Molecular epidemiology of rotavirus gastroenteritis in Japan during 2014-2015: Characterization of re-emerging G2P[4] after rotavirus vaccine introduction.

Rotavirus vaccines have been available in Japan since 2011. This study conducted to monitor the trend of group A rotavirus (RVA) genotypes 3 years after vaccine introduction. A total of, 539 fecal samples were collected from children with acute gastroenteritis in six regions during July 2014-June 2015. Among them, 178 samples (33.0%) were positive for RVA. The most predominant genotype was G1P[8] (35.9%) followed by G2P[4] (26.4%), G9P[8] (21.3%), G3P[8] (4.5%), and G3P[9] (4.5%). The detection rate of G2P[4] was increased soon after vaccine introduction. Sequence analyses of VP7 and VP4 genes of the representative G2P[4] strains were found to be clustered in sub-lineage IVa of lineage IV. It is noteworthy that one amino acid substitution in the antigenic epitope (Q114P) of VP4 gene was found in representative G2P[4] strains of the current study. However, it is unclear whether the change in antigenic epitope is due to the effect of vaccination or due to natural variation, warranting further continuous monitoring of rotavirus evolution after vaccine introduction.

A 3-Month-Old Child with Acute Gastroenteritis with Enterovirus D68 Detected from Stool Specimen.

Enterovirus D68 (EV-D68) is known to be causative agent of mild to severe upper and lower respiratory illnesses in sporadic cases and outbreaks. We present a case report of a 3-month-old child with acute gastroenteritis who visited a pediatric clinic in Kyushu area in Japan in 2015. A stool sample collected from the patient was screened for diarrheal viruses by multiplex RT-PCR. The result showed that the sample was positive only for enterovirus, and EV-D68 clade B3 was identified by sequence analysis of the viral protein 1 gene. This study supports an association between EV-D68 infection and acute gastroenteritis.

Molecular Epidemiological Traits of Group A Rotaviruses in Japanese Children During Transitional Period of Rotavirus Vaccine Implementation, 2011 - 2014.

BACKGROUND: Group A rotavirus (RVA) vaccines have been introduced in Japan since 2011. To investigate the molecular epidemiological traits of RVA during the transitional period of rotavirus vaccine implementation in Japan, this study was undertaken by following up three-decade long surveillance conducted in the same regions. METHODS: RVA were screened and genotyped by RT-PCR from diarrheal samples collected from non-hospitalized patients in six localities (Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga) during 2011 - 2014. Selected samples were sequenced to elucidate the evolutionary trend. RESULTS: Among 1858 specimens, the detection rate of RVA declined to 4.0% in 2013 - 2014 from 17.9% in 2011 - 2012 and 22.1% in 2012 - 2013. G1P[8] was the most predominant genotype in the first two years accounting for more than half, and G9P[8] showed the highest detection rate as 35.0% in the last year. Interestingly, the proportional rate of G2 strains in the studied period increased from 0% to 25%. VP6 genotyping revealed that DS-1 like reassortant G1P[8] strains were detected all over Japan and their prevalence fluctuated greatly from 35.0% to 89.5%. Sequence analysis of VP6 showed that strains in the current strains were closely related but distinct from the original reference strains, namely Wa and DS-1. CONCLUSIONS: The detection rates of RVA, their GP combinations, prevalence of reassortant strains varied greatly after the introduction of rotavirus vaccines in Japan. Continuous monitoring is warranted to refine future vaccine strategy.

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.

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.

Distribution of rotavirus genotypes in Japan from 2015 to 2018: Diversity in genotypes before and after introduction of rotavirus vaccines.

