Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan.

ABSTRACT: During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

Contrasting Results from Two Commercial Kits Testing for the Presence of Clostridium perfringens Enterotoxin in Feces from Norovirus-Infected Human Patients.

BACKGROUND: Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. METHODS: We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. RESULTS: CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. CONCLUSIONS: An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases.

Evolutionary Analysis of the VP1 and RNA-Dependent RNA Polymerase Regions of Human Norovirus GII.P17-GII.17 in 2013-2017.

Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both VP1 and RNA-dependent RNA polymerase (RdRp) regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013-2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 VP1 region diverged around 1949, while the ancestral GII.P17 RdRp region diverged around 2010. The evolutionary rates of the VP1 and RdRp regions were estimated at ~2.7 × 10-3 and ~2.3 × 10-3 substitutions/site/year, respectively. The phylogenetic distances of the VP1 region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the RdRp region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years.

Variation of human norovirus GII genotypes detected in Ibaraki, Japan, during 2012-2018.

BACKGROUND: Human norovirus (HuNoV) is the major cause of viral acute gastroenteritis for all age groups in various countries. HuNoV GII in particular accounted for the majority of norovirus outbreaks, among which GII.4 caused repeated outbreaks for a long time. Besides GII.4, other norovirus genotypes, GII.2, GII.6, and GII.17, have also been prevalent in various contexts in recent years, but few detailed epidemiological studies of them have been performed and are poorly understood. We thus conducted an epidemiological analysis of HuNoV GII in Ibaraki Prefecture, Japan, by performing surveillance in the six seasons from September 2012 to August 2018. RESULTS: HuNoV GI occurred almost sporadically for all genotypes; however, each genotype of GII exhibited its typical epidemiological characteristics. Although the number of outbreaks of GII.4 decreased season by season, it reemerged in 2017/2018 season. The timing of the epidemic peak in terms of number of cases for GII.17 differed from that for the other genotypes. The patients age with GII.2 and GII.6 were younger and outbreak of GII.17 occurred frequently as food poisoning. Namely, the primarily infected outbreak group differed for each genotype of HuNoV GII. Moreover, the viral load of patients differed according to the genotype. CONCLUSIONS: Various HuNoV genotypes including GII.2, GII.4, GII.6, and GII.17 were shown to be associated with various types of outbreak sites (at childcare and educational facilities, involving cases of food poisoning, and at elderly nursing homes) in this study. These genotypes emerged in recent years, and their prevalence patterns differed from each other. Moreover, differences in outbreak sites and viral load of patients among the genotypes were identified.

High prevalence of hepatitis E virus infection among domestic pigs in Ibaraki Prefecture, Japan.

BACKGROUND: Hepatitis E virus (HEV) is prevalent in pigs and may serve as a reservoir for human infection. However, data on HEV infections in pigs in Ibaraki Prefecture, Japan, are limited. Here, we clarified the process and course of HEV in naturally infected pigs. Serum (n = 160) and liver (n = 110) samples were collected from pigs at the slaughterhouse. Furthermore, serum samples were collected from 45 breeding sows and serum and feces samples were collected from 7 piglets once a week (raised until 166 days of age). HEV antigen and antibodies were evaluated, and the genotype was identified based on molecular phylogenetic tree analysis. RESULTS: The samples collected from the slaughterhouse revealed that few pigs were HEV carriers but most possessed anti-HEV antibodies. Most breeding sows possessed antibodies, and the piglets excreted HEV on the farm at approximately 10 weeks of age. One pig was initially infected, and in a few weeks, the other pigs living in the same sty became infected. CONCLUSIONS: Most pigs in Ibaraki Prefecture were with HEV. On the farm, most piglets were infected with HEV by the time they reached slaughter age. We confirmed that HEV infection is successively transmitted among piglets living in the same sty.

Molecular Evolution of the Protease Region in Norovirus Genogroup II.

Noroviruses are a major cause of viral epidemic gastroenteritis in humans worldwide. The protease (Pro) encoded in open reading frame 1 (ORF1) is an essential enzyme for proteolysis of the viral polyprotein. Although there are some reports regarding the evolutionary analysis of norovirus GII-encoding genes, there are few reports focused on the Pro region. We analyzed the molecular evolution of the Pro region of norovirus GII using bioinformatics approaches. A time-scaled phylogenetic tree of the Pro region constructed using a Bayesian Markov chain Monte Carlo method indicated that the common ancestor of GII diverged from GIV around 1680 CE [95% highest posterior density (HPD), 1607-1749]. The GII Pro region emerged around 1752 CE (95%HPD, 1707-1794), forming three further lineages. The evolutionary rate of GII Pro region was estimated at more than 10-3 substitutions/site/year. The distribution of the phylogenetic distances of each genotype differed, and showed genetic diversity. Mapping of the negative selection and substitution sites of the Pro structure showed that the substitution sites in the Pro protein were mostly produced under neutral selection in positions structurally adjacent to the active sites for proteolysis, whereas negative selection was observed in residues distant from the active sites. The phylodynamics of GII.P4, GII.P7, GII.P16, GII.P21, and GII.P31 indicated that their effective population sizes increased during the period from 2005 to 2016 and the increase in population size was almost consistent with the collection year of these genotypes. These results suggest that the Pro region of the norovirus GII evolved rapidly, but under no positive selection, with a high genetic divergence, similar to that of the RNA-dependent RNA polymerase (RdRp) region and the VP1 region of noroviruses.

