Predominance of DS-1-like G8P[8] rotavirus reassortant strains in children hospitalized with acute gastroenteritis in Thailand, 2018-2020.

Rotavirus A (RVA) is an important cause of acute gastroenteritis (AGE) in children. This study aims to investigate the molecular epidemiology of RVA in children hospitalized with AGE in Chiang Rai, Thailand in 2018-2020 by reverse transcription polymerase chain reaction. Of 302 samples, RVA was detected in 11.6% (35 samples): 11.3% (19/168) in 2018-2019 and 11.9% (16/134) in 2019-2020. G8P[8] was the predominant genotype at 68.4% in 2018-2019 and 81.2% in 2019-2020. G1P[8] (15.8%), G2P[4] (5.3%), G3P[8] (10.5%) in 2018-2019, and G9P[8] (18.8%) in 2019-2020 were also detected. Whole-genome analysis of G8P[8] revealed a DS-1-like genetic backbone: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetically, the VP7 genes of G8P[8] clustered in a major lineage with previously published 51 DS-1-like G8P[8] reference strains, closely related to 13 G8P[8] strains from Thailand and China. These G8P[8] strains contained two unique amino acid substitutions (A125S and N147D) in the VP7 antigenic epitopes. In addition, the VP1 and NSP2 genes of G8P[8] clustered in lineages separated from the DS-1-like G8P[8] reference strains with a high genetic divergence but were closely related to G1P[8], G2P[4], G3P[8], or G9P[8]. Several amino acid differences were observed in the VP7 and VP8* antigenic epitopes of G8P[8] compared with RVA vaccine strains. Homology modeling confirmed that these different amino acid residues were located on the surface-exposed area of the structure. Taken together, the genetic analysis clearly defines the Chiang Rai DS-1-like G8P[8] strains as a novel reassortant strain that might have evolved genetically through reassortment events and consequently received their VP1 and NSP2 genes from locally cocirculating-RVA genotypes.

Emergence of G9P[8] rotaviruses in children with acute gastroenteritis in Thailand, 2015-2016.

Human group A rotavirus is a major contagious virus causing gastroenteritis in children. Molecular epidemiological study of group A rotavirus infections in hospitalized children was performed by multiplex RT-PCR during 2015-2016 in Chiang Rai, Thailand. G- and P-genotypes of positive rotavirus samples were further analyzed by one-step and two-step multiplex RT-PCR methods. Among 270 fecal specimens tested, rotavirus was the most prevalent (33.7%), followed by norovirus GII (4.1%), adenovirus (3%), and astrovirus (1.5%). Infection was common in patients aged 12-23 months (45.1%) and occurred mostly in children under 3 years of age (85.7%). The highest peak was in a hot month, March (64.8%). G9P[8] emerged as the most predominant (79.1%), followed by G3P[8] (13.2%), G1P[8] (3.3%), and mixed G-types (4.4%). Interestingly, Chiang Rai G9 strains were clustered within a distinct lineage VII including G9 recently reported since 2010-2015. G9-VII also contained four to five unique amino acid substitutions in the VP7 proteins compared with those of the G9 candidate vaccine strain RVA/Human-tc/IND/116E/1985/G9P[11] and the prototype RVA/Human-wt/USA/WI61/1983/G9P[8], defining the G9-VII as a novel variant. G3 strains were closely related to the "new G3P[8] reassortant variant" with an equine-like VP7 gene that emerged in several countries. This study contributes to the understanding of the genetic diversity, providing scientific support for future vaccine strategies to reduce the morbidity and mortality.

Molecular epidemiology of norovirus associated with gastroenteritis and emergence of norovirus GII.4 variant 2012 in Japanese pediatric patients.

