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Background: Stool consistency is an important diagnostic criterion in both research and clinical medicine and is often used to define diarrheal disease., Methods: We examine the pediatric enteric virome across stool consistencies to evaluate differences in richness and community composition using fecal samples collected from children aged 0 to 5 years participating in a clinical trial in the Amhara region of Ethiopia. The consistency of each sample was graded according to the modified Bristol Stool Form Scale for children (mBSFS-C) before a portion of stool was preserved for viral metagenomic analysis. Stool samples were grouped into 29 pools according to stool consistency type. Differential abundance was determined using negative-binomial modeling., Results: Of 446 censused children who were eligible to participate, 317 presented for the study visit examination and 269 provided stool samples. The median age of children with stool samples was 36 months. Species richness was highest in watery-consistency stool and decreased as stool consistency became firmer (Spearman's r = - 0.45, p = 0.013). The greatest differential abundance comparing loose or watery to formed stool was for norovirus GII (7.64, 95% CI 5.8, 9.5) followed by aichivirus A (5.93, 95% CI 4.0, 7.89) and adeno-associated virus 2 (5.81, 95%CI 3.9, 7.7)., Conclusions: In conclusion, we documented a difference in pediatric enteric viromes according to mBSFS-C stool consistency category, both in species richness and composition.

Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of N = 12 complete chicken PBVs (ChPBV) segments (N = 5 of S1 and N = 7 of S2, Acc. Nos.: MH425579-90) were determined using viral metagenomic and RT-PCR techniques from a single cloacal sample of a diarrheic chicken. The identified ChPBV segments are unrelated to each other and distant from all of the currently known PBVs. In silico sequence analyses revealed the presence of conserved prokaryotic Shine-Dalgarno-like (SD-like) sequences upstream of the three presumed open reading frames (ORFs) of the S1 and a single presumed ORF of the S2 segments. According to the results of expression analyses in E. coli using 6xHis-tagged recombinant ChPBV segment 1 construct and Western blot these SD-like sequences are functional in vivo suggesting that S1 of study PBVs can contain three ORFs and supporting the bacteriophage-nature of PBVs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A divergent rotavirus I was detected using viral metagenomics in the feces of a cat with diarrhea. The eleven segments of rotavirus I strain Felis catus encoded non-structural and structural proteins with amino acid identities ranging from 25 to 79% to the only two currently sequenced members of that viral species both derived from canine feces. No other eukaryotic viral sequences nor bacterial and protozoan pathogens were detected in this fecal sample suggesting the involvement of rotavirus I in feline diarrhea.

Viral metagenomics of feces collected from 58 Peruvian children with unexplained diarrhea revealed several small circular ssDNA genomes. Two genomes related to sequences previously reported in feces from chimpanzees and other mammals and recently named smacoviruses were characterized and then detected by PCR in 1.7 % (1/58) and 19 % (11/58) of diarrheal samples, respectively. Another three genomes from a distinct small circular ssDNA viral group provisionally called pecoviruses encoded Cap and Rep proteins with <35 % identity to those in related genomes reported in human, seal, porcine and dromedary feces. Pecovirus DNA was detected in 15.5 % (9/58), 5.9 % (3/51) and 3 % (3/100) of fecal samples from unexplained diarrhea in Peru, Nicaragua and Chile, respectively. Feces containing these ssDNA genomes also contained known human enteric viral pathogens. The cellular origins of these circular ssDNA viruses, whether human cells, ingested plants, animals or fungal foods, or residents of the gut microbiome, are currently unknown.

In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1, KT880670) were identified by viral metagenomic and RT-PCR methods from a single specimen of a diarrheic chicken suffering from a total of eight picornavirus co-infections, in Hungary. The complete genomes of six picornaviruses were determined and their genomic and phylogenetic characteristics and UTR RNA structural models analyzed in details. Picornaviruses belonged to genera Sicinivirus (the first complete genome), Gallivirus, Tremovirus, Avisivirus and "Orivirus" (two potential genotypes). In addition, the unassigned phacoviruses were also detected in multiple samples of chickens in the USA. Multiple co-infections promote and facilitate the recombination and evolution of picornaviruses and eventually could contribute to the severity of the diarrhea in chicken, in one of the most important food sources of humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Viruses with small circular ssDNA genomes encoding a replication initiator protein can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two such viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCV-SL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing metagenomics datasets revealed closely related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cyclovirus and gemycircularvirus reported here include humans or other cellular sources in or on the human body remains to be determined., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A novel gyrovirus genome found in the feces of an adult with diarrhea is described. The genome shows the three expected main ORFs encoding a structural protein (VP1), nonstructural protein (VP2), and Apoptin protein (VP3), which shared identities of 41, 42, and 38 % with those of the most closely related gyrovirus proteins, respectively. Given the high divergence in its genome, this gyrovirus may be considered the prototype for a new viral species (GyV9) in the Gyrovirus genus. Because the closest relatives of this gyrovirus infect chicken, a possible dietary origin for the presence of this virus in human feces is discussed.

A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus.

Group A rotavirus is a major cause of severe gastroenteritis in children and young animals. During a retrospective analysis of samples collected from Paraguayan children under 5 years old with diarrhea, and previously negative for rotavirus and norovirus, we detected the presence of bovine rotavirus sequences by viral metagenomics. Nucleic acid was extracted direct from stool sample and determined to be G8P[1]. The genomic analyzes revealed that the strain presents an Artiodactyl-like genome (G8-P[1]-I2-R2-C2-M1-Ax-N2-T6-E12-H3) suggesting a direct animal-to-human transmission., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: A significant fraction of cases of diarrhea, a leading cause of childhood mortality worldwide, remain unexplained., Objectives: To identify viruses in unexplained cases of diarrhea using an unbiased metagenomics approach., Study Design: Viral nucleic acids were enriched from the feces from 48 cases of unexplained diarrhea from Burkina Faso, sequenced, and compared against all known viral genomes., Results: The full genome of a highly divergent astrovirus was sequenced in a sample co-infected with parechovirus 1. RT-PCR identified a single astrovirus infection in these 48 patients indicating a low prevalence. Human astrovirus-BF34 was most closely related to mamastrovirus species 8 and 9 also found in human with which it shared 62%, 74%, and 57% amino acid identities over its protease, RNA dependent RNA polymerase and capsid proteins, respectively., Conclusions: Burkina Faso astrovirus is proposed as prototype for a novel species in the genus Mamastrovirus, here tentatively called Mamastrovirus 20, representing the fifth human astrovirus species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Parvoviruses cause a variety of mild to severe symptoms or asymptomatic infections in humans and animals. During a viral metagenomic analysis of feces from children with acute diarrhea in Burkina Faso, we identified in decreasing prevalence nucleic acids from anelloviruses, dependoviruses, sapoviruses, enteroviruses, bocaviruses, noroviruses, adenoviruses, parechoviruses, rotaviruses, cosavirus, astroviruses, and hepatitis B virus. Sequences from a highly divergent parvovirus, provisionally called bufavirus, were also detected whose NS1 and VP1 proteins showed <39% and <31% identities to those of previously known parvoviruses. Four percent of the fecal samples were PCR positive for this new parvovirus, including a related bufavirus species showing only 72% identity in VP1. The high degree of genetic divergence of these related genomes from those of other parvoviruses indicates the presence of a proposed new Parvoviridae genus containing at least two species. Studies of the tropism and pathogenicity of these novel parvoviruses will be facilitated by the availability of their genome sequences.

Polyomaviruses are small circular DNA viruses associated with chronic infections and tumors in both human and animal hosts. Using an unbiased deep sequencing approach, we identified a novel, highly divergent polyomavirus, provisionally named MX polyomavirus (MXPyV), in stool samples from children. The ∼5.0 kB viral genome exhibits little overall homology (<46% amino acid identity) to known polyomaviruses, and, due to phylogenetic variation among its individual proteins, cannot be placed in any existing taxonomic group. PCR-based screening detected MXPyV in 28 of 834 (3.4%) fecal samples collected from California, Mexico, and Chile, and 1 of 136 (0.74%) of respiratory samples from Mexico, but not in blood or urine samples from immunocompromised patients. By quantitative PCR, the measured titers of MXPyV in human stool at 10% (weight/volume) were as high as 15,075 copies. No association was found between the presence of MXPyV and diarrhea, although girls were more likely to shed MXPyV in the stool than boys (p=0.012). In one child, viral shedding was observed in two stools obtained 91 days apart, raising the possibility of chronic infection by MXPyV. A multiple sequence alignment revealed that MXPyV is a closely related variant of the recently reported MWPyV and HPyV10 polyomaviruses. Further studies will be important to determine the association, if any, of MXPyV with disease in humans.

