Monitoring and characterization of oseltamivir-resistant pandemic (H1N1) 2009 virus, Japan, 2009-2010.

To monitor and characterize oseltamivir-resistant (OR) pandemic (H1N1) 2009 virus with the H275Y mutation, we analyzed 4,307 clinical specimens from Japan by neuraminidase (NA) sequencing or inhibition assay; 61 OR pandemic (H1N1) 2009 viruses were detected. NA inhibition assay and M2 sequencing indicated that OR pandemic (H1N1) 2009 virus was resistant to M2 inhibitors, but sensitive to zanamivir. Full-genome sequencing showed OR and oseltamivir-sensitive (OS) viruses had high sequence similarity, indicating that domestic OR virus was derived from OS pandemic (H1N1) 2009 virus. Hemagglutination inhibition test demonstrated that OR and OS pandemic (H1N1) 2009 viruses were antigenically similar to the A/California/7/2009 vaccine strain. Of 61 case-patients with OR viruses, 45 received oseltamivir as treatment, and 10 received it as prophylaxis, which suggests that most cases emerged sporadically from OS pandemic (H1N1) 2009, due to selective pressure. No evidence of sustained spread of OR pandemic (H1N1) 2009 was found in Japan; however, 2 suspected incidents of human-to-human transmission were reported.

Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay.

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity.

Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549).

RSV (respiratory syncytial virus)-induced pneumonia and bronchiolitis may be associated with hyperresponsive conditions, including asthma. Eosinophilic proteins such as MBP (major basic protein) may also be associated with the pathophysiology of asthma. To elucidate the roles of RSV infection and MBP in the pathogenesis of pneumonia with hyperresponsiveness, we investigated the effects of RSV infection and MBP on A549 (alveolar epithelial) cells. CPE (cytopathic effects) in A549 cells were observed by microscopy. Apoptosis and cell death was evaluated by flow cytometric analysis and modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We also measured 15 types of cytokines and chemokines in A549 cell supernatants. Although RSV alone did not affect the CPE of A549, high concentrations of MBP resulted in cell death within 24 h. Combinations of RSV and MBP synergistically induced cell death. In A549 cells infected with RSV alone, the release of GM-CSF (granulocyte-macrophage colony-stimulating factor) was significantly enhanced compared with control cells (no infection). In the cells treated with MBP alone, the production of IL (interleukin)-2, 4, 5, 7, 10, 12, 13, 17, IFN (interferon)-γ, GM-CSF, G-CSF (granulocyte colony-stimulating factor) and MIP (macrophage inflammatory protein)-1ß was significantly increased compared with control cells. Notably, the levels of GM-CSF and IL-17 in RSV/MBP-treated cells were significantly higher than those treated with MBP alone. These results suggest that MBP synergistically enhanced the release of various cytokines/chemokines and the cell death of RSV-infected A549 cells, indicating that MBP may be closely associated with the pathophysiology of allergic reactions in bronchiolitis/pneumonia due to RSV.

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

To monitor oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y in neuraminidase (NA) in Japan during 2 influenza seasons, we analyzed 3,216 clinical samples by NA sequencing and/or NA inhibition assay. The total frequency of ORVs was 2.6% (45/1,734) during the 2007-08 season and 99.7% (1,477/1,482) during the 2008-09 season, indicating a marked increase in ORVs in Japan during 1 influenza season. The NA gene of ORVs in the 2007-08 season fell into 2 distinct lineages by D354G substitution, whereas that of ORVs in the 2008-09 season fell into 1 lineage. NA inhibition assay and M2 sequencing showed that almost all the ORVs were sensitive to zanamivir and amantadine. The hemagglutination inhibition test showed that ORVs were antigenetically similar to the 2008-09 vaccine strain A/Brisbane/59/2007. Our data indicate that the current vaccine or zanamivir and amantadine are effective against recent ORVs, but continuous surveillance remains necessary.

A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir.

BACKGROUND: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. RESULTS: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). CONCLUSION: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.

[Current situation in human infections with highly pathogenic avian influenza viruses].

Highly pathogenic avian influenza (HPA1) virus of H5N1 subtype has spread worldwide and continues to cause outbreaks in poultry and sporadic human infections since 2003. As of 9 April 2010, 493 confirmed human cases including 292 fatal cases were reported from 15 countries. Despite emergence of the pandemic A (H1N1)2009 virus, pandemic threats posed by H5N1 subtype virus still remain. H7 subtype avian influenza viruses also pose a similar threat to humans as H5N1 subtype virus. We provide here an updated overview on the human infections of HPA1 viruses in the world.

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10.

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8×10(-3)substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance>0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.

Host Adaptation and the Alteration of Viral Properties of the First Influenza A/H1N1pdm09 Virus Isolated in Japan.

A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions.

Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C.

Human rhinovirus species C (HRV-C) was recently discovered, and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2, and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years, and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10(-3) substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2, and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.

Genetic analysis of human rhinovirus species A to C detected in patients with acute respiratory infection in Kumamoto prefecture, Japan 2011-2012.

