Rotavirus genotype and Vesikari score of outpatients in Japan in the vaccine era.

BACKGROUND: Group A rotaviruses (RVs) are a major cause of severe gastroenteritis among infants and young children. In Japan, RV vaccines were introduced in 2011, leading to a reduction in severe gastroenteritis cases. Studies are required to assess the effectiveness of the vaccines and their effect on the prevalence of RV genotypes. METHODS: Fecal samples were collected from outpatients with RV gastroenteritis in a pediatric clinic in Sapporo, Japan, from 2010 to 2016. GPI genotypes were determined using reverse-transcription polymerase chain reaction. Clinical information and immunization records were obtained from outpatients after 2013. GPI genotypes and clinical features were compared between patients with and without a RV vaccine history. RESULTS: In total, 270 cases were genotyped. G1P[8]I1 (Wa-like G1P[8]) strains were dominant from 2010 to 2012. G1P[8]I2 (DS-1-like G1P[8]) strains appeared in 2012 and dominated in 2013 to 2015. G2P[4]I2 and G9P[8]I1 strains increased every 3 years (G2P[4]I2: 2011 and 2014, G9P[8]I1: 2010, 2013 and 2016). After the 2013 season, 137 cases were collected, 24 of which were vaccinated. Cases requiring drip infusion were fewer in the vaccination group than in the non-vaccination group (16.7% vs 52.2%). No patients required hospitalization in the vaccination group compared with 10.6% in the non-vaccination group. A severe Vesikari score was less common in the vaccination group than in the non-vaccination group (33.3% vs 78.8%). There was no significant difference in the GPI genotype distribution between the two groups. CONCLUSION: Rotaviruses vaccine effectiveness, regardless of GPI genotype, was confirmed in terms of alleviation of disease severity.

Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures.

UNLABELLED: Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE: Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Characterization of the NSP4 gene of group A human rotavirus G1P[8] strains circulating in Sapporo, Japan from 1987 to 2000.

The genetic diversity of the NSP4 gene of rotavirus G1P[8] strains obtained in Sapporo was analyzed, Japan from 1987 to 2000. Sixty-four strains, which were distributed across the whole study period, were included. All G1P[8] NSP4 genes detected in this study belonged to genotype E1, which divided into at least three lineages. The Sapporo rotavirus G1P[8] isolates were found in each lineage. The mean estimated substitution rate was 1.40 × 10(-3) nucleotide substitutions per site per year, which was comparable to that of the G1P[8] VP7 gene. Comparison of the deduced NSP4 amino acid sequences showed genetic diversity at the center of antigenic site II, but not in the enterotoxic domain. This report represents the first investigation of the genetic diversity and evolution of group A rotavirus NSP4 genes in Asia.

Longitudinal analysis of VP7 gene of group A human rotavirus G2P[4] strains circulating in the pre-vaccine era in Sapporo, Japan from 1991 to 2011.

Sequence analysis of the VP7 gene in 23 group A human rotavirus G2P[4] strains obtained during 1991-2011, that is, the pre-vaccine era, in Sapporo, Japan showed considerable genetic diversity, mainly in variable regions. Recent G2P[4] epidemic strains were located in sublineage IVa with a distinctive substitution of D96N. This study provides background data on the genetic variability of G2P[4] rotavirus-VP7 gene prior to the widespread use of rotavirus vaccines in Japan.

Phylogenetic and computational structural analysis of VP7 gene of group a human rotavirus G1P[8] strains obtained in Sapporo, Japan from 1987 to 2000.

Many studies indicate that G1P[8] genotypes are the most prevalent rotavirus strains worldwide. Although two vaccines have been licensed and their value proven in many countries, continuous surveillance for genetic evolution of circulating rotavirus strains before and after the introduction of the vaccines is desirable. G and P typing were carried out on all field strains isolated during 1987-2000 in Sapporo, Japan. Phylogenetic analysis for the VP7 gene of rotavirus G1P[8] strains was performed. Amino acid substitutions were mapped on the predicted three-dimensional VP7 protein image. G1P[8] genotype predominated. One hundred thirteen strains with G1P[8] genotype were analyzed. Phylogenetic studies of the VP7 gene classified these strains into three lineages. The mean estimated substitution rate was 7.25 × 10(-4) nucleotide substitutions per site per year. One predominant lineage contained the mutant strains which had VP7 amino acid substitutions at residue 91 and 212 that is in the neutralization domains. They were estimated to locate in or near intersubunit boundary of VP7 trimer. It is suggested that the most prevalent G1P[8] lineage strains in Sapporo obtained some survival advantages by changing the neutralization domains of VP7.

Full sequence analysis of the original Sapporo virus.

In this study, the full-length genome sequence of the prototype of sapovirus, namely Sapporo virus (SV82), was identified. Sapporo virus RNA was extracted from a fecal sample, amplified by RT-PCR and the PCR products sequenced directly and analyzed. Sequence analysis showed that Sapporo virus consists of 7433 nucleotides and has three open reading frames. The Sapporo strain shows 91.7% nucleotide sequence identity to the Manchester virus. Phylogenic analysis has also revealed the closeness of Sapporo virus to other sapovirus/genogroup I strains. Basic information on the evolutionary history of sapovirus analysis is provided here.

