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

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.

Spatiotemporal Distribution and Evolution of the A/H1N1 2009 Pandemic Influenza Virus in Pigs in France from 2009 to 2017: Identification of a Potential Swine-Specific Lineage

“…Viral RNA extraction from 200 l of nasal swab supernatants as well as an amplification step were performed according to the manufacturer's instructions. RNA extracts that were found to be positive for the IAV M gene were then subjected to real-time RT-PCR assays developed for the specific identification of the HA and NA genes from the H1N1pdm virus in swine samples (51,52). The LSI VetMAX swine influenza A/H1N1/2009-H1 detection kit (Life Technologies, Carlsbad, CA, USA) and the Adiavet A/H1N1 (2009) real-time kit (Bio-X Diagnostics, Rochefort, Belgium) were used interchangeably to amplify the H1pdm gene, whereas the LSI VetMAX swine influenza A/H1N1/2009-N1 detection kit (Life Technologies, Carlsbad, CA, USA) was used to amplify the N1pdm gene (51).…”

Molecular and serological prevalence of influenza A viruses in poultry and poultry farmers in the Ashanti region, Ghana

“…RNA was isolated from swabs (QIAamp viral RNA mini kit, Germany) and tested for influenza A Matrix-specific gene by qRT-PCR [22]. All positive samples were subjected to direct subtyping of all AIV subtypes [23,24]. Additionally, human samples were tested for seasonal influenza viruses of subtypes H1 and H3 by qRT-PCR [24].…”

“…All positive samples were subjected to direct subtyping of all AIV subtypes [23,24]. Additionally, human samples were tested for seasonal influenza viruses of subtypes H1 and H3 by qRT-PCR [24]. Viral isolation in embryonated chicken eggs and MDCK cells of positive samples was attempted.…”

Exposure of domestic swine to influenza A viruses in Ghana suggests unidirectional, reverse zoonotic transmission at the human–animal interface

“…Enzootic swIAVs of subtypes H1N1, H1N2 and H3N2 of different origins (avian or human) and genetic constellations circulate at variable frequencies in swine populations worldwide with the occasional emergence of other subtypes restricted temporally and geographically (Corzo et al, 2013;Harder et al, 2013;Zhu et al, 2011). For instance, H1N1 that circulates in European swine is a purely avian-derived virus transmitted into the swine population in 1979, while in North America, several H1N1 virus lineages of different origins are circulating (Henritzi et al, 2016).…”

“…The transmission of the 2009 human pandemic IAV H1N1pdm09in a reverse zoonotic mode from human into swine populations has further led to the emergence of a plethora of unique reassortants in swine, some of which have been characterized to have zoonotic and prepandemic propensity (Fobian et al, 2015;Henritzi et al, 2016).…”