A molecular epidemiological study on common diarrheal viruses was conducted in Ho Chi Minh City, Vietnam between October 2002 and September 2003. Fecal samples were collected from 1,010 hospitalized children with acute gastroenteritis. Those samples were screened for groups A, B, and C rotavirus, adenovirus, genogroups I and II norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) by RT-multiplex PCR, and the positive specimens were characterized further by ELISA, nested PCR, or sequencing. Among the diarrheal viruses detected, group A rotavirus was the most common, with a proportion of 67.4%, whereas NoV GII, adenovirus, SaV, and HAstV were also found in 5.5, 3.2, 0.8, and 0.6%, respectively. It is noteworthy that the group C rotavirus was first reported in Vietnam, with a proportion of 0.5% in this study. Fifty-six of 1,010 (5.5%) samples were found positive with more than one viral agent, in which 25 samples contained both group A rotavirus and NoV GII. Group A rotavirus could be identified throughout year with the peaks in both the dry and rainy season, whereas other viruses prevailed mainly in the rainy season. G-typing for the group A rotavirus showed that genotype 1 was still the most prevailing (33.0%), but interestingly, serotype 9 was emergent and became the third most common rotavirus G-type in these samples (13.7%). The four most common G-P combinations globally, G1P[8], G2P[4], G3P[8], and G4P[8] were found in 46.8% of rotavirus-positive samples, and it is of interest that one unusual rotavirus G9P[19] strain was first detected in Vietnam. The majority of NoV strains belonged to GII/4, and SaV strains mainly clustered with the Manchester strain (GI/1). Twenty-seven out of 32 adenovirus strains were identified as serotype 41. All HAstVs belonged to genotype 1. The results indicated clearly the impact of viral agents causing gastroenteritis and the importance of vaccination against diarrhea in Vietnam.

Titanium dioxide (TiO2) that had been irradiated with visible light (VL) was demonstrated to inactivate rotavirus, astrovirus, and feline calicivirus (FCV). The virus titers were dramatically reduced after exposure for 24 hrs to the VL-catalytic TiO2. The addition of bovine serum albumin could protect the virus against inactivation by VL-catalytic TiO2 in a dose-dependent manner. This finding implied that the VL-catalytic TiO2 products might somehow interact initially with the viral proteins in the process of virus inactivation. Moreover, we showed partial degradation of the rotaviral dsRNA genome. This was more prominent when the virus was exposed to the VL-catalytic TiO2 treatment for at least 2 days. An attempt was made to elucidate the mechanism underlying the inactivation of the viruses. It was found that upon activation of TiO2 with VL by using a white fluorescent lamp, the reactive oxygen species such as superoxide anions (O2-) and hydroxyl radicals (*OH) were generated in a significant amount after stimulation for 8, 16, and 24 hrs. We therefore assume that virus inactivation by VL-catalytic TiO2 might occur through the generation of O2- and *OH followed by damage to the viral protein and genome. This is the first report, to the best of our knowledge, demonstrating the inactivation of rotavirus, astrovirus and FCV by the presence of TiO2 film under VL as well as describing its mechanism.

A novel reverse transcription-multiplex polymerase chain reaction (RT-multiplex PCR) assay that can detect enteroviruses, hepatitis A and E viruses and influenza A virus from various hosts (avian species, human, swine and horse) was developed. The identification of that group of viruses was performed with the mixture of four pairs of published specific primers (F1 and R1, P3 and P4, 2s and 2as, MMU42 and MMU43) for amplifying viral genomes and specifically generated four different amplicon sizes of 440, 267, 146 and 219 bp for enteroviruses, hepatitis A and E viruses and influenza A virus, respectively. A total of 276 fecal specimens (previously screened for rotavirus, adenovirus, norovirus, sapovirus and astrovirus-negative) from infants and children admitted into hospital with acute gastroenteritis in Ho Chi Minh city, Vietnam during October 2002 and September 2003 were collected and further tested for the presence of those viruses by RT-multiplex PCR. Enteroviruses were identified in 27 specimens and this represented 9.8%. No hepatitis A and E viruses and influenza A virus was found among these subjects. The sensitivity and specificity of RT-multiplex PCR were also assessed and demonstrated the strong validation against RT-monoplex PCR. Taken together, the findings clearly indicated that this novel RT-multiplex PCR is a simple and potential assay for rapid, sensitive, specific and cost-effective laboratory diagnosis to investigate molecular epidemiology of acute gastroenteritis caused by enteroviruses, hepatitis A and E viruses and influenza A virus. This report is the first, to our knowledge, detecting these kinds of viruses in diarrheal feces from infants and children in Vietnam.

