Rapid detection of influenza A pandemic (H1N1) 2009 virus neuraminidase resistance mutation H275Y by real-time reverse transcriptase PCR.
Ontology highlight
ABSTRACT:
SUBMITTER: Hindiyeh M
PROVIDER: S-EPMC2863859 | biostudies-literature | 2010 May
REPOSITORIES: biostudies-literature
Publications
Rapid detection of influenza A pandemic (H1N1) 2009 virus neuraminidase resistance mutation H275Y by real-time reverse transcriptase PCR.
Hindiyeh Musa M Ram Daniela D Mandelboim Michal M Meningher Tal T Hirsh Shira S Robinov Jana J Levy Virginia V Orzitzer Sara S Azar Roberto R Grossman Zehava Z Mendelson Ella E
Journal of clinical microbiology 20100310 5
The emergence of oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus highlights the need for rapid oseltamivir resistance screening. We report the development and validation of high-throughput real-time reverse transcriptase PCR assays for the detection of the H275Y substitution in the neuraminidase 1 gene that can be accomplished in 3 to 4 h. ...[more]
Similar Datasets
Project description:Real-time reverse transcriptase PCR (rRT-PCR) assays have greatly contributed to the detection, control, and prevention of the pandemic influenza A/H1N1 2009 virus. To improve the rRT-PCR assays for detection of pandemic influenza A/H1N1 2009 virus, we evaluated the sensitivity, specificity, and performance of 12 rRT-PCR primer-probe sets [SW (a) to SW (l)] using a panel of virus strains and clinical specimens. These primer-probe sets were derived from published work and designed for detecting the hemagglutinin (HA) or the neuraminidase (NA) gene of the pandemic influenza A/H1N1 2009 virus. A primer-probe set, SW (CDC), developed by the Centers for Disease Control and Prevention (U.S. CDC) to target the HA gene of pandemic influenza A/H1N1 2009 virus, was used as a referee method. Our results demonstrated that although all primer-probe sets in this study had as high as 98.4 to 100% in silico coverage, some of the primer-probe sets had better specificity, sensitivity, and amplification efficiency than others. Two primer-probe sets, SW (h) and SW (l), which target the NA gene of pandemic influenza A/H1N1 2009 virus, were highly sensitive (10(4) copies/reaction), had high detection rates (56/60, P = 0.134, and 59/60, P = 1.000), and showed ideal specificity compared with SW (CDC). In addition, a cocktail of primer-probe sets targeted to the HA and NA genes displayed higher detection sensitivity than primer-probe sets targeting HA or NA alone, indicating that for practical applications, a combination of primer-probes targeting HA and NA genes is the best option for the detection of pandemic influenza A/H1N1 2009 virus.
| S-EPMC3122877 | biostudies-literature
Project description:Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10(-1.3 - -0.7) 50% infectious doses (ID(50)) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation.
| S-EPMC3147828 | biostudies-literature
Project description:Rapid methods for the detection and confirmatory identification of pandemic influenza A virus (also known as pandemic [H1N1] 2009) are of utmost importance. In this study, a conventional reverse transcriptase PCR (RT-PCR) assay for the detection of influenza A virus and the hemagglutinin of swine lineage H1 (swH1) was designed, optimized, and validated. Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal, or throat swab specimens collected early in the outbreak (127 negative specimens, 66 specimens with pandemic [H1N1] 2009 influenza virus, 3 specimens with seasonal [H1N1] influenza A virus, and 2 specimens with seasonal [H3N2] influenza A virus). The performance characteristics of the duplex RT-PCR assay were assessed and compared to those of various detection methods: a monoplex RT-PCR assay at the National Microbiology Laboratory, a real-time RT-PCR assay using a Centers for Disease Control and Prevention protocol, an in-house multiplex RT-PCR assay (targeting influenza A virus, influenza B virus, and respiratory syncytial virus), and a rapid antigen test (the Binax Now Influenza A & B assay). The sensitivity of the duplex RT-PCR assay for influenza A virus detection was 97.2%, whereas the sensitivities were 74.6%, 71.8%, 47.8%, and 12.7% for the other four assays, respectively. The duplex RT-PCR assay was also able to identify swH1 in 94% of the cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmatory identification. Only a limited number of specimens that contained influenza A virus had amounts of virus that fell below the limit of detection of the assay with the swH1 primers. Overall, the duplex RT-PCR assay is a reliable method for the simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus and would be particularly attractive to laboratories without real-time RT-PCR capabilities.
| S-EPMC2786647 | biostudies-literature
Project description: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.
| S-EPMC7173154 | biostudies-literature
Project description:Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the "gold standard" for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10(-6) dilution of 4 x 10(6) 50% tissue culture infective doses/ml when 5 microl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was < or =3.13%, showing good reproducibility. The assays had a wide dynamic range for the detection of pandemic (H1N1) 2009 virus and utilized testing platforms appropriate for high diagnostic throughput with rapid turnaround times. We developed and validated these real-time PCR procedures with the goal that they will be useful for diagnosis and surveillance of pandemic (H1N1) 2009 virus. These findings will contribute to the informed management of this novel virus.
| S-EPMC2772599 | biostudies-literature
Project description:BACKGROUND:Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES:We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN:Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS:121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS:We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.
| S-EPMC7185517 | biostudies-literature
Project description:BackgroundThere is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods.ObjectivesFirst, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections.MethodsRRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v.ResultsThe "perfect match" M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a "gold standard", while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. "Perfect match" M gene RRT PCR had 100% sensitivity and 95.2% specificity for swabs, 93.6% and 98.6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99.1%, respectively, for the swabs, and 100% and 100% for the tissues.ConclusionsTwo RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.
| S-EPMC4634650 | biostudies-literature
Project description:The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents.
| S-EPMC3732439 | biostudies-literature
Project description:BackgroundExposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.Methods and resultsAntibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.ConclusionsThe NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.
| S-EPMC3200314 | biostudies-literature
Project description:Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.
| S-EPMC3349948 | biostudies-literature