In late 2012, an outbreak of acute gastroenteritis due to norovirus variant Sydney_2012 occurred and have been reported from many counties. In this study, we described surveillance study of the incidence of norovirus infections among Japanese pediatric patients in association with gastroenteritis and investigated the antigenic change of the new variant Sydney_2012 circulated in Japanese populations. A total of 2381 fecal specimens collected from children with acute gastroenteritis in Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga from 2009 to 2013 were examined for norovirus and further analyzed molecularly. A high proportion (39.3%) of norovirus positive samples and several genotypes were detected. Norovirus GII.4 dominated over other genotypes (71.4%). The Den_Haag_2006b (43.2%) was detected as the predominant variant and co-circulated with New_Orleans_2009 (17.8%) until March 2012. Subsequently, they were displaced by Sydney_2012. The Sydney_2012 variant has been responsible for the majority of norovirus infections in 2012-2013 (85.7%). Although Sydney_2012 variant has a common ancestor with New_Orleans_2009 variant, analysis of P2 sub-domain showed a high level of diversity in comparison with other variants in four amino acid changes at the antigenic sites. The change in particular residue 393 of new variant may affect HBGA recognition. Analysis of noroviruses circulating in the past 4years revealed a change of predominant variant of norovirus GII.4 in each epidemic season. The change of amino acid in putative epitopes may have led the virus escape from the existing herd immunity and explain the increase of new variant outbreaks.

Genetic analysis and homology modeling of capsid protein of norovirus GII.14.

In this study, a more detailed genetic characterization of the VP1 capsid protein of uncommon norovirus (NoV) GII.14 strains reported previously in Japan and China was performed using sequence analyses and homology modeling technique. The result of genetic comparison with the M7 prototype strain of GII.14 revealed that 10 amino acid mutations were observed at the same positions across the P2 and P1-2 subdomains in both Japanese and Chinese strains. By the homology modeling of the P domain, 7 out of these 10 mutations were predicted to be located on the surface-exposed P2 and P1-2 subdomains. All GII.14 strains had an altered RGD-like motif (RGT → KGT). While the Chinese strains contained 5 random amino acid changes in the S domain and the P2 subdomain, these changes were not detected in the Japanese strains. In addition, the histo-blood group antigen (HBGA)-binding interfaces remain identical to those of the previously determined GII.4 structure (VA387), suggesting the conservation of HBGA binding profile within the GII genogroup. Taken together, this report provides supportive structural data that antigenic drifts that occurred mostly in the P2 and P1-2 subdomains might be sufficient to generate new mutants, thus permitting the GII.14 virus to escape the host pre-existing immunity. These results also suggest the need for comparing the evolutionary profiles and structural models of rare NoV genotypes to an insight into NoV evolution.

Emergence of norovirus GII/4 2006a and 2006b variants in hospitalized children with acute gastroenteritis in Thailand.

BACKGROUND: Norovirus (NoV) is recognized as a significant cause of acute gastroenteritis in infants and young children worldwide. This study investigated the prevalence of NoV infection in hospitalized children with gastroenteritis in Chiang Mai, Thailand in 2006. METHODS: A total of 156 fecal specimens were collected from children with diarrhea admitted to McCormick Hospital in 2006. All fecal specimens were examinedffor NoV by RT-PCR and the genotypes were identified by sequence analysis. RESULTS: A high prevalence of NoV infection was detected (20.5%, 32/156). NoV GII/4 was the most predominant genotype with a prevalence of 87.5% (28/32), while GII/3, GII/6, GII/12, and GII/15 were less common (3.1% each). Among GII/4 strains, 2006b variant (75%, 21/28) emerged as the leading strain and dominated over the Hunter'04-like variant, which was the most common strain in the previous season of 2005. In addition, the 2003, 2004, and 2006a variants were also detected. NoV infections were most commonly observed in the rainy season in Thailand. CONCLUSIONS: This study demonstrated the emergence of GII/4 2006b variants as the major pathogen causing acute gastroenteritis among infants and children at the age of less than 5 years old who admitted to hospital in Chiang Mai, Thailand in 2006. Additionally, other GII/4 variants of 2003, 2004, and 2006a were also reported.

Reemergence of new variant G3 rotavirus in Japanese pediatric patients, 2009-2011.

