Pathogenicity and escape to pre-existing immunity of a new genotype of swine influenza H1N2 virus that emerged in France in 2020.

In 2020, a new genotype of swine H1N2 influenza virus (H1avN2-HA 1C.2.4) was identified in France. It rapidly spread within the pig population and supplanted the previously predominant H1avN1-HA 1C.2.1 virus. To characterize this new genotype which is genetically and antigenically distant from the other H1avNx viruses detected in France, an experimental study was conducted to compare the outcomes of H1avN2 and H1avN1 infections in pigs and evaluate the protection conferred by the only inactivated vaccine currently licensed in Europe containing an HA 1C (clade 1C.2.2) antigen. Infection with H1avN2 induced stronger clinical signs and earlier shedding than H1avN1. The neutralizing antibodies produced following H1avN2 infection were unable to neutralize H1avN1, and vice versa, whereas the cellular-mediated immunity cross-reacted. Vaccination slightly altered the impact of H1avN2 infection at the clinical level, but did not prevent shedding of infectious virus particles. It induced a cellular-mediated immune response towards H1avN2, but did not produce neutralizing antibodies against this virus. As in vaccinated animals, animals previously infected by H1avN1 developed a cross-reacting cellular immune response but no neutralizing antibodies against H1avN2. However, H1avN1 pre-infection induced a better protection against the H1avN2 infection than vaccination, probably due to higher levels of non-neutralizing antibodies and a mucosal immunity. Altogether, these results showed that the new H1avN2 genotype induced a severe respiratory infection and that the actual vaccine was less effective against this H1avN2-HA 1C.2.4 than against H1avN1-HA 1C.2.1, which may have contributed to the H1avN2 epizootic and dissemination in pig farms in France.

Successive Inoculations of Pigs with Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) and Swine H1N2 Influenza Virus Suggest a Mutual Interference between the Two Viral Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (swIAV) are major pathogens of the porcine respiratory disease complex, but little is known on their interaction in super-infected pigs. In this study, we investigated clinical, virological and immunological outcomes of successive infections with PRRSV-1 and H1N2 swIAV. Twenty-four specific pathogen-free piglets were distributed into four groups and inoculated either with PRRSV at study day (SD) 0, or with swIAV at SD8, or with PRRSV and swIAV one week apart at SD0 and SD8, respectively, or mock-inoculated. In PRRSV/swIAV group, the clinical signs usually observed after swIAV infection were attenuated while higher levels of anti-swIAV antibodies were measured in lungs. Concurrently, PRRSV multiplication in lungs was significantly affected by swIAV infection, whereas the cell-mediated immune response specific to PRRSV was detected earlier in blood, as compared to PRRSV group. Moreover, levels of interferon (IFN)-α measured from SD9 in the blood of super-infected pigs were lower than those measured in the swIAV group, but higher than in the PRRSV group at the same time. Correlation analyses suggested an important role of IFN-α in the two-way interference highlighted between both viral infections.

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.

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.

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.

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.

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.

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.

Detection of Listeria monocytogenes in raw and pasteurized liquid whole eggs and characterization by PFGE.

Listeria monocytogenes has been recognized as a human pathogen for decades and is known to be an important foodborne pathogen. There have been no documented foodborne L. monocytogenes illnesses due to the consumption of eggs or egg products, even though the bacterium has been isolated from faeces, body fluid, and oviducts of asymptomatic laying hens. In order to describe L. monocytogenes contamination of egg products, 144 liquid whole egg samples were collected from 3 different egg-breaking plants during 3 sampling periods. L. monocytogenes detection was performed on raw samples stored at 2 degrees C for two days (D+2) and on pasteurized samples stored at 2 degrees C at D+2 and at shelf-life date (SLD). L. monocytogenes was detected in 25 of the 144 raw egg samples collected, in 4 of the 144 pasteurized egg samples at D+2 and in 2 of the 144 ones analysed at SLD. Contamination of raw egg products appeared to be season dependant and was higher during summer and winter than during autumn. One hundred and ninety-six L. monocytogenes isolates were collected and serotyped; 3 serovars were demonstrated. The dominant serovar was L. monocytogenes 1/2a which was presented by 94.4% of the isolates. Typing of 196 L. monocytogenes isolates was carried out by macrorestriction of the genomic DNA with ApaI and AscI enzymes followed by pulsed field gel electrophoresis (PFGE). A large diversity was observed with 21 genotypes of L. monocytogenes, even for a given manufacturer. Nevertheless, most of the egg product samples were contaminated by one genotype, except for five samples which were contaminated by two or three distinct genotypes. The genotypes seem to be specific to each manufacturer. No cluster of L. monocytogenes was found to recur in the different plants over successive seasons.

Replication, pathogenesis and transmission of pandemic (H1N1) 2009 virus in non-immune pigs.

The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella serotype Enteritidis, Typhimurium and Infantis isolates obtained from whole liquid eggs.

Salmonella is a well-documented pathogen known to occur in a wide range of foods, especially poultry products. The most frequently reported food-sources of human infection are eggs and egg products. In this study, in order to describe Salmonella contamination of egg products, 144 liquid egg samples were collected from 3 different egg-breaking plants during the 3 sampling periods. Salmonella detection was performed on raw samples stored at 2 degrees C for 2 days (D+2) and on pasteurised samples stored at 2 degrees C at D+2 and at shelf-life date. Salmonella was detected in 130 of the 144 raw egg samples collected and in 11 of the 288 pasteurised egg samples analysed. 740 Salmonella isolates were collected and serotyped: 14 serovars were demonstrated. A great diversity, particularly during summer, was noted. The dominant serovars were S. Enteritidis, S. Typhimurium and S. Infantis, mainly found in whole raw egg products. Typing of 325 isolates of S. Enteritidis, 54 isolates of S. Typhimurium and 58 isolates of S. Infantis was carried out by macrorestriction of the genomic DNA with XbaI and SpeI enzymes followed by pulsed field gel electrophoresis (PFGE). The Salmonella Enteritidis isolates could be grouped into 3 clusters. Cluster 1 was predominant at all 3 egg-breaking companies during the different sampling periods. This cluster seemed to be adapted to the egg-breaking plants. Cluster 2 was linked to plant 1 and cluster 3 to plant 3. Two main clusters of Salmonella Typhimurium were demonstrated. Cluster A was mainly found at plant 2 during autumn. Plant 3 was contaminated by all the Salmonella Typhimurium genotypes but in a more sporadic manner during the three seasons studied. Plant 1 seemed to be less contaminated by Salmonella Typhimurium than the others. Three clusters and 2 genotypes of Salmonella Infantis were shown. The main cluster, cluster alpha, consisted of 75% of the S. Infantis isolates and was mainly found during summer at plants 1 and 3. Plant 2 seemed to be less contaminated by S. Infantis. In this study, molecular typing demonstrated that, although certain clusters were common to all three companies, specific clusters, notably of S. Enteritidis were present at each plant.