ARS | Publication request: Real-Time Rt-Pcr Assays for the Detection and Differentiation of North American Swine Influenza Viruses

Submitted to: Journal Of Veterinary Diagnostic Investigation
Publication Acceptance Date: March 1, 2004
Publication Date: September 1, 2004
Citation: Richt, J.A., Lager, K.M., Clouser, D.F., Spackman, E., Suarez, D.L., Yoon, K.-J. 2004. Real-Time Rt-Pcr Assays For The Detection And Differentiation Of North American Swine Influenza Viruses. Journal Of Veterinary Diagnostic Investigation. 16:367-373.

Interpretive Summary: Swine influenza (SI) is an acute respiratory disease of swine caused by type A influenza viruses. Before 1998, mainly "classical" H1N1 SI viruses (SIV) were isolated from swine in the U.S. Since then, antigenetically distinct reassortant H3- and H1-SIVs have been identified as causative agents of respiratory disease in pigs on U.S. farms. Improvement in SIV diagnostics is needed in light of the recently observed rapid evolution of H1 and H3 swine influenza viruses and their potential threat to human health. To address this need, real-time RT-PCR assays for the detection of SIVs were developed. We established a highly sensitive M-gene based RT-PCR assay which is able to detect the H1- and H3-subtypes of SIVs with a sensitivity per reaction of approximately 2 copies of in vitro generated M-specific negative-sense RNA molecules and approximately 0.05 TCID50 in nasal swabs of experimentally SIV-infected pigs. This RT-PCR assay can be performed within several hours and showed a sensitivity of 94% and a specificity of 85% when compared to virus isolation. In addition, we have designed H1-, H3-, N1-, and N2-specific primer and probe sets in order to differentiate between different SIVs subtypes. The H and N-specific primer and probes sets were less sensitive than the M-specific assay. They were, however, found to be specific for their respective viral gene, and able to distinguish between their respective SIV-subtypes. These real-time PCR assays will allow rapid on-site pathogen detection and will have application for SIV surveillance and disease management.

Technical Abstract: Swine influenza (SI) is an acute respiratory disease of swine caused by type A influenza viruses. Before 1998, mainly "classical" H1N1 SI viruses (SIV) were isolated from swine in the U.S. Since then, antigenetically distinct reassortant H3- and H1-SIVs have been identified as causative agents of respiratory disease in pigs on U.S. farms. Improvement in SIV diagnostics is needed in light of the recently observed rapid evolution of H1 and H3 swine influenza viruses and their zoonotic potential. To address this need, real-time RT-PCR assays for the detection of SIVs were developed. We established a highly sensitive M-gene based RT-PCR assay which is able to detect the H1- and H3-subtypes of SIVs with a sensitivity per reaction of approximately 2 copies of in vitro generated M-specific negative-sense RNA molecules and approximately 0.05 TCID50 in nasal swabs of experimentally SIV-infected pigs. This RT-PCR assay can be performed within several hours and showed a sensitivity of 94% and a specificity of 85% when compared to virus isolation. In addition, we have designed H1-, H3-, N1-, and N2-specific primer and probe sets in order to differentiate between different SIVs subtypes. The H and N-specific primer and probes sets were less sensitive than the M-specific assay. They were, however, found to be specific for their respective viral gene, and able to distinguish between their respective SIV-subtypes.