Serological Evidence of Backyard Pig Exposure to Highly Pathogenic Avian Influenza H5N8 Virus during 2016-2017 Epizootic in France.

In autumn/winter 2016-2017, HPAI-H5N8 viruses belonging to the A/goose/Guandong/1/1996 (Gs/Gd) lineage, clade 2.3.4.4b, were responsible for outbreaks in domestic poultry in Europe, and veterinarians were requested to reinforce surveillance of pigs bred in HPAI-H5Nx confirmed mixed herds. In this context, ten pig herds were visited in southwestern France from December 2016 to May 2017 and serological analyses for influenza A virus (IAV) infections were carried out by ELISA and hemagglutination inhibition assays. In one herd, one backyard pig was shown to have produced antibodies directed against a virus bearing a H5 from clade 2.3.4.4b, suggesting it would have been infected naturally after close contact with HPAI-H5N8 contaminated domestic ducks. Whereas pigs and other mammals, including humans, may have limited sensitivity to HPAI-H5 clade 2.3.4.4b, this information recalls the importance of implementing appropriate biosecurity measures in pig and poultry farms to avoid IAV interspecies transmission, a prerequisite for co-infections and subsequent emergence of new viral genotypes whose impact on both animal and human health cannot be predicted.

Genetic and Antigenic Evolution of European Swine Influenza A Viruses of HA-1C (Avian-Like) and HA-1B (Human-Like) Lineages in France from 2000 to 2018.

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1av) and -1B (H1hu), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1avNy and 105 H1huNy strains were submitted to hemagglutination inhibition tests. H1avN1 (66.5%) and H1huN2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named "Δ146-147" harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the "Eurasian avian-like lineage", with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1av/H1hu strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.

Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus.

The surveillance of swine influenza A viruses in France revealed the emergence of an antigenic variant following deletions and mutations that are fixed in the HA-encoding gene of the European human-like reassortant swine H1N2 lineage. In this study, we compared the outcomes of the parental (H1huN2) and variant (H1huN2Δ146-147) virus infections in experimentally-inoculated piglets. Moreover, we assessed and compared the protection that was conferred by an inactivated vaccine currently licensed in Europe. Three groups of five unvaccinated or vaccinated piglets were inoculated with H1huN2 or H1huN2Δ146-147 or mock-inoculated, respectively. In unvaccinated piglets, the variant strain induced greater clinical signs than the parental virus, in relation to a higher inflammatory response that involves TNF-α production and a huge afflux of granulocytes into the lung. However, both infections led to similar levels of virus excretion and adaptive (humoral and cellular) immune responses in blood. The vaccinated animals were clinically protected from both infectious challenges and did not exhibit any inflammatory responses, regardless the inoculated virus. However, whereas vaccination prevented virus shedding in H1huN2-infected animals, it did not completely inhibit the multiplication of the variant strain, since live virus particles were detected in nasal secretions that were taken from H1huN2Δ146-147-inoculated vaccinated piglets. This difference in the level of vaccine protection was probably related to the poorer ability of the post-vaccine antibodies to neutralize the variant virus than the parental virus, even though post-vaccine cellular immunity appeared to be equally effective against both viruses. These results suggest that vaccine antigens would potentially need to be updated if this variant becomes established in Europe.

Assessment of Influenza D Virus in Domestic Pigs and Wild Boars in France: Apparent Limited Spread within Swine Populations Despite Serological Evidence of Breeding Sow Exposure.

In order to assess influenza D virus (IDV) infections in swine in France, reference reagents were produced in specific pathogen free pigs to ensure serological and virological analyses. Hemagglutination inhibition (HI) assays were carried out on 2090 domestic pig sera collected in 2012-2018 in 102 farms. Only 31 sera from breeding sows sampled in 2014-2015 in six farrow-to-finish herds with respiratory disorders contained IDV-specific antibodies. In two of them, within-herd percentage of positive samples (73.3% and 13.3%, respectively) and HI titers (20-160) suggested IDV infections, but virus persistence was not confirmed following new sampling in 2017. All growing pigs tested seronegative, whatever their age and the sampling year. Moreover, PB1-gene RT-qPCR performed on 452 nasal swabs taken in 2015-2018 on pigs with acute respiratory syndrome (137 farms) gave negative results. In Corse, a Mediterranean island where pigs are mainly bred free-range, 2.3% of sera (n = 177) sampled on adult pigs in 2013-2014 obtained low HI titers. Finally, 0.5% of sera from wild boars hunted in 2009-2016 (n = 644) tested positive with low HI titers. These results provide the first serological evidence that sows were exposed to IDV in France but with a limited spread within the swine population.

Virological and epidemiological patterns of swine influenza A virus infections in France: Cumulative data from the RESAVIP surveillance network, 2011-2018.