BACKGROUND: Diversity in group A rotavirus (RVA) strains after introduction of RV-vaccines remains an emerging concern worldwide. In this study, we investigated the prevalence and distribution of RVA genotypes in Japanese children with acute gastroenteritis (AGE) from 2015 to 2018. In addition, a comparison of the genotypes in pre-vaccination (2006-2012) and post-vaccination (2012-2018) periods was conducted to understand the impact of these vaccines on genotype distribution. METHODS: Fecal samples were collected regularly from outpatient clinics in six localities: Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga. RVA were screened and genotyped by RT-PCR and sequence-based genotyping. RESULTS: During the period 2015-2018, RVA was detected in 307 (19.7%) samples out of 1557 specimens: 29.9% (95% CI: 25.8% to 34.3%), 17.9% (95% CI: 14.7% to 21.5%), and 13% (95% CI: 10.3% to 16.0%) were detected RVA-positive in 2015-2016, 2016-2017 and 2017-2018, respectively. The average detection of RVA in pre-vaccination (2006-2012) and post-vaccination (2012-2018) era remained almost similar (18%-20%). The G2P[4]I2 (52.1%, 95% CI: 43.5%-60.6%) remained the most common genotype in 2015-2016, whereas G8P[8]I2 (55.9%, 95% CI: 45.2%-66.2%) dominated in 2016-2017. In 2017-2018, G9P[8]I2 (42.0%, 95% CI: 30.5%-53.9%) prevailed, followed by G9P[8]I1 (23.0%, 95% CI: 14.0%-34.2%). The detection rate of some common genotypes of pre-vaccination era like G1P[8] and G3P[8] has been reduced after introduction of RV-vaccine, whereas genotypes that were sporadic before the introduction of vaccines like G2P[4], G2P[8], G9P[8] and G8P[8] were emerged/reemerged in post-vaccination period. CONCLUSIONS: Our study presented the diversity in circulating RVA genotypes in Japan before and after introduction of RV-vaccines. Sudden emergence of DS-1-like (I2) unusual strains in post-vaccination era remains alarming. Continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy.

Prevalence of ß2-agonist inhalation for outpatients in a pediatric emergency center during enterovirus D68 epidemic.

BACKGROUND: Enterovirus D68 (EV-D68) has been reported to have caused severe bronchial asthma attacks and hospitalization epidemics in Japan in September 2015. OBJECTIVE: To investigate the prevalence of ß2-agonist inhalation in a pediatric emergency center during a period of increased hospitalization for bronchial asthma, which was suggested to be associated with EV-D68. METHODS: We investigated the prevalence of ß2-agonist inhalation in a pediatric emergency center in Saga city, Japan, from April 2013 to October 2015, and also clarified the trends in bronchial asthma hospitalization in the same area during that time. RESULTS: The prevalence of ß2-agonist inhalation in the pediatric emergency center, September 2015 was highest when EV-D68 became widespread. The monthly average for ß2-agonist inhalation during the study period was 91 cases, but the count in September 2015 was 255 cases. Hospitalized cases of bronchial asthma in September 2015 were increased for age ≥3 years and not increased for age <3 years, but the prevalence of ß2-agonist inhalation at the pediatric emergency center was increased even under the age of 3 years. CONCLUSION: During the epidemic period for EV-D68, cases requiring ß2-agonist inhalation were increased. The EV-D68 epidemic may be related to not only severe cases requiring hospitalization, but also exacerbation of relatively mild symptoms of bronchial asthma.

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.

Three clusters of Saffold viruses circulating in children with diarrhea in Japan.

Saffold virus (SAFV) is a newly discovered human virus in the genus Cardiovirus, family Picornaviridae. The virus was first described from fecal specimens of a child with fever of unknown origin in 2007. A total of 454 fecal specimens were collected from children with diarrhea attended clinics in Japan, 2010-2011, 7 (1.5%) were positive for SAFV. Mixed-infections of SAFV and other enteric viruses (rotavirus, norovirus, and bocavirus) were found in four out of seven cases, while monoinfection by SAFV alone was detected in three cases. In addition to diarrhea, fever and vomiting were observed in three children and mild dehydration in one case. No particular symptoms of cough and rhinorrhea were noted. Analysis of partial VP1 nucleotide sequence of 7 Japanese SAFV strains revealed that 5 SAFV sequences were most closely related with SAFV2 reference strains, but separated into SAFV2-A (3 strains) and SAFV2-B (2 strains). In addition, the other two strains were classified as SAFV3. Our results indicated that SAFVs (SAFV2 and SAFV3) were circulated in children with acute gastroenteritis in Japan during 2010 and 2011 epidemic 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.