We performed detailed genetic analysis of the VP4/VP2 coding region in human rhinovirus species A to C (HRV-ABC) strains detected in patients with a variety of acute respiratory infections in Kumamoto, Japan in the period 2011-12. The phylogenetic tree and evolutionary timescale were obtained by the Bayesian Markov chain Monte Carlo method. Phylogenetic analyses showed that the present HRV-A, -B, and -C strains belonged to 25, 4, and 18 genotypes, respectively. Some new genotypes were confirmed as prevalent strains of HRV-C. An ancestor of the present HRV-ABCs could be dated back to about 20,000 years ago. The present HRV-A and -C strains have wide genetic divergence (pairwise distance >0.2) with rapid evolutionary rates (around 7 × 10(-4) to 4 × 10(-3)substitutions/site/year). Over 100 sites were found to be under negative selection, while no positively selected sites were found in the analyzed region. No evidence of recombination events was found in this region of the present strains. Our results indicate that the present HRV strains have rapidly evolved and subsequently diverged over a long period into multiple genotypes.

Influenza A(H1N1)pdm09 virus and asthma.

Respiratory viral infection is a major cause of asthma exacerbations in both children and adults. Among the respiratory viruses, influenza virus is a particularly important pathogen due to its enormous morbidity and mortality in annual epidemics. The swine-origin influenza A virus, designated as A(H1N1)pdm09, emerged in the spring of 2009 and caused the first influenza pandemic in the 21st century. With the emergence of the novel A(H1N1)pdm09 virus, numerous epidemiologic studies detected asthma as a frequent comorbid condition in patients infected with this virus. Here we review recent reports regarding asthma in patients infected with influenza A(H1N1)pdm09 virus, and we discuss the utility of influenza vaccines and antivirals.

Anti-influenza virus activity of tricin, 4',5,7-trihydroxy-3',5'-dimethoxyflavone.

BACKGROUND: We examined the anti-influenza virus activity of tricin, 4',5,7-trihydroxy-3',5'-dimethoxyflavone, against five viruses: A/Solomon islands/3/2006 (H1N1), A/Hiroshima/52/2005 (H3N2), A/California/07/2009 (H1N1pdm), A/Narita/1/2009 (H1N1pdm) and B/Malaysia/2506/2004 strains in vitro and against A/PR/8/34 virus in vivo. METHODS: The effect of tricin was studied by an infectious virus yield reduction assay. The anti-influenza virus mechanism of the tricin was examined by western blot analysis, real-time reverse transcriptase PCR analysis, haemagglutination inhibition (HI) assay and neuraminidase (NA) inhibition assay. The anti-influenza virus efficacy of tricin was further examined in a murine influenza virus infection model. RESULTS: Tricin of 3.3 to 30 µM significantly reduced seasonal A (H1N1), (H3N2) viruses, novel A (H1N1pdm) virus, as well as B virus in a dose-dependent manner. The 50% effective concentrations of tricin were 3.4 µM for seasonal A (H3N2) virus, 4.9 µM for B virus and 8.2 µM for A/Narita (H1N1pdm) virus. Tricin decreased the expression of haemagglutinin (HA) protein and matrix (M) protein, and messenger RNA expression of HA and M of influenza virus in the infected cells. Tricin exhibited little or no effects on influenza virus HI and NA activities. In the mouse infection model, tricin was significantly effective in reducing body weight loss, and also effective in prolonging survival times of infected mice. CONCLUSIONS: Tricin was indicated to possess anti-influenza virus activity and to ameliorate body weight loss and survival rate of influenza-A-virus-infected mice. Tricin is a novel compound with potential anti-influenza virus activity in vitro and in vivo.

Phylogenetic and cluster analysis of human rhinovirus species A (HRV-A) isolated from children with acute respiratory infections in Yamagata, Japan.

We performed phylogenetic and cluster analysis of human rhinovirus species A (HRV-A) isolated from 76 children with acute respiratory infection in Yamagata prefecture, Japan during the period 2003-2007. Phylogenetic trees based on the nucleotide and amino acid sequences of the VP4/VP2 coding region showed that the present strains could be classified into 11 and 8 clusters, respectively. The homology among the present strains ranged from 66.6% to 100% at the nucleotide level and 84.7% to 100% at the amino acid level. The interspecies distance (mean+/-standard deviation) was calculated to be 0.235+/-0.048 at the nucleotide level and 0.076+/-0.033 at the amino acid level. In addition, the phylogenetic trees created based on the nucleotide and amino acid sequences showed that HRV-A strains belonging to some clusters were associated with both upper respiratory infection and wheezy bronchiolitis, while other strains were associated with upper respiratory infection alone. These results suggest that the present HRV-A isolates had a wide nucleotide divergence and were associated with acute respiratory infection, including upper respiratory infection and wheezy bronchiolitis, in Yamagata prefecture, Japan during the investigation period.

Molecular evolutionary analysis of the influenza A(H1N1)pdm, May-September, 2009: temporal and spatial spreading profile of the viruses in Japan.

BACKGROUND: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009. METHODS/PRINCIPAL FINDINGS: We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer. CONCLUSIONS: Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan.

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

Antigenic and genetic analysis of the hemagglutinin of approximately 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.

Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses.

Annual influenza epidemics in humans affect 5-15% of the population, causing an estimated half million deaths worldwide per year [Stohr K. Influenza-WHO cares. Lancet Infectious Diseases 2002;2(9):517]. The virus can infect this proportion of people year after year because the virus has an extensive capacity to evolve and thus evade the immune response. For example, since the influenza A(H3N2) subtype entered the human population in 1968 the A(H3N2) component of the influenza vaccine has had to be updated almost 30 times to track the evolution of the viruses and remain effective. The World Health Organization Global Influenza Surveillance Network (WHO GISN) tracks and analyzes the evolution and epidemiology of influenza viruses for the primary purpose of vaccine strain selection and to improve the strain selection process through studies aimed at better understanding virus evolution and epidemiology. Here we give an overview of the strain selection process and outline recent investigations into the global migration of seasonal influenza viruses.