 to December, 2013 were screened for the presence of Aichi virus 1 by RT semi-nested PCR. Out of 923 fecal specimens tested, Aichi virus 1 was detected with the prevalence of 2.6% (24/923). Of these, 0.3% (3/923) was genotype A and 2.3% (21/923) were genotype B. It is interesting to note that the genotype A showed the nucleotide sequence closely related to the Aichi virus reference strain isolated from sewage in Tunisia, while genotype B was most closely related to other human Aichi virus B reference strains. The results suggest that Aichi virus 1 of both genotypes A and B are circulating in pediatric patients in Thailand. J. Med. Virol. 89:234-238, 2017. © 2016 Wiley Periodicals, Inc.

Detection of pepper mild mottle virus as an indicator for drinking water quality in Hanoi, Vietnam, in large volume of water after household treatment

ABSTRACTThe aims of this study were to examine the removal of bacteria and viruses by household point-of-use (POU) treatments and to apply a previously developed large-volume virus concentration method (∼20 L). First, the removal of microbes by household POU treatment was investigated in the laboratory. Second, the prevalence of viruses in drinking water sources for households and the removal efficiency of microbes by POU treatments in two suburban communities in Hanoi, Vietnam, were investigated. Indigenous pepper mild mottle virus (PMMoV) was used as the main target together with adenovirus, Aichi virus, enterovirus, F-specific bacteriophage genogroup 1, and Escherichia coli to investigate the removal efficiency of household treatments. The results from laboratory and field survey were compared. From the laboratory study, ceramic membranes were not effective for removing viruses and bacteria from water; pathogen reduction was less than 1.5 log10. By contrast, reverse osmosis (RO) devices reduced microbes by 3 to > 5 log10. In a field study, PMMoV was found to be the most prevalent waterborne virus. Household sand filtration was ineffective for removing E. coli, total coliforms and PMMoV; the reduction was less than 1 order of magnitude. Boiling the water and then filtering it with a ceramic membrane reduced E. coli by 3 orders of magnitude, but this was not effective for removing PMMoV. RO filtration was one of the promising methods for removing E. coli, total coliforms and PMMoV to below their detection limits in most of the samples studied. The removal of E. coli, total coliforms and PMMoV was >2.3, >4 and >3 log10, respectively. The laboratory results of virus removal efficiency by POU devices agreed with the field study. Due to the prevalence and characteristics of PMMoV, it is a strong candidate for an indigenous indicator to investigate the viral removal efficiency of household POU treatments.

A non-enteric adenovirus A12 gastroenteritis outbreak in Rio de Janeiro, Brazil.

A gastroenteritis outbreak that occurred in 2013 in a low-income community in Rio de Janeiro was investigated for the presence of enteric viruses, including species A rotavirus (RVA), norovirus (NoV), astrovirus (HAstV), bocavirus (HBoV), aichivirus (AiV), and adenovirus (HAdV). Five of nine stool samples (83%) from patients were positive for HAdV, and no other enteric viruses were detected. Polymerase chain reaction products were sequenced and subjected to phylogenetic analysis, which revealed four strains and one strain of non-enteric HAdV-A12 and HAdV-F41, respectively. The HAdV-A12 nucleotide sequences shared 100% nucleotide similarity. Viral load was assessed using a TaqMan real-time PCR assay. Stool samples that were positive for HAdV-A12 had high viral loads (mean 1.9 X 107 DNA copies/g stool). All four patients with HAdV-A12 were < 25 months of age and had symptoms of fever and diarrhoea. Evaluation of enteric virus outbreaks allows the characterisation of novel or unique diarrhoea-associated viruses in regions where RVA vaccination is routinely performed.