No abstract

Our data indicate that NV can cause severe gastroenteritis and is an important etiologic agent in hospitalized cases, whereas SV causes mild gastroenteritis in Japanese children.

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. IMPORTANCESerial 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. R otaviruses, which form one genus within the family Reoviridae, are divided into at least seven species/groups (A to G/H) (1, 2). Although group A to C rotaviruses have been detected in humans with diarrhea, group A rotavirus is the single most important etiologic agent causing severe diarrhea in infants and young children worldwide, resulting in approximately 453,00 deaths (37% of deaths attributable to diarrhea and 5% of all deaths) among children Ͻ5 years of age in 2008 (3). In the United States alone, rotavirus (RV) infections are estimated to cause approximately 20 to 30 deaths, 50,000 to 67,000 hospitalizations, 390,000 to 410,000 physician visits, and a more than $890 million to $1 billion societal cost annually (4, 5). Thus, the introduction of a RV vaccine capable of alleviating this enormous health burden has been an important global public health goal.The RV genome consisting of 11 segments of double-stranded RNA (dsRNA) encodes six structural protein...

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.

Norwalk virus and Sapporo virus (SV) were approved as type species of the genus Norwalk-like viruses and the genus Sapporo-like viruses, respectively, in the family Caliciviridae. Nested polymerase chain reaction (PCR), using newly designed primers in the RNA-dependent RNA polymerase region, was developed to detect and differentiate viruses in the three genetic groups of SV based on the relative size of the PCR products obtained. In addition, a booster nested PCR that performs nested PCR in a single tube was introduced to reduce the chance of contamination during the procedure of standard nested PCR. The specificity of the newly developed PCR was confirmed by testing 77 stool specimens and 16 tissue culture fluids derived from growth of unrelated viruses. The sensitivity of the nested PCR was compared with the conventional PCR using Sapp35/Sapp36 primer pair by testing the three cDNA clones obtained from viruses in the SV/SV82, the SV/London92, and the SV/Parkville virus, respectively. This assay can detect SV in a more sensitive way than the conventional PCR and Southern hybridization. Sensitive and suitable methods to detect and differentiate SV are required to obtain accurate epidemiological data on these viruses and the standard and booster nested PCR should be a very useful tool for this purpose.

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. J. Med. Virol. 84:832–838, 2012. © 2012 Wiley Periodicals, Inc.

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.

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.