The molecular epidemiology and characterization of rotaviruses obtained from non-hospitalized children with acute gastroenteritis in five different prefectures (Hokkaido, Saga, Tokyo, Osaka, and Kyoto) from July 2009 to June 2011 was investigated. Among 831 fecal specimens tested, rotavirus was found in 165 specimens (19.9%). The rotavirus detection rate in 2010-2011 (23.3%) was higher than those in 2009-2010 (16.0%). The highest prevalence of rotavirus was found in children aged 12 to 23 months. Rotavirus could be detected throughout the 8 month period with a peak in April. We found that G3P[8] was the most prevalent genotype (54.5%), followed by G1P[8] (29.1%), G9P[8] (9.1%), G3P[4] (3.0%), G2P[4] (2.5%), G1P[4] (1.2%), and G4P[8] (0.6%), respectively. Interestingly, G3 strains emerged as the most predominant genotype and replaced G1 rotavirus which had been reported as the most predominant genotype in the previous studies. Phylogenetic analysis revealed that G3 rotavirus strains were closely related to the "new variant G3" 5091 strain, which emerged in Japan in 2003-2004. A significant increase in the prevalence of rotavirus G3 found in this study indicates that rotavirus G3 strain is the major cause of infection in five geographical areas of Japan and may distribute globally in the near future.

Emergence of a new norovirus GII.6 variant in Japan, 2008-2009.

Norovirus (NoV) is recognized as one of the most common causative agents of diarrhea disease in young children. A total of 187 fecal specimens collected from non-hospitalized children with acute gastroenteritis in Shizuoka, Japan during July 2008 to June 2009 were investigated for the presence of diarrhea viruses by a multiplex RT-PCR. Diarrhea viruses were overall detected in 158 of 187 (84.5%). Of the viruses detected, NoV was the most prevalent (55.6%). Most of the NoV sequences belonged to GII.4 (53.8%). NoV GII.6 emerged as the second most common strain (40.4%). The full-length capsid sequences of five representative Shizuoka GII.6 strains were compared with all 12 GII.6 strains available in GenBank database between 1990 and 2009. At least three distinct GII.6 subclusters (a-c) appeared in different parts of the world. Shizuoka GII.6 strains formed their own subcluster c, distinct from other complete GII.6 reference sequences. The Shizuoka strains had significant amino acid divergence, particularly in the P2 domain up to 10.9-17.5% and contained eight unique mutations in the P domains, compared with subcluster a and b viruses. The homology model showed that the eight mutations were predicted to be located at the surface-exposed P1 and P2 domains. The data suggest the emergence of a new NoV GII.6 variant in Shizuoka, with a high level of genetic variation.

Detection and genetic characterization of norovirus infections in children with acute gastroenteritis in Japan, 2007-2009.

BACKGROUND: Noroviruses are a major cause of epidemic gastroenteritis in children and adults. The aim of the present study was to investigate the molecular epidemiology of norovirus gastroenteritis in Japan. METHODS: A total of 954 fecal specimens collected from infants and children with acute gastroenteritis from five different regions (Tokyo, Sapporo, Saga, Osaka, and Maizuru) of Japan during 2007-2009 were identified by multiple RT-PCR and semi-nested PCR. RESULTS: Norovirus was detected in a relatively high detection rate (26.6%; 254 of 954). Of the identified NoV, 9.5% (91 of 954) were positive by semi-nested PCR. Norovirus GII (97.3%) was more prevalent than GI (2.7%). Norovirus infections were very common in the patients aged 12-23 months (44.5%; 113 of 254). Winter month seasonality supported norovirus infection in Japan. All 7 GI sequences (100%) detected only in 2007-2008 clustered with Chiba 407 known as GI.4 genotype. Most of the norovirus GII sequences in 2007-2008 belonged to GII.4 (77.9%), followed by GII.14 (11.9%), and GII.3 and GII.6 (5.1% each). In 2008-2009, norovirus sequences were classified into eight distinct genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII12, and GII.14). GII.4/2006b variant was responsible for 100% among the detected GII.4 strains in both seasons. Interestingly, GII.6/GII.14 recombinant strains emerged, for the first time in Japanese children, as the second prevalent genotype (11.9%) in 2007-2008 and then dropped rapidly to 2.3% in a year after. In addition, GII.b/GII.3 and GII.4/GII.3 recombinant strains that had been described previously were also found in this study. CONCLUSIONS: This is the first report to demonstrate the co-circulation of the predominant GII.4/2006b variant and the emerging GII.6/GII.14 recombinant strains and supports the importance of norovirus as a causative agent of diarrhea in Japanese children with acute gastroenteritis.

Comparison of the rapid methods for screening of group a rotavirus in stool samples.

In this study, the sensitivity and specificity of three immunochromatography (IC) test kits for rapid detection of group A rotavirus were compared and evaluated with stool samples collected from children, who suffered from acute gastroenteritis during February to June, 2009 in Japan. A total of 86 stool samples were tested and compared with a reference RT-PCR method. The sensitivity among IP-Rota V, Dipstick Eiken ROTA and ROTA-ADENO test kits were 97.2, 95.8 and 88.7%, while the specificity were 100, 93.3 and 100%, respectively. It was demonstrated that the IC kits evaluated in this study could be used as an alternative method for the rapid screening of group A rotavirus in fecal specimens, especially during acute gastroenteritis outbreak season.

Detection and genetic characterization of rotavirus infections in non-hospitalized children with acute gastroenteritis in Japan, 2007-2009.

The molecular epidemiology of rotavirus infections in non-hospitalized children in five different regions (Sapporo, Saga, Tokyo, Osaka, and Maizuru) of Japan during 2007-2009 was investigated. Overall, rotavirus was detected in 156 out of 1008 (15.5%) specimens. The rotavirus infection in 2007-2008 (19.3%) was higher than those in 2008-2009 (12.1%). G1P[8] was the most prevalent (62.8%), followed by G3P[8] (21.8%), G9P[8] (14.7%), and G2P[4] (0.7%). Interestingly, the number of G3P[8] strains increased threefold from the former season (2006-2007) from 7.3% to 21.8%, whereas G2P[4] and G9P[8] decreased from 11.4% to 0.7% and 20.3% to 14.7%, respectively. In the phylogenetic analysis, G3 rotaviruses were closely related to "the new variant G3" 5091 strain, which previously emerged in Japan and China. G9 viruses isolated in 2007-2008 were genetically close to the Thai strain, while those isolated in 2008-2009 had a close relationship with Chinese strains. G1 viruses appeared to be more similar to the recently reported G1 strain in China. Nucleotide sequence analysis of 33 P[8]-nontypeable strains revealed 5 nucleotide mismatches at the primer binding site. Based on previously reported (2003-2007) and current (2007-2009) data of rotavirus surveillance in the five areas of Japan, it was revealed that in Sapporo, Osaka, and Maizuru, G1P[8] and G3P[8] were detected at high frequencies, ranging from 47.2 to 57.7% and 31.7 to 47.4%, respectively. In Tokyo, G1P[8] (47.4%) was the predominant strain, followed by G9P[8] (20.6%), whereas in Saga, G3P[8] (38.9%) and G9P[8] (36.1%) were identified as the most dominant types. None of G9P[8] was detected in Sapporo. This study highlights the genetic diversity and the significance of rotavirus diarrhea in Japan.

Human bocavirus infection in children with acute gastroenteritis in Japan and Thailand.

A total of 329 fecal specimens, which had been known to be negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus, and which were collected from infants and children with acute gastroenteritis in Japan and Thailand during 2005-2008 were screened for human bocavirus (HBoV). HBoV was detected by PCR with a primer pair that amplified the NP1 region of its genome and was genotyped by sequencing of the VP1/VP2 region. Of the 329 samples tested, 6 (1.8%) were positive for HBoV. Of these, five samples were collected from Japan and one sample was from Thailand, and the detection rates of HBoV in each country were 2% and 1.2%, respectively. For the detected HBoV, the capsid VP1/VP2 gene of all HBoV strains was successfully sequenced. Four Japanese HBoV strains studied were clustered into group 1, while the remaining Japanese strain and a unique Thai strain belonged to group 2. No severe acute gastroenteritis associated with HBoV was noted. This study provides better understanding on the epidemiology of HBoV infections in children with acute gastroenteritis in Japan and Thailand.

