Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

Dinah Henritzi,Jesper S Krog,Emanuela Foni,Chiara Chiapponi,Elke Starick,Ian H Brown,Na Zhao,Bernd Hoffmann,Scott M Reid,Timm C Harder,Lars Erik Larsen,Silke Wacheck,Gaelle Simon,Martin Beer,Peter Schmid

doi:10.1111/irv.12407

Abstract

BackgroundA diversifying pool of mammalian‐adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood.ObjectivesEfficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost‐effective large‐scale analysis.MethodsNew SIV haemagglutinin (HA) and neuraminidase (NA) subtype‐ and lineage‐specific multiplex real‐time RT‐PCRs (RT‐qPCR) have been developed and validated with reference virus isolates and clinical samples.ResultsA diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M‐gene‐specific influenza A virus RT‐qPCR. In a second step, positive samples are examined by tetraplex HA‐ and triplex NA‐specific RT‐qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian‐derived), “hu” (European human‐derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT‐qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT‐PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT‐qPCR subtyping.ConclusionsThese new multiplex RT‐qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

Highlights

Influenza A virus (IAV) infections have a significant clinical impact on domestic swine populations
This is due to the expression of cellular glycan moieties in the porcine upper respiratory tract that act as receptors for viral attachment by the haemagglutinin protein (HA) of both avian and mammalian influenza A viruses (IAV).[4,5,6]
We present two multiplex RT-qPCRs to facilitate the detection and differentiation of four and three lineages of HA and of NA gene segments, respectively, of Swine influenza viruses (SIV) subtypes which currently cocirculate in European swine populations

Summary

Introduction

Influenza A virus (IAV) infections have a significant clinical impact on domestic swine populations. This is due to the expression of cellular glycan moieties (alpha 2-6 or 2-3-linked terminal sialic acids, respectively) in the porcine upper respiratory tract that act as receptors for viral attachment by the haemagglutinin protein (HA) of both avian and mammalian IAV.[4,5,6] simultaneous infection of porcine cells with mammalian/human and avian IAV is possible Such double infections open possibilities for an exchange of genome segments between these viruses (reassortment) and progeny viruses with mixed parental genotypes (reassortants) potentially leading to the emergence of new phenotypic properties. Conclusions: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe

Objectives

Methods

Conclusion