The global spread of rotavirus G10 strains: Detection in Ghanaian children hospitalized with diarrhea.

From October 2003 through September 2004, a total of 289 stool samples were collected from children <5 years of age who had severe diarrhea at admission to or when visiting the emergency department at the Navrongo War Memorial Hospital in rural Ghana during a study on rotavirus disease burden. Rotavirus antigen was detected in 115 stool samples (39.8%) tested for rotavirus. Four rotavirus-positive samples were found to bear G10P[6] specificity by reverse-transcription polymerase chain reaction, polymerase chain reaction-enzyme-linked immunosorbent assay, and oligonucleotide microarray hybridization. Two of these strains further exhibited serotype G10 specificity by neutralization and subgroup II specificity by enzyme immunoassay and possessed long electropheretic patterns by polyacrylamide gel electrophoresis. Their VP7 genes shared a much closer nucleotide identity with other African human G10 strains (>97%) than with human G10 strain from Asia or South America (<86%) or animal strains (<85%). The VP8* genes of the Ghanaian G10 strains exhibited >94% identity to that of human P[6] virus strains and belonged to the P[6] lineage 1a. The deduced VP7 amino acid sequence showed that the Ghanaian strains were more closely related to human G10 strains than to animal G10 strains. The possession of the typical human subgroup II specificity and the P[6] specificity (frequently found in Ghana and the rest of Africa) and the marked similarity in the VP7 antigenic sites suggest that these G10 strains may have evolved through genetic reassortment between bovine and human strains.

Sustained reduction in rotavirus-coded hospitalizations in children aged <5 years after introduction of self-financed rotavirus vaccines in Japan.

This is an extension of our previous study, which evaluated the incidence of seasonal rotavirus gastroenteritis (RVGE) hospitalizations in children aged <5 years from 2009 to 2015 in Japan. Here, we evaluated the incidence of RVGE hospitalizations in children aged <10 years during the rotavirus season (January‒June) from 2009 to 2017 in Japan, before and after the monovalent and pentavalent rotavirus vaccines were introduced in November 2011 and July 2012, using the same health insurance claims database and study methods. In children aged <5 years, the incidence of RVGE hospitalizations greatly declined in 2014 after vaccine introduction, consistent with our previous findings, and the decline was sustained until 2017. However, in children aged ≥5‒<10 years, no apparent trend for a continuous decline in RVGE hospitalizations was observed during the study period. Improved RV vaccination coverage may lead to a further reduction in severe RVGE in Japan.

The VP7 genes of two G9 rotaviruses isolated in 1980 from diarrheal stool samples collected in Washington, DC, are unique molecularly and serotypically.

In a retrospective study of archival diarrheal stool samples collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, DC, we detected three genotype G9P[8] viruses in specimens collected in 1980, which represented the earliest human G9 viruses ever isolated. The VP7 genes of two culture-adapted 1980 G9 viruses were phylogenetically related closely to the lineage 2 G9 virus VP7 gene. Unexpectedly, however, the VP7s of the 1980 G9 viruses were more closely related serotypically to lineage 3 VP7s than to lineage 2 VP7, which may be supported by amino acid sequence analyses of the VP7 proteins.

Acrylamide concentration affects the relative position of VP7 gene of serotype G2 strains as determined by polyacrylamide gel electrophoresis.

BACKGROUND: During the course of development and characterization of various rotavirus reassortants, we found that the relative position of the gene encoding neutralization and protective antigen VP7 of certain rotavirus strains in a PAGE gel was influenced by the concentration of acrylamide. OBJECTIVES: We investigated systematically various factors that affected the relative position of the rotavirus VP7 gene in a PAGE gel. STUDY DESIGN: We analyzed dsRNAs of selected rotavirus strains bearing G1, G2, G3 or G9 specificity by PAGE at varying concentrations of acrylamide. RESULTS: We demonstrated that the relative position of the VP7 gene of three G2 strains varied depending upon the concentration of acrylamide in a PAGE gel, which occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus gene background; and the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this phenomenon when the PAGE running conditions were varied. CONCLUSIONS: The concentration of acrylamide in a PAGE gel was the major factor that influenced the relative position of the VP7 gene of G2 rotavirus strains (i.e., VP7 gene coding assignment by PAGE).

Decline of rotavirus-coded hospitalizations in children under 5 years: A report from Japan where rotavirus vaccines are self-financed.

