Wisoot Chan-it

Detection of Rare G3P[19] Porcine Rotavirus Strains in Chiang Mai, Thailand, Provides Evidence for Origin of the VP4 Genes of Mc323 and Mc345 Human Rotaviruses

ABSTRACT Among 175 fecal specimens collected from diarrheic piglets during a surveillance of porcine rotavirus (PoRV) strains in Chiang Mai, Thailand, 39 (22.3%) were positive for group A rotaviruses. Of these, 33.3% (13 of 39) belonged to G3P[19], which was a rare P genotype seldom reported. Interestingly, their VP4 nucleotide sequences were most closely related to human P[19] strains (Mc323 and Mc345) isolated in 1989 from the same geographical area where these PoRV strains were isolated. These P[19] PoRV strains were also closely related to another human P[19] strain (RMC321), isolated from India in 1990. The VP4 sequence identities with human P[19] were 95.4% to 97.4%, while those to a porcine P[19] strain (4F) were only 87.6 to 89.1%. Phylogenetic analysis of the VP4 gene revealed that PoRV P[19] strains clustered with human P[19] strains in a monophyletic branch separated from strain 4F. Analysis of the VP7 gene confirmed that these strains belonged to the G3 genotype and shared 97.7% to 98.3% nucleotide identities with other G3 PoRV strains circulating in the regions. This close genetic relationship was also reflected in the phylogenetic analysis of their VP7 genes. Altogether, the findings provided peculiar evidence that supported the porcine origin of VP4 genes of Mc323 and Mc345 human rotaviruses.

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. J. Med. Virol. 84: 1089–1096, 2012.

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.

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.

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. J. Med. Virol. 83:331–336, 2011.

Multiple Combinations of P[13]-Like Genotype with G3, G4, and G5 in Porcine Rotaviruses

ABSTRACT 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.

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. J. Med. Virol. 81:176–182, 2009.

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. J. Med. Virol. 83:286–290, 2011.

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.

Seasonal pattern and genotype distribution of sapovirus infection in Japan, 2003–2009

Sapovirus, a member of the family Caliciviridae, is one of the major causative agents of viral gastroenteritis affecting all age groups. A total of 3232 faecal specimens collected from infants and children with gastroenteritis in five different regions of Japan during 2003-2009 were examined for sapovirus by reverse transcription-polymerase chain reaction. Sapoviruses were detected in 131 (4·05%) patients with the peak observed mainly in the cold season (November-March) in Japan during 2003-2009. During the last 6 years, sapovirus GI/1 was the predominant strain in Japan followed by GIV, GII/3, GII/6, GII/2, GII/12 and GI, respectively.