Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform

Nicole Bakkegård Goecke*, Jesper Schak Krog, Charlotte Kristiane Hjulsager, Kerstin Skovgaard, Timm C. Harder, Solvej Ø. Breum, Lars E. Larsen

*Corresponding author for this work

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Abstract

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.
Original languageEnglish
Article number165
JournalFrontiers in Cellular and Infection Microbiology
Volume8
Number of pages12
ISSN2235-2988
DOIs
Publication statusPublished - 2018

Bibliographical note

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Cite this

@article{80e2e604f6ab4ec1bfaef40dbb037975,
title = "Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform",
abstract = "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.",
author = "Goecke, {Nicole Bakkeg{\aa}rd} and Krog, {Jesper Schak} and Hjulsager, {Charlotte Kristiane} and Kerstin Skovgaard and Harder, {Timm C.} and Breum, {Solvej {\O}.} and Larsen, {Lars E.}",
note = "This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.",
year = "2018",
doi = "10.3389/fcimb.2018.00165",
language = "English",
volume = "8",
journal = "Frontiers in Cellular and Infection Microbiology",
issn = "2235-2988",
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Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform. / Goecke, Nicole Bakkegård; Krog, Jesper Schak; Hjulsager, Charlotte Kristiane; Skovgaard, Kerstin; Harder, Timm C.; Breum, Solvej Ø.; Larsen, Lars E.

In: Frontiers in Cellular and Infection Microbiology, Vol. 8, 165, 2018.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform

AU - Goecke, Nicole Bakkegård

AU - Krog, Jesper Schak

AU - Hjulsager, Charlotte Kristiane

AU - Skovgaard, Kerstin

AU - Harder, Timm C.

AU - Breum, Solvej Ø.

AU - Larsen, Lars E.

N1 - This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

PY - 2018

Y1 - 2018

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.

U2 - 10.3389/fcimb.2018.00165

DO - 10.3389/fcimb.2018.00165

M3 - Journal article

VL - 8

JO - Frontiers in Cellular and Infection Microbiology

JF - Frontiers in Cellular and Infection Microbiology

SN - 2235-2988

M1 - 165

ER -