Clyde Dapat

New Genotypes within Respiratory Syncytial Virus Group B Genotype BA in Niigata, Japan

ABSTRACT Phylogenetic analysis of respiratory syncytial virus (RSV) group B genotype BA strains from the 2002-2003 to 2009-2010 seasons collected in Niigata, Japan, revealed four distinct clusters, designated new BA genotypes BA7, BA8, BA9, and BA10. These new genotypes were not associated with large outbreaks in the community.

Emergence of H274Y oseltamivir-resistant A(H1N1) influenza viruses in Japan during the 2008–2009 season

BACKGROUND A substantial increase in oseltamivir-resistant A(H1N1) influenza viruses was reported in Europe in late 2007. OBJECTIVES To monitor the antiviral susceptibility profile of human A(H1N1) influenza viruses in Japan during the 2007-2008 and 2008-2009 seasons. STUDY DESIGN Viruses were obtained from respiratory samples of patients with influenza collected in Japan between December 2007 and April 2008 (n=1046) and between December 2008 and April 2009 (n=1789). Oseltamivir resistance was determined by an H274Y-specific real-time PCR cycling probe assay and a neuraminidase inhibition assay. Amantadine resistance was assessed by sequencing the M2 gene. Sequencing of the hemagglutinin and NA genes was performed to infer phylogenetic relationships between different strains. RESULTS Three of 687 (0.4%) A(H1N1) viruses from the 2007-2008 season and 745 of 745 (100%) viruses from the 2008-2009 season carried the NA-H274Y substitution and demonstrated a >300-fold reduction in oseltamivir susceptibility. All oseltamivir-resistant viruses from the 2008-2009 season possessed an A193T substitution in the receptor-binding domain of the hemagglutinin. Amantadine resistance was detected in 431 of 687 (62.7%) and 0 of 745 (0.0%) of the A(H1N1) viruses from the 2007-2008 and 2008-2009 seasons, respectively. CONCLUSIONS A dramatic surge in oseltamivir-resistant A(H1N1) viruses possessing the NA-H274Y substitution was detected in Japan during the 2008-2009 season. The emergence of oseltamivir-resistant viruses was facilitated by mutations in the viral genome. Intensified surveillance, including phenotypic assays and sequencing of the hemagglutinin, neuraminidase, and M2 gene would allow monitoring of the spread and evolution of drug-resistant influenza virus variants.

Genetic Characterization of Human Influenza Viruses in the Pandemic (2009–2010) and Post-Pandemic (2010–2011) Periods in Japan

Background Pandemic influenza A(H1N1) 2009 virus was first detected in Japan in May 2009 and continued to circulate in the 2010–2011 season. This study aims to characterize human influenza viruses circulating in Japan in the pandemic and post-pandemic periods and to determine the prevalence of antiviral-resistant viruses. Methods Respiratory specimens were collected from patients with influenza-like illness on their first visit at outpatient clinics during the 2009–2010 and 2010–2011 influenza seasons. Cycling probe real-time PCR assays were performed to screen for antiviral-resistant strains. Sequencing and phylogenetic analysis of the HA and NA genes were done to characterize circulating strains. Results and Conclusion In the pandemic period (2009–2010), the pandemic influenza A(H1N1) 2009 virus was the only circulating strain isolated. None of the 601 A(H1N1)pdm09 virus isolates had the H275Y substitution in NA (oseltamivir resistance) while 599/601 isolates (99.7%) had the S31N substitution in M2 (amantadine resistance). In the post-pandemic period (2010–2011), cocirculation of different types and subtypes of influenza viruses was observed. Of the 1,278 samples analyzed, 414 (42.6%) were A(H1N1)pdm09, 525 (54.0%) were A(H3N2) and 33 (3.4%) were type-B viruses. Among A(H1N1)pdm09 isolates, 2 (0.5%) were oseltamivir-resistant and all were amantadine-resistant. Among A(H3N2) viruses, 520 (99.0%) were amantadine-resistant. Sequence and phylogenetic analyses of A(H1N1)pdm09 viruses from the post-pandemic period showed further evolution from the pandemic period viruses. For viruses that circulated in 2010–2011, strain predominance varied among prefectures. In Hokkaido, Niigata, Gunma and Nagasaki, A(H3N2) viruses (A/Perth/16/2009-like) were predominant whereas, in Kyoto, Hyogo and Osaka, A(H1N1)pdm09 viruses (A/New_York/10/2009-like) were predominant. Influenza B Victoria(HA)-Yamagata(NA) reassortant viruses (B/Brisbane/60/2008-like) were predominant while a small proportion was in Yamagata lineage. Genetic variants with mutations at antigenic sites were identified in A(H1N1)pdm09, A(H3N2) and type-B viruses in the 2010–2011 season but did not show a change in antigenicity when compared with respective vaccine strains.