OBJECTIVES: To estimate the trend in incidence of rotavirus gastroenteritis (RVGE) hospitalization among children aged <5 years in Japan during pre- and post-vaccine periods (2009-2011 and 2012-2015). STUDY DESIGN: This retrospective observational study used a health insurance claims database (constructed by Japan Medical Data Center Co., Ltd.). Rotavirus vaccine became commercially available in 2011. We analyzed data of all children aged <5 years between January 2009 and December 2015. We estimated the incidence rate (IR) of RVGE hospitalization per 1000 person-years from 2009 to 2015 and incidence rate ratio (IRR) of post-vaccine years compared with the averaged pre-vaccine years. IRs and IRRs were also estimated by age group. Primary analysis was limited to the rotavirus season (January to June) of each year. RESULTS: The IR was 6.3-9.3 in pre-vaccine years, 2.3 in 2014, and 3.0 in 2015; the decline was estimated to be 71% in 2014 and 61% in 2015 (p<0.01). By age group, reduction in hospitalizations began in 2013 among children <1 year old, followed by children aged 1 to <5 years in 2014. In the 2014 season, a 65% reduction in RVGE hospitalization was observed in children aged 36 to <60 months, although this age group was unlikely to be vaccinated. CONCLUSIONS: A substantial decline of RVGE hospitalization in 2014 and its persistence was observed among children aged <5 years in Japan after introduction of rotavirus vaccine, although not included in the national immunization program. Indirect effects of rotavirus vaccination were suggested in the 2014 season.

Detection of enteric viruses in rectal swabs from children with acute gastroenteritis attending the pediatric outpatient clinics in Sapporo, Japan.

BACKGROUND: Gastroenteritis is a world-wide disorder. Numerous studies to identify causative viral agents have been reported for hospitalized patients but there are only a few for outpatients with mild symptoms who are usually managed in the outpatient clinics. OBJECTIVES: Our aim was to clarify the epidemiological and clinical characteristics of acute gastroenteritis in children who visited the outpatient clinics with various complaints suggestive of gastroenteritis. STUDY DESIGN: From December 2003 to December 2005, 877 rectal swabs were collected from patients attending outpatient clinics in Sapporo, Japan. Viral genomes of major five enteric viruses (rotavirus, norovirus, adenovirus, astrovirus and sapovirus) and bocavirus were investigated by RT-PCR or PCR. RESULTS: At least one viral agent was found in 326 (37.2%) cases of the 877 studied. Rotaviruses were the most prevalent and were detected in 143 (16.3%) followed by norovirus in 116 (13.2%), adenovirus in 42 (4.8%), astrovirus in 40 (4.6%) and sapovirus in 15 (1.7%) cases. Bocavirus was detected in only 4 (0.5%) cases. Frequent diarrhea and frequent vomiting were prominent in rotavirus and norovirus infection, respectively. CONCLUSIONS: The prevalence of each enteric virus in outpatients resembled that previously estimated in hospitalized patients, although the detection rate of rotavirus was slightly low. The contribution of bocavirus appears to be small.

Development and validation of DNA microarray for genotyping group A rotavirus VP4 (P[4], P[6], P[8], P[9], and P[14]) and VP7 (G1 to G6, G8 to G10, and G12) genes.

Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.

Virological, serological, and clinical features of an outbreak of acute gastroenteritis due to recombinant genogroup II norovirus in an infant home.

Norovirus (NV) is an important cause of acute nonbacterial gastroenteritis worldwide. Recently, several sporadic cases due to naturally occurring recombinant NVs have been reported. In January 2000, there was an outbreak of gastroenteritis in an infant home in Sapporo, Japan. Of 34 residents of the home that were less than 2 years old, 23 developed gastrointestinal symptoms and NV infection was confirmed by conventional reverse transcription-PCR to detect the RNA polymerase region of genogroup II NV. In this virus, the RNA polymerase region shared 86% nucleotide identity with Hawaii virus but only 77% with Mexico virus; however, its capsid region shared only 70% identity with Hawaii virus but 90% with Mexico virus. On the other hand, both regions shared a higher 96% nucleotide identity with Arg320 virus, which was found in Mendoza, Argentina, in 1995 and considered to be a recombinant of Hawaii and Mexico viruses. The findings indicate that the virus involved in the outbreak was similar and may have evolved from the Arg320 virus. Clinically the cases were more severe than those of previously reported sporadic or outbreak cases of NV infection.

Detection and differentiation of Norwalk virus by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.

We have developed a reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (RT-PCR-ELISA), using genetic cluster-specific probes in a microtiter plate format, for the detection and differentiation of Norwalk virus (NV) in stool samples. The specificity of the RT-PCR-ELISA was confirmed by testing 76 stool specimens and 15 tissue culture fluids derived from growths of unrelated viruses. The sensitivity of the RT-PCR-ELISA was compared with conventional PCR and Southern hybridization by testing the four cDNA clones derived from the RNA-dependent RNA polymerase region of the NV68 (NV/GI) virus and viruses in the NV/GII/P1B, the NV/GII/P2A, and the NV/GII/P2B cluster. This assay was as sensitive as the conventional RT-PCR with Southern hybridization regardless of primer pairs and probes used in the experiments. However, the actual sensitivity of this method was higher when clinical stool samples were examined because this assay examines all the samples irrespective of the RT-PCR results. The RT-PCR-ELISA format is simple, time saving, and suitable for testing many samples. It should be reliable for large-scale epidemiological studies of NV.