Miho Kobayashi

Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan

We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p‐distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10−3 substitutions/site per year). The p‐distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV‐A emerged approximately10 years ago and spread to some countries with a high evolution rate.

Molecular epidemiological study of human rhinovirus species A, B and C from patients with acute respiratory illnesses in Japan

Recent studies suggest that human rhinovirus species A, B and C (HRV-ABCs) may be associated with both the common cold and severe acute respiratory illnesses (ARIs) such as bronchiolitis, wheezy bronchiolitis and pneumonia. However, the state and molecular epidemiology of these viruses in Japan is not fully understood. This study detected the genomes of HRV-ABCs from Japanese patients (92 cases, 0-36 years old, mean±sd 3.5±5.0 years) with various ARIs including upper respiratory infection, bronchiolitis, wheezy bronchiolitis, croup and pneumonia between January and December 2010. HRV-ABCs were provisionally type assigned from the pairwise distances among the strains. On phylogenetic trees based on the nucleotide sequences of the VP4/VP2 coding region, HRV-A, -B and -C were provisionally assigned to 14, 2 and 12 types, respectively. The present HRV-A and -C strains had a wide genetic diversity (>30 % divergence). The interspecies distances were 0.230±0.063 (mean±sd, HRV-A), 0.218±0.048 (HRV-B) and 0.281±0.105 (HRV-C), based on nucleotide sequences, and 0.075±0.036 (HRV-A), 0.049±0.022 (HRV-B) and 0.141±0.064 (HRV-C) at the deduced amino acid level. Furthermore, HRV-A and -C were the predominant species and were detected throughout the seasons. The results suggested that HRV-A and -C strains have a wide genetic divergence and are associated with various ARIs in Japan.

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997-2002) and that of genotype ON1 in 2005 (95% HPD; 2000-2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03×10(-3) vs. 4.61×10(-3) substitutions/site/year, p<0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.

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 the capsid gene in human norovirus genogroup II.

We studied the molecular evolution of the capsid gene in all genotypes (genotypes 1–9) of human norovirus (NoV) genogroup I. The evolutionary time scale and rate were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also performed selective pressure analysis and B-cell linear epitope prediction in the deduced NoV GI capsid protein. Furthermore, we analysed the effective population size of the virus using Bayesian skyline plot (BSP) analysis. A phylogenetic tree by MCMC showed that NoV GI diverged from the common ancestor of NoV GII, GIII, and GIV approximately 2,800 years ago with rapid evolution (about 10−3 substitutions/site/year). Some positive selection sites and over 400 negative selection sites were estimated in the deduced capsid protein. Many epitopes were estimated in the deduced virus capsid proteins. An epitope of GI.1 may be associated with histo-blood group antigen binding sites (Ser377, Pro378, and Ser380). Moreover, BSP suggested that the adaptation of NoV GI strains to humans was affected by natural selection. The results suggested that NoV GI strains evolved rapidly and date back to many years ago. Additionally, the virus may have undergone locally affected natural selection in the host resulting in its adaptation to humans.

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.

The impact of Saffold cardiovirus in patients with acute respiratory infections in Yamagata, Japan

From the 1 Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi-shi, Gunma, 2 Yamagata Prefectural Institute of Public Health, Yamagata-shi, Yamagata, 3 Yamanobe Pediatric Clinic, Higashimurayama-gun, Yamagata, 4 Pathogen Genomics Center, National Institute of Infectious Diseases, shinjuku-ku, Tokyo, 5 Department of Virology III, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, 6 Department of Molecular Biodefence Research, Yokohama City University Graduate School of Medicine, Yokohama-shi, Kanagawa, and 7 Infectious Diseases Surveillance Center, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan

Stability of Chlamydophila pneumoniae in a harsh environment without a requirement for acanthamoebae

The effect of actual interactions between Chlamydophila pneumoniae and amoebae (Acanthamoeba) on the survival of C. pneumoniae was investigated. C. pneumoniae and amoebae were detected in 75 soil samples by IFU assay and PCR. Although C. pneumoniae could not be cultured, the DNA prevalence of C. pneumoniae and amoebae in natural soil was 20% and 92% (no correlation between the prevalence of DNA was observed). The viability of C. pneumoniae spiked in autoclaved soil was assessed by IFU assay and RT‐PCR. Although the number of infective progeny decreased for three days, transcripts of C. pneumoniae were detected for up to 98 days independently of amoebae. The stability of C. pneumoniae in liquid medium was also assessed by IFU assay and transmission electron microscopy. The bacteria could survive at 15°C for 14 days independently of amoebae. Bacteria cultured without amoebae were confirmed to have normal structures. Thus, the presence of amoebae has no effect on C. pneumoniae survival, and the bacteria can survive in the absence of host cells for an extended period of time.