Detection of human parechovirus in stool samples collected from children with acute gastroenteritis in Japan during 2007-2008.

Of 477 stool specimens, which had been screened for rotavirus, adenovirus, norovirus, sapovirus and astrovirus, collected from infants and children with acute gastroenteritis in pediatric clinics encompassing five localities (Sapporo, Tokyo, Maizuru, Osaka, and Saga) in Japan from July 2007 to June 2008, 247 negative samples (51.7%) were subjected to screening for human parechovirus. Human parechovirus (HPeV) was detected by RT-PCR using a primer pair to amplify 5'UTR region of its genome and was genotyped by sequencing of the VP1 gene. HPeV was detected in 20 of 247 specimens tested, and the detection rate was found to be 8.1%. Seventeen of the 20 strains that tested positive for HPeV were sequenced successfully the VP1 gene. The majority of the HPeV strains (n = 15) could be identified as HPeV1, and the remaining 2 strains could be typed as HPeV3. By phylogenetic and identical matrix analyses of HPeV VP1 sequences, HPeV1 should be divided into two lineages, and all of the Japanese studied HPeV1 strains belong to the lineage 2 accordingly. This is the first report of the circulation of HPeV, especially HPeV1 in Japan.

A novel RT-multiplex PCR for detection of Aichi virus, human parechovirus, enteroviruses, and human bocavirus among infants and children with acute gastroenteritis.

A novel reverse transcription-multiplex polymerase chain reaction assay was developed to detect Aichi virus, human parechovirus, enteroviruses, and human bocavirus. A mixture of four pairs of published specific primers, 6261 and 6779, ev22(+) and ev22(-), F1 and R1, 188F and 542R, was used to amplify the viral genomes and specifically generate four different amplicon sizes of 519, 270, 440, and 354 bp for Aichi virus, human parechovirus, enteroviruses, and human bocavirus, respectively. A total of 247 fecal specimens previously screened for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus-negative, collected from infants and children with acute gastroenteritis in Japan from July 2007 to June 2008, were tested further for the presence of the four viruses, Aichi virus, human parechovirus, enteroviruses, and human bocavirus, by RT-multiplex PCR. The total detection rate of these viruses was 26.7% (66 out of 247 samples). Of these, HPeV, EVs, and HBoV were identified in 20, 41, and 5 specimens. No Aichi virus was found among these subjects. The sensitivity and specificity of RT-multiplex PCR were assessed and demonstrated a strong validation against RT-monoplex PCR. This is the first report of detecting these types of viruses in fecal samples from infants and children with acute gastroenteritis by RT-multiplex PCR.

Immunochromatography test for rapid detection of norovirus in fecal specimens.

An immunochromatography (IC) assay for rapid detection of norovirus (NoV) was evaluated with fecal samples collected from children who suffered from acute gastroenteritis during the winter season of 2007-2008 in Japan. A total of 75 fecal specimens were tested for NoV by the newly developed IC kit and by a gold standard RT-PCR method. The sensitivity, specificity, and agreement of this IC kit were 75.4%, 100%, and 80%, respectively. In addition, phylogenetic analysis revealed that the majority of NoV circulating in Japan during 2007-2008 belonged to the new variant GII/4 2006b genetic cluster. It was demonstrated that the IC kit evaluated in this study could detect these new variant NoV strains, which emerged recently in Japan. Therefore, it is suggested that this NoV IC kit could be used as an alternative method for the screening of NoV in fecal specimens, especially during the season of acute gastroenteritis outbreak.

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain.