Viral gastroenteritis is one of the most common illnesses in humans worldwide and it has a great impact on people. Recently, we reported three RT-multiplex PCR assays termed A, B and C that could detect three groups of diarrheal viruses; group A, B and C rotaviruses and adenovirus [Phan et al., J Med Virol 2004; 74:173-9]; norovirus GI and GII, sapovirus and astrovirus [Yan et al., J Virol Meth 2003; 114:37-44]; enteroviruses, hepatitis A and E viruses and influenza A virus [Phan et al., Arch Virol 2005; 1175-85], respectively. In the present study, we developed a novel protocol with a small volume of reaction mixture for RT and PCR products (only 8 microl and 11 microl, respectively) that can amplify genomes of target viruses simultaneously. A total of 100 fecal specimens from infants and children with acute gastroenteritis in Birobiclzhan city, Eastern Russia, were collected during November 2003 and March 2004 and tested for the presence of those viruses by the novel RT-multiplex PCR protocol. Group A rotavirus was the most prevalent (67%), followed by norovirus GII (4%), group C rotavirus (1%), and sapovirus (1%). Interestingly, one fecal specimen turned out to be positive for hepatitis A virus. The sensitivity and specificity of RT-multiplex PCR assays with a novel protocol demonstrated a strong validation against the previously published RT-multiplex PCR. The findings clearly indicated that this novel protocol is simple and cost-effective to investigate the molecular epidemiology of acute gastroenteritis caused by diarrheal viruses. This report is the first, to our knowledge, detecting hepatitis A virus in feces from diarrheal infants and children in Eastern Russia.

A total of 557 fecal specimens from infants and children with acute gastroenteritis in five places (Maizuru, Tokyo, Sapporo, Saga and Osaka) in Japan from July 2002 to June 2003 were tested for the presence of diarrheal viruses by RT-PCR, PRHA, RNA-PAGE and latex agglutination methods. Of these, 56.4% (314) were found positive for diarrheal viruses. Among them, group A rotavirus was the most prevalent (43.6%, 137 of 314) followed by norovirus (29.9%, 94 of 314), adenovirus (7.6%, 24 of 314), group C rotavirus (6.4%, 20 of 314), sapovirus (5.1%, 16 of 314) and astrovirus (1.6%, 5 of 314), respectively. A high rate (7.4%, 19 of 314) of viral mixed infections, including one triple infection (adenovirus, norovirus and astrovirus) was demonstrated. Norovirus infection that usually has a peak during November and January in Japan was detected year-round and highest in September in our study. Norovirus was subjected to molecular genetic analysis by sequencing. The results clearly indicated that norovirus group II was a dominant genogroup (94.3%, 100 of 106). It is noteworthy that noroviruses detected in this study were classified into 8 genotypes (GI/1, GI/4, GII/2, GII/3, GII/4, GII/5, GII/6 and GII/12). Of these, NVGII/4 was the predominant genotype, followed by NVGII/6, and these presented 75.6% (80 of 106) and 11.3% (12 of 106), respectively. Another interesting feature in our study was the sudden appearance and disappearance of SaitamaU16-like strains belonging to NVGII/6 in the short period (January 2003 to June 2003). Our findings confirmed the presence of many diarrheal viruses co-circulating among Japanese infants and children and showed the great genetic diversity among norovirus.

A total of 3,577 fecal specimens from infants and children with acute gastroenteritis in Japan, Korea and Vietnam during 1998 and 2001 were tested for adenovirus by the ELISA method. Of these, adenovirus was detected in 158 (4.4%). The detection rate of adenovirus was highest in Korea (8.7%, 20/231) followed by 5.0% (100/1,991) in Japan and 2.8% (38/1,355) in Vietnam. All adenoviruses were further serotyped by PCR-RFLP. The diversity of adenovirus serotypes including Ad2, 3, 5, 8, 31, 40 and 41 was demonstrated. Worth of note was a decrease in the rate of isolation of Ad40 (7.6%, 12/158) and a concomitant increase of Ad41 (63.9%, 101/158) to become the predominant serotype. Another interesting feature of the study was the presence of Ad3 (4.0% and 30%) and Ad8 (8.0% and 20%) in Japan and Korea, respectively, which is mainly associated with keratoconjunctivitis worldwide. Our result underscored the importance of adenovirus in association with acute gastroenteritis in Asian countries.

