Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs

Authors: SLOMKA, MAREK - Veterinary Laboratories Agency (VLA); DENSHAM, ANSTICE - Veterinary Laboratories Agency (VLA); COWARD, VIVIEN - Veterinary Laboratories Agency (VLA); ESSEN, STEVE - Veterinary Laboratories Agency (VLA); BROOKES, SHARON - Veterinary Laboratories Agency (VLA); IRVINE, RICHARD - Veterinary Laboratories Agency (VLA); Spackman, Erica; RIDGEON, JONATHAN - Veterinary Laboratories Agency (VLA); GARDNER, REBECCA - Veterinary Laboratories Agency (VLA); HANNA, AMANDA - Veterinary Laboratories Agency (VLA); Suarez, David; BROWN, IAN - Veterinary Laboratories Agency (VLA)

Submitted to: Influenza and Other Respiratory Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2010
Publication Date: 8/18/2010
Citation: Slomka, M., Densham, A., Coward, V.J., Essen, S., Brookes, S.M., Irvine, R.M., Spackman, E., Ridgeon, J., Gardner, R., Hanna, A., Suarez, D.L., Brown, I. 2010. Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses. 4:277-293.

Interpretive Summary: Upon the emergence of a novel H1N1 influenza strain in the spring of 2009, it was necessary to develop diagnostic tests for poultry and swine. To accomplish this tests for the virus genetic material that can identify influenza viruses in general and another test that specifically identifies the 2009 H1N1 strain was developed. The performance of the new test was evaluated for specificity and sensitivity with both archived and clinical specimens and was found to be excellent. This new test is rapid and sensitive and may be used with numerous species to detect and diagnose infection with the 2009 H1N1 influenza virus.

Technical Abstract: BACKGROUND. Requirement to detect pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by RealTime real time reverse transcription (RRT) PCR methods. Objectives. First, modify an existing M gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Secondly, design an H1 RRT PCR to specifically detect H1N1v infections. METHODS. RRT PCR assays tested laboratory isolates of SIV (n=51; 37 European and 14 North American), H1N1v (n=2) and avian influenza virus (AIV; n=43). Diagnostic sensitivity and specificity were calculated for swabs (n=133) and tissues (n=114) collected from field and experimentally-infected pigs that included infections with SIVs and H1N1v. RESULTS. C "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 to 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 revealed sensitivity and specificity of 100% and 95.2% respectively for the swabs, and 93.5% and 98.5% for the tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99.1% respectively for the swabs, and 100% and 100% for the tissues. CONCLUSIONS. The two RRT PCRs were carefully validated for the purposes of (i) generic detection of SIV (influenza A) and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection in pigs.