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One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.

ABSTRACT: Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R(2)=0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.

SUBMITTER: Nakauchi M

PROVIDER: S-EPMC7173154 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.

Nakauchi Mina M Yasui Yoshihiro Y Miyoshi Tatsuya T Minagawa Hiroko H Tanaka Tomoyuki T Tashiro Masato M Kageyama Tsutomu T

Journal of virological methods 20101026 1

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PC ...[more]

PMID: 21029748

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Project description:Reassortment of gene segments between coinfecting influenza A viruses (IAVs) facilitates viral diversification and has a significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have cocirculated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous coinfections with pH1N1 and H3N2 viruses were compared to those obtained following coinfection with homologous, genetically tagged, pH1N1 viruses as a control. High genotype diversity was observed among progeny of both coinfections; however, diversity was more limited following heterologous coinfection. Pairwise analysis of genotype patterns revealed that homologous reassortment was random while heterologous reassortment was characterized by specific biases. pH1N1/H3N2 reassortant genotypes produced under single-cycle coinfection conditions showed a strong preference for homologous PB2-PA combinations and general preferences for the H3N2 NA, pH1N1 M, and H3N2 PB2 except when paired with the pH1N1 PA or NP. Multicycle coinfection results corroborated these findings and revealed an additional preference for the H3N2 HA. Segment compatibility was further investigated by measuring chimeric polymerase activity and growth of selected reassortants in human tracheobronchial epithelial cells. In guinea pigs inoculated with a mixture of viruses, parental H3N2 viruses dominated but reassortants also infected and transmitted to cage mates. Taken together, our results indicate that strong intrinsic barriers to reassortment between seasonal H3N2 and pH1N1 viruses are few but that the reassortants formed are attenuated relative to parental strains.IMPORTANCE The genome of IAV is relatively simple, comprising eight RNA segments, each of which typically encodes one or two proteins. Each viral protein carries out multiple functions in coordination with other viral components and the machinery of the cell. When two IAVs coinfect a cell, they can exchange genes through reassortment. The resultant progeny viruses often suffer fitness defects due to suboptimal interactions among divergent viral components. The genetic diversity generated through reassortment can facilitate the emergence of novel outbreak strains. Thus, it is important to understand the efficiency of reassortment and the factors that limit its potential. The research described here offers new tools for studying reassortment between two strains of interest and applies those tools to viruses of the 2009 pandemic H1N1 and seasonal H3N2 lineages, which currently cocirculate in humans and therefore have the potential to give rise to novel epidemic strains.

Project description:BackgroundTwo subtypes of influenza A currently circulate in humans: seasonal H3N2 (sH3N2, emerged in 1968) and pandemic H1N1 (pH1N1, emerged in 2009). While the epidemiological characteristics of the initial wave of pH1N1 have been studied in detail, less is known about its infection dynamics during subsequent waves or its severity relative to sH3N2. Even prior to 2009, few data was available to estimate the risk of severe outcomes following infection with one circulating influenza strain relative to another.MethodsWe analyzed antibodies in quadruples of sera from individuals in Hong Kong collected between July 2009 and December 2011, a period that included three distinct influenza virus epidemics. We estimated infection incidence using these assay data and then estimated rates of severe outcomes per infection using population-wide clinical data.ResultsCumulative incidence of infection was high among children in the first epidemic of pH1N1. There was a change towards the older age group in the age distribution of infections for pH1N1 from the first to the second epidemic, with the age distribution of the second epidemic of pH1N1 more similar to that of sH3N2. We found no serological evidence that individuals were infected in both waves of pH1N1. The risks of excess mortality conditional on infection were higher for sH3N2 than for pH1N1, with age-standardized risk ratios of 2.6 [95% CI: 1.8, 3.7] for all causes and 1.5 [95% CI: 1.0, 2.1] for respiratory causes throughout the study period.ConclusionsOverall increase in clinical incidence of pH1N1 and higher rates of severity in older adults in post pandemic waves were in line with an age-shift in infection towards the older age groups. The absence of repeated infection is good evidence that waning immunity did not cause the second wave. Despite circulating in humans since 1968, sH3N2 is substantially more severe per infection than the pH1N1 strain. Infection-based estimates of individual-level severity have a role in assessing emerging strains; updating seasonal vaccine components; and optimizing of vaccination programs.

Project description:BACKGROUND:The mortality burden of the 2009 A/H1N1 pandemic remains unclear in many countries due to delays in reporting of death statistics. We estimate the age- and cause-specific excess mortality impact of the pandemic in France, relative to that of other countries and past epidemic and pandemic seasons. METHODS:We applied Serfling and Poisson excess mortality approaches to model weekly age- and cause-specific mortality rates from June 1969 through May 2010 in France. Indicators of influenza activity, time trends, and seasonal terms were included in the models. We also reviewed the literature for country-specific estimates of 2009 pandemic excess mortality rates to characterize geographical differences in the burden of this pandemic. RESULTS:The 2009 A/H1N1 pandemic was associated with 1.0 (95% Confidence Intervals (CI) 0.2-1.9) excess respiratory deaths per 100,000 population in France, compared to rates per 100,000 of 44 (95% CI 43-45) for the A/H3N2 pandemic and 2.9 (95% CI 2.3-3.7) for average inter-pandemic seasons. The 2009 A/H1N1 pandemic had a 10.6-fold higher impact than inter-pandemic seasons in people aged 5-24 years and 3.8-fold lower impact among people over 65 years. CONCLUSIONS:The 2009 pandemic in France had low mortality impact in most age groups, relative to past influenza seasons, except in school-age children and young adults. The historical A/H3N2 pandemic was associated with much larger mortality impact than the 2009 pandemic, across all age groups and outcomes. Our 2009 pandemic excess mortality estimates for France fall within the range of previous estimates for high-income regions. Based on the analysis of several mortality outcomes and comparison with laboratory-confirmed 2009/H1N1 deaths, we conclude that cardio-respiratory and all-cause mortality lack precision to accurately measure the impact of this pandemic in high-income settings and that use of more specific mortality outcomes is important to obtain reliable age-specific estimates.

Dataset Information

One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.

Publications

One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.

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