Swine influenza A viruses (swIAVs) cause acute respiratory syndromes in pigs and may also infect humans. Following the 2009 pandemic, a network was established in France to reinforce swIAV monitoring. This study reports virological and epidemiological data accumulated through passive surveillance conducted during 1,825 herd visits from 2011 to 2018. Among them, 887 (48.6 %) tested swIAV-positive. The proportion of positive cases remained stable year-on-year and year-round. The European avian-like swine H1N1 (H1avN1) virus was the most frequently identified (69.6 %), and was widespread across the country. The European human-like reassortant swine H1N2 (H1huN2) virus accounted for 22.1 % and was only identified in the north-western quarter and recently in the far north. The 2009 pandemic H1N1 (H1N1pdm) virus (3.6 %) was detected throughout the country, without settling in areas of higher pig densities. Its proportion increased in winter, during the seasonal epidemics in humans. The European human-like reassortant swine H3N2 as well as H1avN2 viruses were identified sporadically. In up to 30 % of swIAV-positive cases, pigs exhibited clinical signs of high intensity, regardless of the viral subtype and vaccination program. The recurrent pattern of the disease, i.e., an endemic infection at the herd level, was reported in 41% of cases and mainly affected post-weaning piglets (OR = 5.11 [3.36-7.76]). Interestingly, the study also revealed a significant association between the recurrent pattern and sow vaccination (OR = 1.96 [1.37-2.80]). Although restricted to the studied pig population, these results bring new knowledge about swIAV dynamics and infection patterns in pig herds in France.

Virus persistence in pig herds led to successive reassortment events between swine and human influenza A viruses, resulting in the emergence of a novel triple-reassortant swine influenza virus.

This report describes the detection of a triple reassortant swine influenza A virus of H1avN2 subtype. It evolved from an avian-like swine H1avN1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.

Bidirectional Human-Swine Transmission of Seasonal Influenza A(H1N1)pdm09 Virus in Pig Herd, France, 2018.

In 2018, a veterinarian became sick shortly after swabbing sows exhibiting respiratory syndrome on a farm in France. Epidemiologic data and genetic analyses revealed consecutive human-to-swine and swine-to-human influenza A(H1N1)pdm09 virus transmission, which occurred despite some biosecurity measures. Providing pig industry workers the annual influenza vaccine might reduce transmission risk.

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.

Serological survey of influenza A viruses in domestic and wild Suidae in Corsica (France), a Mediterranean island environment.

Corsica is a mountainous French island in the north-western Mediterranean Sea. It is a rural area, where pig farming is a major economic activity. Although no acute respiratory outbreaks due to swine influenza A viruses (swIAVs) have ever been reported in this free-ranging pig breeding system, influenza A viruses (IAVs) could be circulating within this pig population. A serological study was conducted as a first approach to domestic pigs and wild boars. Serum samples from 543 pigs raised on 91 different farms were collected during the 2013-2014 slaughtering season, and 279 sera from wild boars were obtained over four hunting seasons (between 2009 and 2014). They were first analysed by ELISA and then IAV positive and doubtful sera were subjected to haemagglutination inhibition tests using antigens representative of the four major enzootic swIAV lineages in Europe, i.e. avian-like swine H1N1 (H1avN1), pandemic-like swine H1N1 (H1N1pdm), H1N2 and H3N2. According to the ELISA results, 26.4% (CI95%: 17.7-36.7%) of herds had at least one positive animal (positive or doubtful by ELISA) and 12.4% (CI95%: 7.8-19.8%) of the pigs tested positive. Using the test characteristics (sensitivity and specificity), the true seroprevalence among Corsican pigs was estimated to be 16.4% (95% CI: 9.9-26.3). Antibodies directed against two different viral lineages were identified: H1N1pdm (in 66.2% and 45.8% of the IAV positive pigs and farms respectively) and H1avN1 (15.0% and 20.8% respectively). Evidence of exposure to viruses from two distinct lineages were detected on a single farm but in two different animals. Among the wild boars, 1.4% (CI95%: 0.4-3.6%) tested positive by ELISA and antibodies against the same two viruses were detected. Altogether, these results suggest that swIAVs from at least two different lineages are circulating among Corsican pigs, i.e. the H1N1pdm virus, probably introduced during the 2009 pandemic, and the H1avN1 virus, which is the most frequent swIAV in Europe. The low frequency of positive results observed in the Corsican wild boars hunted suggests that they would not play a major role in IAV dispersion dynamics on the island.

Maternally-derived antibodies do not inhibit swine influenza virus replication in piglets but decrease excreted virus infectivity and impair post-infectious immune responses.

