Mika Saitoh

Detection, Quantitation, and Phylogenetic Analysis of Noroviruses in Japanese Oysters

ABSTRACT Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 102 copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008-2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63×10⁻³ and 4.56×10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.

Detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type 1 isolates from patients with acute respiratory infection between 2002 and 2009 in Yamagata prefecture, Japan.

BackgroundHuman parainfluenza virus type 1 (HPIV1) causes various acute respiratory infections (ARI). Hemagglutinin-neuraminidase (HN) glycoprotein of HPIV1 is a major antigen. However, the molecular epidemiology and genetic characteristics of such ARI are not exactly known. Recent studies suggested that a phylogenetic analysis tool, namely the maximum likelihood (ML) method, may be applied to estimate the evolutionary time scale of various viruses. Thus, we conducted detailed genetic analyses including homology analysis, phylogenetic analysis (using both the neighbor joining (NJ) and ML methods), and analysis of the pairwise distances of HN gene in HPIV1 isolated from patients with ARI in Yamagata prefecture, Japan.ResultsA few substitutions of nucleotides in the second binding site of HN gene were observed among the present isolates. The strains were classified into two major clusters in the phylogenetic tree by the NJ method. Another phylogenetic tree constructed by the ML method showed that the strains diversified in the late 1980s. No positively selected sites were found in the present strains. Moreover, the pairwise distance among the present isolates was relatively short.ConclusionsThe evolution of HN gene in the present HPIV1 isolates was relatively slow. The ML method may be a useful phylogenetic method to estimate the evolutionary time scale of HPIV and other viruses.

Molecular epidemiology of the attachment glycoprotein (G) gene in respiratory syncytial virus in children with acute respiratory infection in Japan in 2009/2010

This study performed a detailed genetic analysis of the glycoprotein (G) gene of respiratory syncytial virus (RSV) detected in 50 Japanese children with acute respiratory infection (ARI) in the 2009/2010 season. A phylogenetic tree constructed by the neighbour-joining method showed that 34 and 16 of the RSV strains could be classified into subgroups A and B, respectively. Strains belonging to subgroups A and B were further subdivided into GA2 and BA, respectively. The nucleotide and deduced amino acid sequence identities were relatively high among these strains (>90%). The deduced amino acid sequences implied that a relatively high frequency of amino acid substitutions occurred in the C-terminal 3rd hypervariable region of the G protein in these strains. In addition, some positively selected sites were estimated. The results suggest that RSV with genotypes GA2 and BA was associated with ARI in Japanese children in 2009/2010.

Molecular evolution of haemagglutinin ( H ) gene in measles virus

We studied the molecular evolution of the haemagglutinin (H) gene (full length) in all genotypes (24 genotypes, 297 strains) of measles virus (MeV). The gene’s evolutionary timescale was estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also analysed positive selection sites. The MCMC tree indicated that the MeV H gene diverged from the rinderpest virus (same genus) about 250 years ago and that 24 MeV genotypes formed 3 lineages dating back to a 1915 ancestor (95% highest posterior density [HPD] 1882–1941) with relatively rapid evolution (mean rate: 9.02 × 10−4 substitutions/site/year). The 3 lineages diverged in 1915 (lineage 1, 95% HPD 1882–1941), 1954 (lineage 2, 95% HPD 1937–1969), and 1940 (lineage 3, 95% HPD 1927–1952). These 24 genotypes may have diverged and emerged between the 1940s and 1990s. Selective pressure analysis identified many negative selection sites on the H protein but only a few positive selection sites, suggesting strongly operated structural and/or functional constraint of changes on the H protein. Based on the molecular evolution of H gene, an ancestor MeV of the 24 genotypes emerged about 100 years ago and the structure of H protein has been well conserved.

Molecular Evolution of Hemagglutinin (H) Gene in Measles Virus Genotypes D3, D5, D9, and H1

We studied the molecular evolution of H gene in four prevalent Asian genotypes (D3, D5, D9, and H1) of measles virus (MeV). We estimated the evolutionary time scale of the gene by the Bayesian Markov Chain Monte Carlo (MCMC) method. In addition, we predicted the changes in structure of H protein due to selective pressures. The phylogenetic tree showed that the first division of these genotypes occurred around 1931, and further division of each type in the 1960–1970s resulted in four genotypes. The rate of molecular evolution was relatively slow (5.57×10−4 substitutions per site per year). Only two positively selected sites (F476L and Q575K) were identified in H protein, although these substitutions might not have imparted significant changes to the structure of the protein or the epitopes for phylactic antibodies. The results suggested that the prevalent Asian MeV genotypes were generated over approximately 30–40 years and H protein was well conserved.

Detection and Phylogenetic Analysis of Norovirus in Corbicula fluminea in a Freshwater River in Japan

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 104 copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters.

Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3Cpro and 3Dpol coding regions performed. To assess time‐scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p‐distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10−3 substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77–0.94. The p‐distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

Novel Reassortant Influenza A(H1N2) Virus Derived from A(H1N1)pdm09 Virus Isolated from Swine, Japan, 2012

We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time.

Development of an assay for the detection and quantification of the measles virus nucleoprotein (N) gene using real-time reverse transcriptase PCR

We developed a new quantification method for the measles virus (MeV) nucleoprotein (N) gene using real-time reverse transcriptase PCR. This method allowed us to quantify 10(1)-10(7) copies per reaction (corresponding to 5x10(-1)-5x10(5) copies microl(-1)) of the MeV N gene. We also quantified the MeV N gene from the throat swabs of 22 patients with measles as well as the MeV genotypes A, D3, D5, D9 and H1 in viral suspensions derived from MeV-infected cells. As a result, 3.9x10(3)-5.2x10(6) copies ml(-1) and 7.4x10(7)-2.0x10(8) copies ml(-1) of the MeV genomes (N gene) were detected in the throat swabs and viral suspensions, respectively. No other viruses (enteroviruses, respiratory syncytial virus, human metapneumovirus or mumps virus) were detected in the assay. The results suggest that this method is applicable to the detection and quantification of some genotypes of MeV.