A total of 236 fecal specimens collected from infants and children with gastroenteritis in Maizuru city, Japan from July 2002 to June 2003, were tested for the presence of rotaviruses, noroviruses, sapoviruses, astroviruses, and adenoviruses by RT-PCR, PAGE, RPHA, and latex agglutination methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (32.2%; 76 of 236) followed by norovirus GII (21.2%; 50 of 236), group C rotavirus (10.2%; 24 of 236), adenovirus (3.8%; 9 of 236), sapovirus (2.5%; 6 of 236), astrovirus (1.3%; 3 of 236), and norovirus GI (0.8%; 2 of 236), respectively. It is noteworthy that group C rotavirus infection was apparently confined only within the period of 5 months (December 2002 through April 2003). This pattern of infection implied that the outbreak of group C rotavirus in these patients, which was the first outbreak of gastroenteritis attributed to group C rotavirus in Maizuru city. Moreover, about half (12 of 24) of group C rotavirus infected cases were confined to infants and young children less than 3 years old. Another interesting feature of the study was the demonstration of the mixed infections with group C rotavirus and group A rotavirus, as well as group C rotavirus and norovirus GII in 20.8% (5 of 24) and 8.3% (2 of 24), respectively. This is the first report of gastroenteritis associated with the mixed infections with group C rotavirus and other viral enteropathogens such as norovirus. The results indicate that group C rotavirus could infect not only older children and adults but also infants and young children under 3 years old.

Rotavirus, adenovirus, norovirus, sapovirus and astrovirus are considered to be significant global enteropathogens associated with sporadic cases and outbreaks of acute gastroenteritis. Therefore, a rapid and sensitive assay is preferred to screen for the presence of these viruses in diarrheal fecal specimens. In a previous study, we developed a reverse transcription single-round multiplex polymerase chain reaction (RT-smPCR) assay for the simultaneous detection of norovirus (genogroup I, genogroup II), sapovirus and astrovirus in fecal specimens (Yan et aL, 2003). Recently, we developed another RT multiplex PCR for one-step amplification of all subgenera A to F adenoviruses, and group A and C rotaviruses. In this study, a total of 207 fecal specimens collected from children with acute gastroenteritis between December 2001 and April 2003 in Yunnan Province, China were examined for the presence of adenoviruses, and group A and C rotaviruses, by RT-multiplex PCR. The detection rate of these three viruses was 55.1% (114 out of 207 specimens), among which adenovirus and group A and C rotaviruses were identified in 11, 101 and 1 fecal specimen, respectively. Furthermore, one specimen was found to be positive for co-infection with adenovirus and group A rotavirus. An epidemic of acute gastroenteritis was also identified as peaking mainly in October and November. Taken together, our results clearly indicate that this novel assay provides a potentially rapid and convenient tool for epidemiologic investigation of diarrhea caused by adenovirus and group A and C rotaviruses.

Fecal specimens from 517 infants and young children admitted to the Civil Karachi Hospital, Dow Medical College, Karachi city, Pakistan with acute gastroenteritis from 1990 to 1994 were collected and screened by RT-PCR for human astrovirus (AstV), norovirus (NV), and sapovirus (SV). The specific epidemiological data for illness caused by these viruses in Pakistan are not available. AstV, NV, and SV were detected in 58, 51, and 17 of 517 fecal specimens, and this represented 11.2, 9.9, and 3.2%, respectively. An outbreak of gastroenteritis attributable to AstV serotype 1 was identified during September and October 1990. Moreover, one specimen with a triple mixed infection between AstV (serotypes 1 and 3) and NV GII was found. NV and SV were subjected to molecular analysis by sequencing. One of the sequenced specimens positive for SV turned out to be similar to a strain tentatively called a genogroup IV. The result underscores the importance of these viruses in association with acute gastroenteritis in Karachi city, Pakistan., (Copyright 2004 Wiley-Liss, Inc.)