Cold-passaged isolates and bat-swine influenza a chimeric viruses as modified live-attenuated vaccines against influenza a viruses in pigs.

Swine influenza A virus (swIAV) infections in pig populations cause considerable morbidity and economic losses. Frequent reverse zoonotic incursions of human IAV boost reassortment opportunities with authentic porcine and avian-like IAV in swine herds potentially enhancing zoonotic and even pre-pandemic potential. Vaccination using adjuvanted inactivated full virus vaccines is frequently used in attempting control of swIAV infections. Accelerated antigenic drift of swIAV in large swine holdings and interference of maternal antibodies with vaccine in piglets can compromise these efforts. Potentially more efficacious modified live-attenuated vaccines (MLVs) bear the risk of reversion of MLV to virulence. Here we evaluated new MLV candidates based on cold-passaged swIAV or on reassortment-incompetent bat-IAV-swIAV chimeric viruses. Serial cold-passaging of various swIAV subtypes did not yield unambiguously temperature-sensitive mutants although safety studies in mice and pigs suggested some degree of attenuation. Chimeric bat-swIAV expressing the hemagglutinin and neuraminidase of an avian-like H1N1, in contrast, proved to be safe in mice and pigs, and a single nasal inoculation induced protective immunity against homologous challenge in pigs. Reassortant-incompetent chimeric bat-swIAV vaccines could aid in reducing the amount of swIAV circulating in pig populations, thereby increasing animal welfare, limiting economic losses and lowering the risk of zoonotic swIAV transmission.

Surveillance of European Domestic Pig Populations Identifies an Emerging Reservoir of Potentially Zoonotic Swine Influenza A Viruses.

Swine influenza A viruses (swIAVs) can play a crucial role in the generation of new human pandemic viruses. In this study, in-depth passive surveillance comprising nearly 2,500 European swine holdings and more than 18,000 individual samples identified a year-round presence of up to four major swIAV lineages on more than 50% of farms surveilled. Phylogenetic analyses show that intensive reassortment with human pandemic A(H1N1)/2009 (H1pdm) virus produced an expanding and novel repertoire of at least 31 distinct swIAV genotypes and 12 distinct hemagglutinin/neuraminidase combinations with largely unknown consequences for virulence and host tropism. Several viral isolates were resistant to the human antiviral MxA protein, a prerequisite for zoonotic transmission and stable introduction into human populations. A pronounced antigenic variation was noted in swIAV, and several H1pdm lineages antigenically distinct from current seasonal human H1pdm co-circulate in swine. Thus, European swine populations represent reservoirs for emerging IAV strains with zoonotic and, possibly, pre-pandemic potential.

A newly developed tetraplex real-time RT-PCR for simultaneous screening of influenza virus types A, B, C and D.

BACKGROUND: Human- or avian-to-swine transmissions have founded several autonomously circulating influenza A virus (IAV) lineages in swine populations that cause economically important respiratory disease. Little is known on other human influenza virus types, like B (IBV) and C (ICV) in European swine, and of the recently detected novel animal influenza virus type D (IDV). OBJECTIVES: Development of a cost-effective diagnostic tool for large-scale surveillance programmes targeting all four influenza virus types. METHODS: An influenza ABCD tetraplex real-time RT-PCR (RT-qPCR) was developed in the frame of this study. A selection of reference virus strains and more than 4000 porcine samples from a passive IAV surveillance programme in European swine with acute respiratory disease were examined. RESULTS: Two IBV, a single IDV but no ICV infections were identified by tetraplex RT-qPCR. IBV and IDV results were confirmed by conventional RT-PCR and partial sequence analysis. CONCLUSIONS: The tetraplex RT-qPCR proved fit for purpose as a sensitive, specific and high-throughput tool to study influenza virus transmission at the human-animal interface. Complementing close-meshed active virological and serological surveillance is required to better understand the true incidence and prevalence of influenza virus type B, C and D infections in swine.

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.

The H1N1 influenza virus responsible for the most recent pandemic in 2009 (H1N1pdm) has spread to swine populations worldwide while it replaced the previous seasonal H1N1 virus in humans. In France, surveillance of swine influenza A viruses in pig herds with respiratory outbreaks led to the detection of 44 H1N1pdm strains between 2009 and 2017, regardless of the season, and findings were not correlated with pig density. From these isolates, 17 whole-genome sequences were obtained, as were 6 additional hemagglutinin (HA)/neuraminidase (NA) sequences, in order to perform spatial and temporal analyses of genetic diversity and to compare evolutionary patterns of H1N1pdm in pigs to patterns for human strains. Following mutation accumulation and fixation over time, phylogenetic analyses revealed for the first time the divergence of a swine-specific genogroup within the H1N1pdm lineage. The divergence is thought to have occurred around 2011, although this was demonstrated only through strains isolated in 2015 to 2016 in the southern half of France. To date, these H1N1pdm swine strains have not been related to any increased virulence in swine herds and have not exhibited any antigenic drift compared to seasonal human strains. However, further monitoring is encouraged, as diverging evolutionary patterns in these two species, i.e., swine and humans, may lead to the emergence of viruses with a potentially higher risk to both animal and human health.IMPORTANCE Pigs are a "mixing vessel" for influenza A viruses (IAVs) because of their ability to be infected by avian and human IAVs and their propensity to facilitate viral genomic reassortment events. Also, as IAVs may evolve differently in swine and humans, pigs can become a reservoir for old human strains against which the human population has become immunologically naive. Thus, viruses from the novel swine-specific H1N1pdm genogroup may continue to diverge from seasonal H1N1pdm strains and/or from other H1N1pdm viruses infecting pigs and lead to the emergence of viruses that would not be covered by human vaccines and/or swine vaccines based on antigens closely related to the original H1N1pdm virus. This discovery confirms the importance of encouraging swine IAV monitoring because H1N1pdm swine viruses could carry an increased risk to both human and swine health in the future as a whole H1N1pdm virus or gene provider in subsequent reassortant viruses.

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.

BACKGROUND: A 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. OBJECTIVES: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. METHODS: New 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. RESULTS: A 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. CONCLUSIONS: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

Riems influenza a typing array (RITA): An RT-qPCR-based low density array for subtyping avian and mammalian influenza a viruses.

Rapid and sensitive diagnostic approaches are of the utmost importance for the detection of humans and animals infected by specific influenza virus subtype(s). Cascade-like diagnostics starting with the use of pan-influenza assays and subsequent subtyping devices are normally used. Here, we demonstrated a novel low density array combining 32 TaqMan(®) real-time RT-PCR systems in parallel for the specific detection of the haemagglutinin (HA) and neuraminidase (NA) subtypes of avian and porcine hosts. The sensitivity of the newly developed system was compared with that of the pan-influenza assay, and the specificity of all RT-qPCRs was examined using a broad panel of 404 different influenza A virus isolates representing 45 different subtypes. Furthermore, we analysed the performance of the RT-qPCR assays with diagnostic samples obtained from wild birds and swine. Due to the open format of the array, adaptations to detect newly emerging influenza A virus strains can easily be integrated. The RITA array represents a competitive, fast and sensitive subtyping tool that requires neither new machinery nor additional training of staff in a lab where RT-qPCR is already established.