Genetic Makeup of Amantadine-Resistant and Oseltamivir-Resistant Human Influenza A/H1N1 Viruses

ABSTRACT The emergence and widespread occurrence of antiviral drug-resistant seasonal human influenza A viruses, especially oseltamivir-resistant A/H1N1 virus, are major concerns. To understand the genetic background of antiviral drug-resistant A/H1N1 viruses, we performed full genome sequencing of prepandemic A/H1N1 strains. Seasonal influenza A/H1N1 viruses, including antiviral-susceptible viruses, amantadine-resistant viruses, and oseltamivir-resistant viruses, obtained from several areas in Japan during the 2007-2008 and 2008-2009 influenza seasons were analyzed. Sequencing of the full genomes of these viruses was performed, and the phylogenetic relationships among the sequences of each individual genome segment were inferred. Reference genome sequences from the Influenza Virus Resource database were included to determine the closest ancestor for each segment. Phylogenetic analysis revealed that the oseltamivir-resistant strain evolved from a reassortant oseltamivir-susceptible strain (clade 2B) which circulated in the 2007-2008 season by acquiring the H275Y resistance-conferring mutation in the NA gene. The oseltamivir-resistant lineage (corresponding to the Northern European resistant lineage) represented 100% of the H1N1 isolates from the 2008-2009 season and further acquired at least one mutation in each of the polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), hemagglutinin (HA), and neuraminidase (NA) genes. Therefore, a reassortment event involving two distinct oseltamivir-susceptible lineages, followed by the H275Y substitution in the NA gene and other mutations elsewhere in the genome, contributed to the emergence of the oseltamivir-resistant lineage. In contrast, amantadine-resistant viruses from the 2007-2008 season distinctly clustered in clade 2C and were characterized by extensive amino acid substitutions across their genomes, suggesting that a fitness gap among its genetic components might have driven these mutations to maintain it in the population.

Epidemiology of Human Influenza A and B Viruses in Myanmar from 2005 to 2007

Objectives: To perform genetic analysis of influenza A and B viruses in Myanmar from 2005 to 2007 and to determine the prevalence of amantadine-resistant influenza A viruses. Methods: Phylogenies of the HA and NA genes were analyzed and mutations in M2 that confer resistance to amantadine were screened. Results: Influenza in Myanmar exhibited seasonality, which coincided during the rainy season from June to August. Out of 2,618 samples, 76 influenza A and 132 influenza B viruses were isolated. Phylogenetic analysis showed that in 2005, 11 A/H1N1 isolates formed one cluster with A/Solomon Islands/3/2006 and were amantadine-sensitive strains. One A/H3N2 isolate was amantadine-resistant harboring S31N mutation in M2 and possessing S193F and D225N substitutions in HA (clade N), similar to A/Wisconsin/67/2005. No viruses were isolated in 2006 due to sample storage failure. In 2007, all 64 A/H3N2 isolates were amantadine-resistant and similar to A/Brisbane/10/2007. For influenza B, 3 Yamagata-lineage and 17 Victoria-lineage isolates were detected in 2005 and 112 Victoria-lineage viruses were isolated in 2007. All Victoria-lineage isolates were reassortants possessing NA derived from the Yamagata lineage. Conclusion: Continuous surveillance in tropical countries is important for elucidating the seasonality of influenza and determining the molecular characteristics of circulating strains.

Reduced effectiveness of oseltamivir in children infected with oseltamivir-resistant influenza A (H1N1) viruses with His275Tyr mutation.

Background: Little is known about whether neuraminidase inhibitors are effective for children infected with oseltamivir-resistant influenza A(H1N1) viruses. Methods: Children aged 15 years and younger having influenza-like illness and who visited outpatient clinics within 48 hours of fever onset were enrolled from 2006–2007 to 2008–2009 influenza seasons in Japan. Patients received oseltamivir, zanamivir, or no treatment after screening by a rapid antigen test. Nasopharyngeal swabs were collected before antiviral therapy and were used for virus isolation. Oseltamivir resistance was determined by detection of the H275Y mutation in neuraminidase, and susceptibility test using neuraminidase inhibition assay. Daily body temperature was evaluated according to drug type and susceptibility by univariate and multivariate analyses. Results: Of 1647 patients screened, 238 oseltamivir-resistant H1N1 cases (87 oseltamivir-treated, 64 zanamivir-treated, and 87 nontreated) and 110 oseltamivir-susceptible cases (60 oseltamivir-treated and 50 nontreated) were evaluated. In oseltamivir-resistant cases, fever on days 4 to 5 after the start of treatment was significantly higher in oseltamivir-treated and nontreated than in zanamivir-treated patients (P < 0.05). In oseltamivir-susceptible cases, fever was significantly lower in oseltamivir-treated than nontreated on days 3 to 6 (P < 0.01). Similar findings were obtained for duration of the fever and proportion of recurrent fever. Reduced effectiveness of oseltamivir was more prominent in children 0 to 6 years old than in those 7 to 15 years old. Multiple logistic regression analysis showed that lower age, nontreatment, and oseltamivir treatment of oseltamivir-resistant patients were factors associated with the duration of the longer fever. Conclusions: Infection with oseltamivir-resistant viruses significantly reduced the effectiveness of oseltamivir, and this tendency was more apparent in younger children.

