Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are ß-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.

Characterization of human Influenza Viruses in Lebanon during 2010-2011 and 2011-2012 post-pandemic seasons.

OBJECTIVE: To genetically characterize human influenza viruses and their susceptibilities to antivirals during two post-pandemic seasons in Lebanon. METHODS: Influenza virus was isolated from nasopharyngeal swabs that were obtained from patients with influenza-like illness during 2010-2012 and further analyzed both phenotypically and genotypically. RESULTS: During the 2010-2011 season, both 2009 pandemic H1N1 (H1N1p) and B viruses co-circulated with equal prevalence, while the H3N2 virus predominated during the 2011-2012 season. All H3N2 and H1N1 viruses were resistant to amantadine. Importantly, all viruses of the influenza A and B types were susceptible to the neuraminidase (NA) inhibitors oseltamivir, zanamivir, peramivir, and laninamivir. Nonetheless, all 2011-2012 H1N1p isolates had three mutations (V241I, N369K, and N386S) in the NA gene that were suggested to be permissive of the H275Y mutation, which confers resistance to oseltamivir. We also detected one H1N1p virus during the 2010-2011 season with a 4-fold decrease in susceptibility to oseltamivir due to an NA-S247N mutation. This isolate was phylogenetically distinct from other H1N1p viruses that were isolated in other regions. CONCLUSIONS: Influenza A viruses with reduced susceptibility to oseltamivir and mutations permissive for acquiring NA resistance-conferring mutation with minimal burden on their fitness were isolated in Lebanon.

Effectiveness of trivalent influenza vaccine among children in two consecutive seasons in a community in Japan.

Influenza vaccination is considered the single most important medical intervention for the prevention of influenza. The dose of trivalent influenza vaccine in children was increased almost double since 2011/12 season in Japan. We estimated the influenza vaccine effectiveness for children 1-11 years of age using rapid test kits in Isahaya City, involving 28,884 children-years, over two consecutive influenza seasons (2011/12 and 2012/13). Children were divided into two groups, vaccinated and unvaccinated, according to their vaccination record, which was obtained from an influenza registration program organized by the Isahaya Medical Association for all pediatric facilities in the city. There were 14,562 and 14,282 children aged from 1-11 years in the city in 2011 and 2012 respectively. In the 2011/12 season, the overall vaccine effectiveness in children from 1-11 years of age, against influenza A and B were 23% [95% confidence interval (CI): 14%-31%] and 20% [95% CI: 8%-31%], respectively. In the 2012/13 season, vaccine effectiveness against influenza A and B was 13% (95% CI: 4%-20%) and 9% (95% CI: -4%-21%), respectively. The vaccine effectiveness was estimated using the rapid diagnosis test kits. Age-stratified estimation showed that vaccine effectiveness was superior in younger children over both seasons and for both virus types. In conclusion, the trivalent influenza vaccine has a significant protective effect for children 1-11 years of age against influenza A and B infection in the 2011/12 season and against influenza A infection in the 2012/13 season in a community in Japan.

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.

Delayed emergence of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A(H1N1)pdm09 viruses in Myanmar.

The prevalence and timing of emergence of oseltamivir-resistant seasonal and pandemic influenza A (H1N1) viruses in Myanmar in 2008 and 2009 are described in this report. In 2008, the oseltamivir-resistant seasonal H1N1 virus was detected at a lower rate (6%) and emerged at least 2 months later when compared with neighboring countries. Similarly, the prevalence of pandemic H1N1 virus was low (3%) and the timing of emergence was late (August 2009) in Myanmar. Interestingly, we detected three isolates that were resistant to both amantadine and oseltamivir. Limited movement of people into the country is attributed to the delayed emergence of drug-resistant seasonal and pandemic A(H1N1) viruses.

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.

Phylogenetic analysis of an off-seasonal influenza virus A (H3N2) in Niigata, Japan, 2010.

The objective of this study was to characterize the off-seasonal influenza virus A subtype H3N2, which caused an outbreak in an elderly hospital in Niigata, Japan. Virus isolates were subtyped by the hemagglutination-inhibition test and screened for antiviral drug sensitivity by real-time PCR using cycling probe technology the and 50% inhibitory concentration (IC(50)) method. Whole genome sequencing was performed in order to determine the phylogeny of the outbreak virus. Seven virus isolates were analyzed in this study, and the results showed that all belonged to the influenza virus A (H3N2). These viruses exhibited the S31N mutation in M2, which confers resistance to amantadine. The results of the IC(50) analysis showed that these viruses were sensitive to both oseltamivir and zanamivir. Whole genome analysis revealed that the virus was similar to the A/Perth/16/2009 strain and that it is a triple reassortant virus with a 3+3+2 pattern of segment recombination.

High frequency of repeated infections due to emerging genotypes of human respiratory syncytial viruses among children during eight successive epidemic seasons in Japan.

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.

New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan.

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.

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.

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.