Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs

Abstract

Swine influenza, apart from its importance in animal health, may also be of public health significance. Although the first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure, this virus was shown to be related genetically to swine influenza viruses (SIV) and easily transmissible to pigs. In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences on the risk of human infection by increasing the reservoir of this virus and the risk of possible emergence of new reassortant viruses with increased virulence for pigs and/or humans. Sensitive tools to monitor and detect rapidly such an infection are therefore mandatory. In this study, five commercial real-time RT-PCR assays developed by manufacturers LSI and Adiagène were assessed and validated, (i) for rapid detection of influenza A viruses, including pH1N1/09, in pig and (ii) for the differentiation of pH1N1/09 in that species. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene. All five kits are ready-to-use, one-step duplex RT-PCR and contain an internal positive control (IPC), appropriate for porcine biological samples, for assessing RNA extraction efficiency and the presence of PCR inhibitors. They have been used successfully by veterinary laboratories and shown to be powerful tools for the diagnosis and epidemiological surveillance of influenza virus infections in pigs.

Introduction

Swine influenza is a highly contagious respiratory viral infection of pigs that has become enzootic in areas densely populated with pigs (Olsen et al., 2006). It is responsible for significant economic losses in affected herds due to morbidity and secondary infections. Swine influenza viruses (SIVs) can also be transmitted to humans, but the infection usually remains inapparent or produces a mild disease (Myers et al., 2007). SIVs are enveloped viruses belonging to the genus Influenza virus A, family Orthomyxoviridae. The genome consists of eight segments of single-stranded RNA of negative polarity. SIVs can evolve by point mutation and/or reassortment, and are typed according to their major antigenic determinants, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Three subtypes (H1N1, H3N2 and H1N2) are circulating in swine populations worldwide and various genetic lineages can be distinguished within each subtype. Thus, North American, European and Asian SIVs differ in their origin, i.e. transmission from birds or from humans, wholly or after single or multiple reassortment events (Kuntz-Simon and Madec, 2009).

The first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure or to a contemporary infection in the swine population (New swine-origin A/H1N1 Influenza Virus Investigation Team, 2009). However, genomic sequence analysis revealed that pH1N1/09 contains a combination of genes of swine origin that had never been identified previously (Garten et al., 2009, Smith et al., 2009). Data from experimental infections of pigs as well as experience of natural outbreaks in swine farms worldwide have shown that porcine populations are fully susceptible to this virus and able to sustain uninterrupted transmission chains (Brookes et al., 2009, Brookes et al., 2010, Howden et al., 2009, Lange et al., 2009, Moreno et al., 2010, Pasma and Joseph, 2010, Pereda et al., 2010, Slomka et al., 2010, Vincent et al., 2010, Weingartl et al., 2010, Welsh et al., 2010). In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences, in terms of the risk of human infection, by increasing the reservoir of this virus. In addition, reassortment could occur with enzootic SIV and lead to the emergence of new subtypes with increased virulence for pigs and/or humans (Vijaykrishna et al., 2010). Therefore, sensitive methods for rapid monitoring and detection of such a transmission are mandatory.

The aim was to implement molecular diagnosis tools, (i) to detect rapidly influenza A viruses, including pH1N1/09, in pigs and (ii) to differentiate pH1N1/09 in that species. Concerning pH1N1/09 subtyping, the objective was to develop sensitive tests capable of identifying and differentiating the pandemic strain from other influenza A viruses, especially SIV from lineages which are enzootic in European pigs, i.e. “avian-like swine H1N1” viruses, “human-like reassortant swine H3N2” and “human-like reassortant swine H1N2” (Kuntz-Simon and Madec, 2009, Kyriakis et al., 2009). Manufacturers were therefore asked to develop real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays that contained an internal positive control (IPC) appropriate for biological samples from pigs to assess the efficiency of RNA extraction and the presence of PCR inhibitors. The validation procedure followed mostly recommendations from OIE (OIE, 2008), which included a proficiency test for local veterinary laboratories belonging to a French surveillance network for swine influenza. The assessment and validation of five commercial, ready-to-use, one-step duplex real-time RT-PCR kits using TaqMan technology are described. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene.