A matrix gene-based multiplex real-time RT-PCR for detection and differentiation of 2009 pandemic H1N1 and other influenza A viruses in North America

Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA 50011, USA.
Influenza and Other Respiratory Viruses (Impact Factor: 2.2). 11/2010; 4(6):405-10. DOI: 10.1111/j.1750-2659.2010.00153.x
Source: PubMed


The emergence in humans of pandemic (H1N1) 2009 (pH1N1) with similarities to swine influenza viruses (SIVs) caused much concern for both human and animal health as potential for interspecies transmission was initially unknown.
The goal of this study was to develop a real-time RT-PCR test for the detection and differentiation of 2009 pH1N1 and endemic influenza A viruses in North America.
Matrix (M) gene sequences from U.S. human pH1N1 cases and U.S. SIVs were aligned to determine a suitable region for an assay target. Primers were selected to amplify all influenza A. Two probes were designed to differentiate pH1N1 (EA matrix) from endemic (NA matrix) SIVs. The assay was validated using the first U.S. pH1N1 strain, 10 human pH1N1-positive specimens and nine U.S. SIV isolates, then evaluated on 165 specimens of swine and other animal origin submitted to the Iowa State University Veterinary Diagnostic Laboratory. Results were compared to other influenza A PCR assays. Sequences from additional pH1N1 strains and contemporary H1N1 SIVs were used to assess robustness of the selected primers and probes for the intended purpose.
The new assay's results from clinical specimens concurred with confirmatory PCR testing. The additional probe designed from sequence analysis improved detection of the NA matrix subtype when added to the reaction mixture.
This assay detects and differentiates pH1N1 and US influenza A viruses in various sample matrices and species. Good bioinformatics support is critical when designing RT-PCR assays and monitoring their performance.

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    • "RNA was extracted with the MagMAX-96 viral RNA isolation kit per manufacturer's instructions (Life Technologies). rt-RT-PCR was performed using QIAGEN QuantiTect RT-PCR (QIAGEN) and primers and probes designed to detect and differentiate the North American lineage M gene and pH1N1-derived Eurasian M gene [17]. For specific detection of the pH1N1 NA gene, primers and probes were designed targeting sequences unique to Eurasian-lineage NA. "
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    ABSTRACT: The pandemic H1N1 (pH1N1) influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV), were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST) cells with swine-derived endemic H1N2 (MN745) and pH1N1 (MN432) yielded two reassortant H1N2 viruses (R1 and R2), both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log(10) TCID(50)/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.
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