Genetic Analysis of Human Norovirus Strains in Japan in 2016-2017.

In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10-3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase Region in Norovirus Genogroup II.

Noroviruses are the leading cause of viral gastroenteritis in humans across the world. RNA-dependent RNA polymerase (RdRp) plays a critical role in the replication of the viral genome. Although there have been some reports on a limited number of genotypes with respect to the norovirus evolution of the RdRp region, no comprehensive molecular evolution examination of the norovirus GII genotype has yet been undertaken. Therefore, we conducted an evolutionary analysis of the 25 genotypes of the norovirus GII RdRp region (full-length), collected globally using different bioinformatics technologies. The time-scaled phylogenetic tree, generated using the Bayesian Markov Chain Monte Carlo (MCMC) method, indicated that the common ancestor of GII diverged from GIV around 1443 CE [95% highest posterior density (HPD), 1336-1542]. The GII RdRp region emerged around 1731 CE (95% HPD, 1703-1757), forming three lineages. The evolutionary rate of the RdRp region of the norovirus GII strains was estimated at over 10-3 substitutions/site/year. The evolutionary rates were significantly distinct in each genotype. The composition of the phylogenetic distances differed among the strains for each genotype. Furthermore, we mapped the negative selection sites on the RdRp protein and many of these were predicted in the GII.P4 RdRp proteins. The phylodynamics of GII.P4, GII.P12, GII.P16, and GII.Pe showed that their effective population sizes increased during the period from 2003 to 2014. Our results cumulatively suggest that the RdRp region of the norovirus GII rapidly and uniquely evolved with a high divergence similar to that of the norovirus VP1 gene.

Phylogeny and Immunoreactivity of Norovirus GII.P16-GII.2, Japan, 2016-17.

During the 2016-17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010-12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

Molecular Evolution of the VP1 Gene in Human Norovirus GII.4 Variants in 1974-2015.

Human norovirus (HuNoV) is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1) gene sequences (466 strains) to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC) method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10-3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1), shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly evolved in a few decades, created various variants, and altered its evolutionary rate and antigenicity.

Molecular evolution of the capsid gene in human norovirus genogroup II.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.

The high prevalence of hepatitis E virus infection in wild boars in Ibaraki Prefecture, Japan.

Hepatitis E virus (HEV) is known as a causative agent of zoonosis and food poisoning. Pigs and some species of wild animals, including wild boar, are known to be a reservoir of HEV. In this study, we investigated the situation regarding HEV infection in wild boars in Ibaraki Prefecture, Japan. Serum, liver and feces samples from 68 animals were collected, and the presence or absence of HEV genomic RNA and HEV antibodies were analyzed. The viral genome was detected in samples from 7 (10.3%) animals, with all HEVs classified as genotype 3, subtype 3b. HEV antibodies were detected in samples from 28 (41%) animals. This report demonstrates for the first time the high prevalence of HEV infection in wild boars in Ibaraki Prefecture.

Characterisation of meticillin-resistant Staphylococcus aureus ST97 and ST5 isolated from pigs in Japan.

The objective of this study was to determine the frequency and clonal types of meticillin-resistant Staphylococcus aureus (MRSA) among slaughter pigs in a top pig-producing area in Japan. In total, 100 nasal swabs were collected from slaughterhouse pigs originating from 21 different farms. MRSA isolates were analysed by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST) and were examined for susceptibility to nine antimicrobial agents (ampicillin, oxacillin, gentamicin, kanamycin, erythromycin, clindamycin, vancomycin, ciprofloxacin and tetracycline). MRSA isolates were obtained from eight swabs (8%), representing three pig farms (14%). Five of the isolates were classified as ST97/spa t1236/SCCmec V and three were classified as ST5/spa t002/atypical SCCmec type. All of the isolates were resistant to ampicillin, oxacillin and tetracycline, and seven isolates (88%) were resistant to clindamycin. The five ST97 MRSA isolates displayed an unusual phenotype (clindamycin-resistant/erythromycin-susceptible). In conclusion, this is the first report of a ST97 MRSA isolate in Japan. The overall prevalence of MRSA is low in pigs, although it appears to be adapting among pigs in Japan owing to the new ST97 and ST5 MRSA strains.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.