TY - JOUR

T1 - Subtyping of swine influenza viruses using a high-throughput real-time PCR platform

AU - Goecke, Nicole B.

AU - Krog, Jesper S.

AU - Hjulsager, Charlotte K.

AU - Skovgaard, Kerstin

AU - Harder, Timm C.

AU - Breum, Solvej

AU - Larsen, Lars E.

PY - 2018/5/22

Y1 - 2018/5/22

N2 - Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

AB - Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.

KW - High-throughput real-time PCR, diagnostics

KW - Real-time PCR

KW - Subtyping

KW - Surveillance

KW - Swine influenza virus

U2 - 10.3389/fcimb.2018.00165

DO - 10.3389/fcimb.2018.00165

M3 - Journal article

C2 - 29872645

AN - SCOPUS:85047566938

VL - 8

JO - Frontiers in Cellular and Infection Microbiology

JF - Frontiers in Cellular and Infection Microbiology

SN - 2235-2988

IS - MAY

M1 - 165

ER -