An unusual strain of human rotavirus G3P[10] (CMH079/05) was detected in a stool sample of a 2-year-old child admitted to the hospital with severe diarrhea in Chiang Mai, Thailand. Analysis of the VP7 gene sequence revealed highest identities with unusual human rotavirus G3 strain CMH222 at 98.7% on the nucleotide and 99.6% on the amino acid levels. Phylogenetic analysis of the VP7 sequence confirmed that the CMH079/05 strain formed a cluster with G3 rotavirus reference strains and showed the closest lineage with the CMH222 strain. Analysis of partial VP4 gene of CMH079/05 revealed highest degree of sequence identities with P[10] rotavirus prototype strain 69M at nucleotide and amino acid levels of 92.9% and 94.6%, respectively. Phylogenetic analysis of the VP4 sequence revealed that CMH079/05 and 69M clustered closely together in a monophyletic branch separated from other rotavirus genotypes. To our knowledge, this is a novel G-P combination of G3 and P[10] genotypes. In addition, analyses of VP6, NSP4, and NSP5/6 genes revealed these uncommon genetic characteristics: (i) the VP6 gene differed from the four other known subgroups; (ii) the NSP4 gene was identified as NSP4 genetic group C, an uncommon group in humans; and (iii) the NSP5/6 gene was most closely related with T152, a G12P[9] rotavirus previously isolated in Thailand. The finding of uncommon G3P[10] rotavirus in this pediatric patient provided additional evidence of the genetic diversity of human group A rotaviruses in Chiang Mai, Thailand.

A simple screening assay for receptor switching of avian influenza viruses.

BACKGROUND: Adaptation of the receptor-binding preference from alpha2,3- to alpha2,6-linked sialic acid is an essential step for an avian influenza virus to transmit efficiently in human population and become a pandemic virus. The currently available assays for receptor-binding preference are complex and not widely available. OBJECTIVES: A simple high-throughput screening assay will facilitate early detection of a potential pandemic virus, which is crucial for the prevention and control of the possible pandemic. We wanted to develop a simple assay to differentiate influenza viruses with alpha2,3- or alpha2,6-linked receptor-binding preference. STUDY DESIGN: The assay employs a specific sialidase (from Salmonella thyphimurium) that can eliminate alpha2,3-linked sialic acid from red blood cells. A reduction of hemagglutination titer indicates alpha2,3-linked receptor preference in this assay. RESULTS: Using a panel of H5N1 avian influenza isolates and H1/H3 human influenza isolates, as well as mutated H5 reverse genetics virus, the assay could accurately differentiate the viruses according to their receptor-binding preference. Furthermore, the assay was sufficiently sensitive to detect a minor variant with alpha2,6-linkage-specificity in a background of alpha2,3-linkage-specific virus. CONCLUSIONS: We have developed a simple screening assay capable of detecting avian influenza viruses that have switched their receptor-binding preference.

Multiple combinations of P[13]-like genotype with G3, G4, and G5 in porcine rotaviruses.

Epidemiological surveillance of porcine rotavirus (PoRV) strains was carried out in Chiang Mai Province, Thailand, from 2002 to 2003, and eight rotavirus isolates could not be completely typed by PCR. Of these, six were G3 and one was G4 and displayed a P-nontypeable genotype, while another isolate was both G and P nontypeable. Analysis of a partial VP4 gene of all eight P-nontypeable strains revealed a high degree of amino acid sequence identities (94.7% to 100%), suggesting that they belonged to the same P genotype. Comparison of the amino acid sequences of two representative strains (namely, strains CMP178 and CMP213) with those of 27 other known P genotypes revealed a high degree of amino acid sequence identity with those of P[13] porcine rotavirus reference strains HP113 and HP140, which were recently isolated in India. However, amino acid sequence comparison with non-P[13] rotavirus strains revealed relatively low identities, ranging from 58.2% to 84.8% for full-length VP4 sequences and 35.1% to 80.6% for VP8* sequences. Phylogenetic analysis revealed that CMP178 and CMP213 clustered together in a monophyletic branch with P[13]-like genotypes HP113 and HP140 which was clearly separated from the other lineages of P[13] or P[22] strains. Altogether, these findings indicate that PoRV strains CMP178 and CMP213 should be considered the P[13]-like VP4 genotype, a rare genotype that has been identified only in pigs. This study provides additional evidence of increasing genetic diversity among group A rotaviruses in nature.