SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY
Location: Virus and Prion Research Unit
Title: One Step Real-Time RT-PCR for 2009 Pandemic H1N1 Matrix Gene Detection in Swine Samples
Submitted to: American Association of Swine Veterinarians Annual Meeting
Publication Type: Proceedings
Publication Acceptance Date: February 1, 2010
Publication Date: March 6, 2010
Citation: Lorusso, A., Faaberg, K.S., Killian, M., Koster, L., Vincent, A.L. 2010. One Step Real-Time RT-PCR for 2009 Pandemic H1N1 Matrix Gene Detection in Swine Samples [abstract]. American Association of Swine Veterinarians. American Association of Swine Veterinarians. p. 369.
Introduction: In the spring of 2009, a novel H1N1 influenza A virus began to spread among humans worldwide (1). Because the virus is now circulating widely in the human population, swine herds are at increased risk of becoming infected. In order to investigate the potential outbreak of the new pandemic virus in pigs, a real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) based on TaqMan technology for the differential detection of the 2009 pandemic H1N1 RNA in clinical specimens was developed.
Materials and Methods: Matrix (M) gene sequences of endemic swine influenza virus (SIV), novel 2009 pandemic H1N1 and sequences from a panel of human and avian type A influenza viruses were aligned and primers designed to amplify a conserved 159 bp region within the aligned M genes. However, the probe was purposely designed using the sequence of A/California/04/2009 (H1N1)v in a lineage-specific region shared by 2009 pandemic H1N1 isolates. The TaqMan probe was dual-labeled with FAM at the 5’ end and TAMRA at the 3’ end. To obtain a standard for the TaqMan assay, an RT-PCR product containing the full-length M gene of A/California/04/2009 (H1N1)v was utilized to produce standard RNA. To estimate the applicability of the test as a diagnostic tool in screening of field specimens, we analyzed field isolates of SIV collected during diagnostic investigations in pigs, and nasal swab and bronchoalveolar lavage samples collected during an experimental in vivo study conducted in two separate groups of 4-week-old pigs inoculated with two pandemic 2009 H1N1 isolates. In order to exclude cross-reactivity between pandemic 2009 H1N1 and other viral respiratory pathogens of pigs, the specificity was evaluated by analysis of isolates of endemic SIV, avian and equine influenza viruses, porcine coronaviruses, PRRSV, PCV-2, porcine adenovirus and porcine parvovirus.
Results and Discussion: The qRT-PCR assay was able to detect the new pandemic H1N1 2009 viral RNA with the ability to differentiate the new lineage from the extant SIV circulating in the U.S. swine population. The assay was shown to be reproducible and linear over a range of 9 orders of magnitude, from 10**1 to 10**9 RNA copies, thus ensuring an accurate measurement of (H1N1) 2009 viral loads in clinical samples. Moreover, specificity was demonstrated as cross-reactivity with endemic swine influenza viruses and other swine viral pathogens was not detected.
References: 1. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., Garten, R.J., Gubareva, L.V., Xu, X., Bridges, C.B., Uyeki, T.M. 2009. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 361: 102.