Maternally-derived antibodies (MDA) reduce piglet susceptibility to swine influenza A virus, but interfere with post-infectious immune responses, raising questions about protection after waning of passive immunity. We therefore analysed the impact of different levels of residual MDA on virus excretion and immune responses in piglets born to vaccinated sows (MDA+) and infected with H1N1 at 5, 7 or 11 weeks of age, in comparison to piglets born to unvaccinated sows (MDA-). Subsequent protection against a second homologous infection occurring 4 weeks after the primo-infection was also investigated. MDA- pigs showed clinical signs, shed the virus, and developed specific immune responses despite some age-dependent differences: 7-week-old pigs were less affected clinically, showed a 2-day delayed excretion peak and excreted less virus than younger pigs. In MDA+ animals, clinical signs increased together with the decrease of MDA levels related to the age at infection-time. Virus shedding was not prevented and genome quantification profiles were similar to those obtained in MDA- piglets. However, viral particles excreted by 5-week-old MDA+ piglets appeared to be less infectious than those shed by MDA- piglets at the same age. Humoral response was affected by MDA as illustrated by the absence of HI and neutralizing response regardless the infection age, but anti-NP/M responses were less affected. Proliferative T cell responses were slightly delayed by high MDA levels. Nevertheless, MDA+ animals were all protected from a second infection, like MDA- piglets. In conclusion, responses of pigs to H1N1 were affected by both the physiological development of animals at infection and the MDA level.

Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis.

BACKGROUND: Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important. RESULTS: Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146-147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the ß-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts. CONCLUSION: The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146-147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.

Maternally-derived antibodies do not prevent transmission of swine influenza A virus between pigs.

A transmission experiment involving 5-week-old specific-pathogen-free (SPF) piglets, with (MDA(+)) or without maternally-derived antibodies (MDA(-)), was carried out to evaluate the impact of passive immunity on the transmission of a swine influenza A virus (swIAV). In each group (MDA(+)/MDA(-)), 2 seeders were placed with 4 piglets in direct contact and 5 in indirect contact (3 replicates per group). Serological kinetics (ELISA) and individual viral shedding (RT-PCR) were monitored for 28 days after infection. MDA waning was estimated using a nonlinear mixed-effects model and survival analysis. Differential transmission rates were estimated depending on the piglets' initial serological status and contact structure (direct contact with pen-mates or indirect airborne contact). The time to MDA waning was 71.3 [52.8-92.1] days on average. The airborne transmission rate was 1.41 [0.64-2.63] per day. The compared shedding pattern between groups showed that MDA(+) piglets had mainly a reduced susceptibility to infection compared to MDA(-) piglets. The resulting reproduction number estimated in MDA(+) piglets (5.8 [1.4-18.9]), although 3 times lower than in MDA(-) piglets (14.8 [6.4-27.1]), was significantly higher than 1. Such an efficient and extended spread of swIAV at the population scale in the presence of MDAs could contribute to swIAV persistence on farms, given the fact that the period when transmission is expected to be impacted by the presence of MDAs can last up to 10 weeks.

European surveillance network for influenza in pigs: surveillance programs, diagnostic tools and Swine influenza virus subtypes identified in 14 European countries from 2010 to 2013.

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events.

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.

Influenza A(H1N1)pdm09 virus in pigs, Réunion Island.

During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended.

Study of influenza A virus in wild boars living in a major duck wintering site.

Wild birds, which are reservoirs of influenza viruses, are believed to be the original source of new influenza viruses-including highly pathogenic ones-that can be transmitted to domestic animals as well as humans and represent a potential epizootic and/or pandemic threat. Despite increasing knowledge on influenza A virus dynamics in wild birds, the viral circulation in wild boars remains largely unknown. This is of particular interest since pigs can be infected with both human and avian viruses; upon co-infection, they can act as a mixing vessel through reassortment, a mechanism that resulted in the emergence of the pandemic H1N1 virus in 2009. The Camargue (Southern France) appears as an ideal study area to investigate inter-species transmission of influenza A viruses from wild birds and possibly humans to wild boars. Indeed, the important local wild boar population shares wetland use with humans and the largest concentration of wintering ducks in France, that are both susceptible to infection by influenza A viruses. Additionally, wild boars occasionally prey on ducks. We conducted a virological and serological survey on wild boars in the Camargue (Southern France) between September 2009 and November 2010. No influenza A virus was detected in the collected nasal swabs (n=315) and no influenza specific antibodies were observed in the serological samples (n=20). As the study was mainly focused on viral excretion, which is limited in time, we cannot exclude that low or occasional influenza A virus circulation took place during the study period. Although, wild boars did not seem to be a key element in the dynamics of influenza A virus circulation in the Camargue, wild boar influenza A virus infections should be more widely studied to determine if the pattern observed here represents the normal situation or an exceptional one.