Rare influenza A (H3N2) variants with reduced sensitivity to antiviral drugs.

In 2007 and 2008 in Myanmar, we detected influenza viruses A (H3N2) that exhibited reduced sensitivity to both zanamivir and amantadine. These rare and naturally occurring viruses harbored a novel Q136K mutation in neuraminidase and S31N mutation in M2.

High Frequency of Repeated Infections Due to Emerging Genotypes of Human Respiratory Syncytial Viruses among Children during Eight Successive Epidemic Seasons in Japan

ABSTRACT In eight successive seasons (2001 to 2009), a total of 726 human respiratory syncytial virus (HRSV) infections from a total of 1,560 children with acute lower respiratory tract illness were identified. Molecular analysis of the attachment (G) protein gene confirmed that 52 (7.8%) children were infected more than once with any of the 3 genotypes of HRSV-A (genotypes GA5, NA1, and NA2) and/or 6 genotypes of HRSV-B (genotypes BA4, BA5, and BA7 to BA10). Repeated infections in 46 cases (82.1%) occurred in the next season, and only one case occurred in the same season (10-day interval). First infections were 33 (63.5%) HRSV-A cases and 19 (36.5%) HRSV-B cases, whereas second infections occurred in 35 (67.3%) HRSV-A cases and 17 (32.7%) HRSV-B cases. Third infections were attributed to 4 (100.0%) HRSV-A cases. Homologous subgroup reinfections were detected in 28 cases, 23 HRSV-A cases and 5 HRSV-B cases (P = 0.005), whereas homologous genotype reinfections were detected only for 5 HRSV-A cases (2GA5 and 3NA2) but not any HRSV-B case. Heterologous subgroup reinfections were detected in 28 cases, 12 cases from HRSV-A-to-HRSV-B reinfections and 16 cases from HRSV-B-to-HRSV-A reinfections. Genotypes NA1 and NA2 had higher numbers of heterologous genotype infections than did other genotypes. Our observations suggest that repeated infections occur more frequently in HRSV-A strains than in HRSV-B strains, and heterologous genotype reinfections occur more frequently than homologous genotype reinfections, especially in the case of the emerging genotypes NA1 and NA2 of HRSV-A strains that circulated in the community during our study period.

Neuraminidase inhibitor susceptibility profile of pandemic and seasonal influenza viruses during the 2009–2010 and 2010–2011 influenza seasons in Japan

Two new influenza virus neuraminidase inhibitors (NAIs), peramivir and laninamivir, were approved in 2010 which resulted to four NAIs that were used during the 2010-2011 influenza season in Japan. This study aims to monitor the susceptibility of influenza virus isolates in 2009-2010 and 2010-2011 influenza seasons in Japan to the four NAIs using the fluorescence-based 50% inhibitory concentration (IC₅₀) method. Outliers were identified using box-and-whisker plot analysis and full NA gene sequencing was performed to determine the mutations that are associated with reduction of susceptibility to NAIs. A total of 117 influenza A(H1N1)pdm09, 59 A(H3N2), and 18 type B viruses were tested before NAI treatment and eight A(H1N1)pdm09 and 1 type B viruses were examined from patients after NAI treatment in the two seasons. NA inhibition assay showed type A influenza viruses were more susceptible to NAIs than type B viruses. The peramivir and laninamivir IC₅₀ values of both type A and B viruses were significantly lower than the oseltamivir and zanamivir IC₅₀ values. Among influenza A(H1N1)pdm09 viruses, the prevalence of H274Y viruses increased from 0% in the 2009-2010 season to 3% in the 2010-2011 season. These H274Y viruses were resistant to oseltamivir and peramivir with 200-300 fold increase in IC₅₀ values but remained sensitive to zanamivir and laninamivir. Other mutations in NA, such as I222T and M241I were identified among the outliers. Among influenza A(H3N2) viruses, two outliers were identified with D151G and T148I mutations, which exhibited a reduction in susceptibility to oseltamivir and zanamivir, respectively. Among type B viruses, no outliers were identified to the four NAIs. For paired samples that were collected before and after drug treatment, three (3/11; 27.3%) H274Y viruses were identified among A(H1N1)pdm09 viruses after oseltamivir treatment but no outliers were found in the laninamivir-treatment group (n=3). Despite widespread use of NAIs in Japan, the prevalence of NAI-resistant influenza viruses is still low.

Identification of Oseltamivir Resistance among Pandemic and Seasonal Influenza A (H1N1) Viruses by an His275Tyr Genotyping Assay Using the Cycling Probe Method

ABSTRACT Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.