Sayaka Takanashi

Detection and Genetic Characterization of Group A Rotavirus Strains Circulating among Children with Acute Gastroenteritis in Japan

ABSTRACT A total of 603 fecal specimens collected from July 2005 to June 2006 from children with acute gastroenteritis, encompassing five different localities in Japan, were screened for group A rotavirus by reverse transcription-PCR. It was found that 117 fecal specimens (19.4%) were positive for group A rotavirus. Rotavirus infection was detected continuously from November to June, with the highest prevalence in April. The G (VP7 genotypes) and P (VP4 genotypes) types were further investigated. The predominant genotype was G1P[8] (70.1%), followed by G3P[8] (17.9%), G9P[8] (6.8%), and G2P[4] (2.6%). A number of unusual G1P[4] combinations were also detected during this study period. A novel nomenclature for G1 is proposed, in which worldwide rotavirus G1 strains are classified into 11 lineages with 17 sublineages. A wide range of amino acid substitutions (up to 34) in VP7 that are specific for G1 lineages and sublineages were identified. Interestingly, only short amino acid motifs located at positions 29 to 75 and 211 to 213 of VP7 defined correctly the phylogenetic G1 lineages and sublineages. Examination of the deduced sequences of antigenic regions of VP7 also revealed multiple particular amino acid substitutions that correlated with the phylogenetic G1 lineages and sublineages. Of note, at least three distinct clusters of rotavirus G1 isolates were cocirculating in the Japanese pediatric population studied.

Detection, genetic characterization, and quantification of norovirus RNA from sera of children with gastroenteritis

BACKGROUND Norovirus (NoV) infection is thought to be confined to the intestines, whereas many reports suggest antigenemia and viremia occur during rotavirus gastroenteritis. OBJECTIVES To detect NoV RNA in sera and cerebrospinal fluids (CSF) from NoV-infected children, and to quantify and genetically characterize the NoV found in these compartments. STUDY DESIGN Semi-nested PCR was conducted on stool, serum and CSF samples from 56 patients with acute gastroenteritis. Positive samples for NoV were analyzed further by sequencing and real-time PCR. RESULTS From 39 patients with NoV RNA in stools, 6 also had NoV RNA in sera and none had NoV RNA in CSF. Genotypes of the NoV in stool and serum from the same patient matched completely. The strains in this study had high homology (98.1-100%) with registered strains in the database. The median viral load in stools of the serum-positive patients was greater than that of the serum-negative patients, but this difference was not statistically significant (9.8 x 10(9)copies/g versus 1.1 x 10(9)copies/g (p=0.117)). CONCLUSIONS NoV RNA appeared in the blood stream in 15% of the patients of NoV gastroenteritis. Although the viral load in stool was not statistically correlated with NoV appearance in serum, genetic analysis indicated that NoV RNA in sera originated from the NoV gastroenteritis.

Characterization of a Broadly Reactive Monoclonal Antibody against Norovirus Genogroups I and II: Recognition of a Novel Conformational Epitope

ABSTRACT Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.

Human Parechovirus Infection in Children Hospitalized with Acute Gastroenteritis in Sri Lanka

ABSTRACT Of 362 fecal specimens collected from infants and children hospitalized with acute gastroenteritis in Sri Lanka from September 2005 to August 2006, 30 (8.3%) were positive for human parechovirus (HPeV). Six different HPeV genotypes, including HPeV1, -3, -4, -5, -10, and -11, were identified, of these, HPeV11 was reported for the first time.

Four-year study of viruses that cause diarrhea in Japanese pediatric outpatients

Acute gastroenteritis continues to be a major public health problem worldwide. A wide variety of viruses associated with diarrhea disease is being reported continually. This study investigated the epidemiological situation of viruses that cause diarrhea in Japanese pediatric patients. This study enrolled a total of 2,381 fecal specimens collected between 2009 and 2013 from Japanese children with acute gastroenteritis. There is currently a 70.4% prevalence of viruses causing diarrhea among these Japanese pediatric outpatients. Norovirus was detected in 39.3% of the patients, whereas the prevalence of rotavirus, human parechovirus, enterovirus, adenovirus, sapovirus, astrovirus, and Aichi virus was 20.1, 6.6, 6.1, 5.6, 4.8, 2.3, and 0.1%, respectively. Co‐infections were observed at the prevalence rates of 13.4 and 0.5% for double infections and triple infections, respectively. Mixed viral infections were found commonly in Japanese outpatients, and the norovirus seemed to play a major role in co‐infections. Viral diarrhea cases were detected mostly in children younger than 3 years of age. The norovirus and rotavirus can be detected throughout the year, with a peak during the cold and dry seasons, whereas other common viruses are found during no specific season. Surveillance data revealed that a wide variety of viruses has caused diarrhea to circulate currently in Japanese pediatric outpatients, with very high detection rates; and norovirus and rotavirus are the most important pathogens. The data obtained from this study are valuable for compiling the overall picture of several viruses that causes diarrhea and associates with acute gastroenteritis in the Japanese pediatric population. J. Med. Virol. 87:1141–1148, 2015.

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.

Novel Human Parechovirus, Sri Lanka

Of 362 fecal samples collected from children with acute gastroenteritis in Sri Lanka during 2005–2006, 30 (8.3%) were positive for human parechovirus (HPeV) by reverse transcription–PCR. A novel HPeV, designated as HPeV10, was identified in 2 samples by sequence analysis of the viral protein 1 gene of the detected HPeVs.

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.

Evaluation of immunochromatography tests for detection of novel GII.17 norovirus in stool samples

A novel GII.17 norovirus has emerged as a major cause of epidemic and endemic acute gastroenteritis in several countries in Asia. We used a small panel of stool samples in which GII.17 virus had been quantified by real-time RT-PCR to evaluate four commercially available norovirus immunochromatography (IC) kits. At least 10(8) copies/mL of GII.17 virus were required by each IC kit for a positive result, which is 1,000-fold more than that reported for these assays for GII.4 viruses.

Multiplex RT-PCR for rapid detection of viruses commonly causing diarrhea in pediatric patients.

Multiplex RT‐PCR method using five sets of panel primers was developed for the detection of diarrheal viruses, including rotavirus A, B, and C, adenovirus, astrovirus, norovirus GI and GII, sapovirus, Aichi virus, parechovirus, enterovirus, cosavirus, bocavirus, and Saffold virus. The sensitivity of the method was evaluated and tested with 751 fecal specimens collected from Japanese children with acute diarrhea. Several kinds of viruses were detected in 528 out of 751 (70.3%) fecal specimens. Mixed‐infection with different viruses in clinical specimens could also be effectively detected. The method proved to be reliable with highly sensitive and specific and useful for routine diagnosis. J. Med. Virol. 89:818–824, 2017.