Tsutomu Kageyama

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

ABSTRACT We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

Genogroup-specific PCR primers for detection of Norwalk-like viruses.

Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.

The global circulation of seasonal influenza A (H3N2) viruses.

Antigenic and genetic analysis of the hemagglutinin of ∼13,000 human influenza A (H3N2) viruses from six continents during 2002–2007 revealed that there was continuous circulation in east and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions were seeded from this network each year. Seed strains generally first reached Oceania, North America, and Europe, and later South America. This evidence suggests that once A (H3N2) viruses leave E-SE Asia, they are unlikely to contribute to long-term viral evolution. If the trends observed during this period are an accurate representation of overall patterns of spread, then the antigenic characteristics of A (H3N2) viruses outside E-SE Asia may be forecast each year based on surveillance within E-SE Asia, with consequent improvements to vaccine strain selection.

Coexistence of Multiple Genotypes, Including Newly Identified Genotypes, in Outbreaks of Gastroenteritis Due to Norovirus in Japan

ABSTRACT Norovirus (NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology 299:225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol. 41:1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.

One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses

Abstract Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R 2 =0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.

Proteolytic Processing of Sapovirus ORF1 Polyprotein

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

Poly(A)- and Primer-Independent RNA Polymerase of Norovirus

ABSTRACT Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3′ genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.

Oseltamivir-resistant influenza viruses A (H1N1) during 2007-2009 influenza seasons, Japan.

Prevalence of these viruses increased during the 2008–09 season.

Full-length GBV-C/HGV genomes from nine Japanese isolates: characterization by comparative analyses

SummaryThe genomes of nine GBV-C/HGV isolates from Japanese chronic hepatitis patients were fully sequenced and characterized. They shared 85% nucleotide sequence homology with previously characterized isolates from the US and West Africa. Homology studies and phylogenetic analyses showed that the Japanese isolates formed a third group distinct from the established groups 1 and 2. The genetic distances between the three groups of GBV-C/HGV were very similar to the distances between the two classical swine fever virus (CSFV) serotypes, which suggested that they might belong to a separate GBV-C/HGV serotype. Plot similarity analysis comparing the three groups exposed relatively conserved terminal non-coding regions. Hairpin structures predicted in the Japanese isolates are probably involved in viral replication. The region coding E1-E2-NS-2 showed the least similarity (80%); in HCV the similarity here is only 50% due to its hypervariablity. NS-3 and NS-5b that respectivity encode the helicase/protease and RNA-dependent RNA polymerase, had a high degree of amino acid homo- logy, suggesting a high degree of functional constraint in this region. The NS-5b nucleotide sequence was highly conserved perhaps because of constraints from RNA secondary structure and/or an open reading frame in the negative strand.

Slow evolutionary rate of GB virus C/hepatitis G virus.

Abstract. With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 × 10−6 per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000–10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection.