# Trends in seroprevalence of influenza A virus infections in pigs in France (2008-2022)

Séverine Hervé, Nicolas Rose, Nicolas Barbier, Stéphane Quéguiner, Stéphane Gorin, Roselyne Fonseca, Gaëtan Pinsard, Gautier Richard, Agnès Jardin, Gaëlle Simon

► To cite this version:

Séverine Hervé, Nicolas Rose, Nicolas Barbier, Stéphane Quéguiner, Stéphane Gorin, et al.. Trends in seroprevalence of influenza A virus infections in pigs in France (2008-2022) . 2025. anses-05144045

HAL Id: anses-05144045

https://anses.hal.science/anses-05144045v1

Preprint submitted on 4 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Trends in seroprevalence of influenza A virus infections in pigs in France (2008-2022)

## Séverine Hervé

Severine.HERVE@anses.fr

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Nicolas Rose

ANSES, Ploufragan-Plouzané-Niort Laboratory, Epidemiology Animal Health and Welfare Unit

## Nicolas Barbier

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Stéphane Quéguiner

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Stéphane Gorin

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Roselyne Fonseca

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Gaëtan Pinsard

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Gautier Richard

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza

## Agnès Jardin

CEVA Santé Animale

## Gaëlle Simon

ANSES, Ploufragan-Plouzané-Niort Laboratory, Swine Virology Immunology Unit, National reference laboratory for Swine Influenza
**Keywords:** Influenza, Pig, Infection, Prevalence, Herd, Survey, Serology, Antibody

**Posted Date:** March 13th, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-5931993/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

Read Full License

**Additional Declarations:** No competing interests reported.
# Abstract

# Background

Swine influenza A viruses (swIAV) are highly contagious zoonotic pathogens that cause an acute respiratory infection in pigs, presenting substantial economic and health risks. This drives the pig industry and stakeholders in animal health to monitor swIAV in livestock. Prior to the 2009 flu pandemic, H1<sub>av</sub>N1 (HA-1C.2.1) and H1<sub>hu</sub>N2 (HA-1B.1.2.3) circulated in pig herds in France. The H1N1<sub>pdm</sub> (HA-1A.2.3.3) lineage became enzootic after its introduction. In 2020, a new H1<sub>av</sub>N2 genotype (HA-1C.2.4) emerged, altering the frequencies of enzootic swIAV lineages. To support our knowledge built on event-based surveillance and secure the exhaustiveness of the information, serological studies were conducted. Three independent surveys were performed nationally in 2008 and in 2018 (before and after the A/H1N1pdm09 pandemic, respectively), and in North West in 2022 (after the H1<sub>av</sub>N2 emergence in Brittany area). These surveys aimed to estimate swIAV prevalence in livestock using ELISA on fattening pigs and hemagglutination inhibition (HI) tests to determine the relative frequencies of different swIAV lineages.

# Results

The national seroprevalence was 91% [83–96]<sub>CI95</sub>, in 2008, and 87% [81–92]<sub>CI95</sub> in 2018. In 2022, seroprevalence in the North-West reached 91% [73–97]<sub>CI95</sub>. At each period, antibodies against several subtypes were detected simultaneously in a quarter of positives batches. In 2008, anti-HA-1C.2.1 antibodies were widespread, while anti-HA-1B.1.2.3 antibodies were found in most regions except in the South-West. H3N2 seroprevalence was very low, restricted to the North-East. By 2018, anti-HA-1C.2.1 antibodies remained the most prevalent and anti-H3 antibodies the weakest, but anti-HA-1B.1.2.3 prevalence had strongly decreased, while anti-HA-1A.3.3.2 antibodies were detected nationwide. In 2022, the North-West showed higher seroprevalence for H1<sub>av</sub>N2 (HA-1C.2.4) than H1N1<sub>pdm</sub> and H1<sub>hu</sub>N2, though still lower than H1<sub>av</sub>N1.

# Conclusions

These surveys reveal high, sustained swIAV seroprevalence, particularly in dense herd areas. They also highlight changing in anti-HA antibodies relative frequencies, reflecting viral emergence and dynamics. Despite challenges in interpreting HI test results, the surveys provided valuable data, uncovering rare events, potential undiagnosed cases, and co-circulating viruses, which may lead to genomic reassortments and new virus emergence.

# Background
Swine influenza is an acute respiratory infection, lasting less than one week and expressed clinically by dyspnea, cough, sneezing, fever and apathy (1). It plays a key role in porcine respiratory disease complex and generates economic losses for the pig industry (2, 3). Longitudinal field studies also revealed permanently infected herds highlighting the complexity of the infection and the difficulties to control it (4, 5). The etiologic agents of this highly contagious disease are swine influenza A viruses (swlAV) which spread within herd by direct contact between pigs and by infectious aerosol (6). Moreover, swlAV are zoonotic viruses with pandemic potential (7, 8). They harbour a segmented genome that undergo the possibility of reassortment by exchanging their segments in case of co-infection of a host cell by viruses of different genetic lineages and potentially different origins. Therefore, pigs can facilitate the emergence of novel IAV strains and are considered as "mixing vessels" (9). In intensive pig herds, many contacts between animals and between pigs and farmworkers occur, thus creating a favourable environment for viral persistence and zoonotic transmissions. Given the animal and public health challenges associated with swlAV, monitoring their presence in livestock is a crucial strategy for enhancing our understanding of the impact of swine influenza. Globally, H1 and H3 subtypes as well as various combinations of N1 and N2 subtypes circulate worldwide in pig herds exhibiting distinct regional variations. In Europe before the 2009 flu pandemic, three subtypes from three genetic lineages circulated in pig herds: H1<sub>av</sub>N1 (HA-1C), H1<sub>hu</sub>N2 (HA-1B) and H3N2 (10, 11). However, in France, the H3N2 virus had not been detected since the late 90s. Following the introduction of the A/H1N1pdm09 virus into the pig population in 2010, viral diversity increased (12, 13). From 2011 to 2018, the situation in France remained quite stable, with H1<sub>av</sub>N1 (HA-1C.2.1) and H1<sub>hu</sub>N2 (HA-1B.1.2.3) still predominating, and the presence of the H1N1<sub>pdm</sub> (HA-1A.3.3.2) lineage and a few other genetic combinations detected sporadically (14–16). In 2020, there was a marked increase in the number of clinical cases investigated, associated with the emergence of an H1<sub>av</sub>N2 (HA-1C.2.4) virus of a genotype newly introduced into Brittany from abroad (17). Since then, this virus has spread throughout North-western France, significantly changing the proportions of the swlAVs circulating previously (18).

The virological surveillance allows an in-depth study of swlAV that are monitored both genetically and antigenically by the national reference laboratory. The swlAV are collected by a national surveillance network (RESAVIP) and other stakeholders of the pig industry. It is an event-based surveillance driven by influenza-like illness clinical signs reported by the farmer to voluntary veterinarians (15). Asymptomatic or pauci-symptomatic cases may escape from this surveillance system. Despite the availability of highly sensitive commercial kits for detecting the viral genome (19), the viral excretion of an infected pig is short, which implies a short sampling timeframe for virus detection. Detecting antibodies produced by the infected host can be a way to overcome the previous limitations. There is also an interest of implementing complementary serological surveys to event-based surveillance to increase information about the burden of swlAV infections in pig populations.

Therefore, this study aimed at i) estimating the prevalence of swlAV infections at the herd level in France and comparing its levels before and after the introduction of the pandemic A/H1N1pdm09 virus, as well as before and after the emergence of a new H1<sub>av</sub>N2 genotype in 2020, ii) assessing the prevalence of the
different swIAV lineages over time, both at the country level and in particular regions. Thus, three independent serological surveys were performed in 2008, 2018 and 2022, respectively, with different approaches but all targeting production farms. For each period, the rules for interpreting the results were adapted according to the knowledge of the reference antigens used in the hemagglunitation inhibition tests.

# Materials and Methods

## *Sampling plans*

The target population was pigs from production herds in hexagonal France (i.e., continental France excluding Corsica) and the studied population was composed by finishing pigs (> 10 weeks old) (table 1). At this stage, most maternally derived antibodies have disappeared, which ensures that the antibodies detected are of post-infectious origin. Three blood sample collections were constituted in 2008 (survey A), 2018 (survey B) and 2022 (survey C), respectively, as follows.

### Survey A (2008)

The sampling plan aimed to cover 95% of national production in hexagonal France by sampling pig batches at slaughterhouses using a cluster sampling design. The number of batches to be sampled at slaughterhouses was determined from the number of pigs slaughtered per year and considering a minimal prevalence between 30% and 45% with a 20% relative precision. The number of pigs per batch was adapted to a minimal within-herd prevalence of the infection of 30% to be detected. In view of the activity of slaughterhouses in the North-West, because of the high density of pig herds in this area, a minimum number of batches had to be drawn at random in other regions, leading to their over-representation. Thus, a total of 186 batches of 10 pigs from 24 slaughterhouses located in 45 administrative counties across the country were sampled from May 2008 to November 2009 (table 1 and figure 1). The sampling area was divided into four major geographical areas defined according to administrative regions: North-East with Hauts-De-France, Grand-Est, Bourgogne-Franche-Comté; North-West with Brittany, Normandy, Pays-de-La-Loire, Centre-Val-De-Loire; South-East with Auvergne-Rhône-Alpes, Provence-Alpes-Côte d'Azur; South-West with Nouvelle-Aquitaine, Occitania.

### Survey B (2018)

The samples were collected directly from farms representative of the French pig production stratified on farm type and region and selected randomly from the national database for pig herd identification (BDPORC (20)). The sample size was adapted for this survey to the estimated prevalence of the different viral subtypes based on data obtained from event-based monitoring in 2016 (15). Therefore, sample size was calculated to allow the estimation of a prevalence between 15% and 20% for each subtype with a 20% relative precision. Thus, 487 herds were randomly selected from farrow-to-finish/post-weaning-finishing/finishing farms located in 58 administrative counties across the country in the four areas
between January and June 2018 (table 1 and figure 1). In each herd, 10 pigs (>10 weeks old) were sampled to detect a minimal within-herd prevalence of the infection of 30%.

## Survey C (2022)

As for survey A, a cluster-sampling scheme was used based on the main slaughterhouses in the area taken as clusters but the survey was restricted to the North-West. The number of sampled farms was determined considering a minimal prevalence of 45% with a 20% relative precision. The number of pigs per batch was adapted to a minimal within-herd prevalence of the disease of 30% to be detected. Thus, 116 batches of 10 pigs from three slaughterhouses located in 13 administrative counties in Brittany and bordering regions (Normandy and Pays-de-La-Loire) were randomly sampled between February and March 2022 (table 1 and figure 1).

Blood samples were collected from pigs at slaughterhouses or farm sites and sera were stored at -20°C until analyses.

<table><caption>Table 1: Study design of the three serological surveys conducted in pig herds in France</caption><thead><tr><th>Year<br>Sampling period Survey ID</th><th>Sampling process for herds</th><th>Number of sampled herds</th><th>Geographic distribution of batches</th><th>within-herd sample size</th></tr></thead><tbody><tr><td><strong>2008</strong><br>2008-05 to 2009-11<br>Survey A</td><td>Cluster-based: 24 slaughterhouses</td><td>N=186</td><td>45 administrative counties</td><td rowspan="3">10 finishing pigs per batch</td></tr><tr><td><strong>2018</strong><br>2018-01 to 2018-06<br>Survey B</td><td>Individual-based: 487 herds</td><td>N=487</td><td>58 administrative counties</td></tr><tr><td><strong>2022</strong><br>2022-02 to 2022-03<br>Survey C</td><td>Cluster-based: 3 slaughterhouses</td><td>N=116</td><td>13 administrative counties</td></tr></tbody></table>
All sera were tested by ELISA (ID Screen® Influenza A Nucleoprotein Swine Indirect, Innovative Diagnostics, France) according to manufacturer's instructions to detect antibodies directed against the swIAV nucleoprotein (NP). A serum was considered positive if its S/P ratio was >0.4, that corresponded to an antibody titer >1053. A batch was identified as positive if at least 1/10 serum tested positive.

## Hemagglutination inhibition tests

ELISA positive serum batches were submitted to hemagglutination inhibition (HI) tests including representative antigens of the enzootic swIAV lineages in France or Europe at the different periods of time i.e., H1<sub>av</sub>N1 (HA clade 1C.2.1), H1<sub>av</sub>N2 (HA clade 1C.2.4), H1N1pdm (HA clade 1A.3.3.2), H1<sub>hu</sub>N2 (HA clade 1B.1.2.3) and/or H3N2 (lineage 1970.1) (Table 2) (11, 12, 14-16, 18).

Sera were treated to inactivate nonspecific hemagglutination inhibitors and to remove non-specific agglutinins.

For surveys A and B, four volumes of *Vibrio cholerae* receptor-destroying enzyme were added to one volume of serum and incubated overnight at 37°C. Then, the sera were incubated with five volumes of 1.5% sodium citrate solution for 30 min at 56°C. Finally, chicken erythrocytes (50%) were added in 1/10 volume of the serum and incubated at 4°C under gentle shaking for 1.5 hours, before removing erythrocytes by centrifugation at 1000g for 10 min at 4°C. This step involved starting the serum dilution range at 1:10 (21).

For survey C, nine volumes of trypsin diluted at 1/27000 were added to one volume of serum and incubated for 30 min at 58°C. 225µl of metaperiodate of sodium (0.01M) was added to the mixture and incubated at room temperature for 15 min. After a contact of 10 min with 25µl of glycerine (10%), a suspension of chicken erythrocytes (50%) was added in 1/20 volume of the serum and stored overnight at 4°C before removing the erythrocytes by centrifugation. The collected supernatant corresponded to a dilution 1:20 of the serum.

HI tests were performed using four hemagglutinating units (HAU) of virus with 0.5% chicken erythrocytes according to standard procedures (22). Two-fold treated serum dilutions were tested starting from 1:10 (surveys A and B) or 1:20 (survey C) dilution, respectively. HI titers were expressed as the reciprocal of the highest dilution of serum inhibiting four HAU. Negative and positive pig sera were included as controls. Negative sera were obtained from specific pathogen free (SPF) pigs, bred in ANSES, Ploufragan, France. Positive sera were hyper-immune sera (HIS) directed against the reference swIAVs. They were produced in SPF pigs in ANSES biosecurity level 3 facilities and involved intranasal inoculation of live virus followed by intramuscular injection of live virus in the presence of adjuvant three weeks later (12, 14). The reproducibility of the HI tests was ascertained by HIS titers obtained in homologous reactions that must be the initial expected titers +/- one dilution.

For the three surveys, a serum was declared positive towards a given reference antigen when its HI antibody titer was ≥ 20. In surveys A and B, HI titers equal to 10 were doubtful.
At the batch level, rules were established to interpret HI test results for each survey. Previously, the European Surveillance Network for Influenza in Pigs (ESNIP) set rules to discriminate between multiple infections and cross-reactions within the HI tests (23). These rules were used for interpretation of survey A, where three antigens were tested. However, some of these rules needed to be updated for interpretation of surveys B and C, for which four antigens were needed to be tested, in line with swIAV genetic and antigenic evolution, making the results even more complex to interpret.

For the three surveys, a batch was declared positive, in the first instance, for the antigen with which the highest frequency of positive animals was observed.

A batch was declared as positive towards a second antigen, and a third antigen, and possibly a fourth antigen if a serum with a HI titer < 20 to the first antigen (negative serum) had an HI titer ≥ 20 (positive serum) towards another antigen. Thus, for survey A, a batch was also positive towards a second antigen if at least one serum had an equal or higher HI titer against other antigens than those against the first one. For surveys B and C, the evaluation of homologous and heterologous reactions between the four tested reference antigens and HIS made it possible to establish other thresholds of positivity adjusted to the tested lineages. Each threshold was defined based on the results of twelve independent cross-HI assays between HIS, negative serum from SPF pig and reference swine antigens (17) (supplementary table). Thus, for surveys B and C, a batch was declared as positive towards a second antigen according to the positive thresholds applied by lineage and detailed in table 3.

Moreover, for survey B, given the number of batches exhibiting doubtful individual results (HI titers equal to 10), a batch was also considered positive towards an antigen when at least 3/10 sera were not strictly negative towards this antigen. For survey A, the doubtful results were not considered to define positive batches, in accordance with initial ESNIP rules.

Finally, batches were declared of undetermined serotype if 10/10 sera obtained HI titer <20 towards all antigens in survey A and C, and at least 8/10 sera obtained HI titer <10 in survey B.

Table 2: swIAV strains used as antigens in hemagglutination inhibition tests depending on the survey
<table><caption>Table 3: HI titer thresholds for positivity at the batch level in surveys B and C.</caption><thead><tr><th>swIAV lineage based on HA subtype and clade</th><th>2008</th><th>2018</th><th>2022</th></tr><tr><td></td><td>Survey A</td><td>Survey B</td><td>Survey C</td></tr></thead><tbody><tr><td>H1<sub>av</sub> (HA clade 1C.2.1)</td><td>A/Sw/Morbihan/0070/05</td><td>A/Sw/France/29-200272-01/2020</td><td>A/Sw/France/29-200272-01/2020</td></tr><tr><td>H1<sub>av</sub> (HA clade 1C.2.4)</td><td>-</td><td>-</td><td>A/Sw/France/35-200154/2020</td></tr><tr><td>H1<sub>pdm</sub> (HA clade 1A.3.3.2)</td><td>-</td><td>A/Sw/France/57-140136/2014</td><td>A/Sw/France/57-140136/2014</td></tr><tr><td>H1<sub>hu</sub> (HA clade 1B.1.2.3)</td><td>A/Sw/Scotland/410440/94</td><td>A/Sw/France/35-110415/2011</td><td>A/Sw/France/35-110415/2011</td></tr><tr><td>H3 (1970 lineage)</td><td>A/Sw/Gent/1/84</td><td>A/Sw/France/59-150357/2015</td><td>-</td></tr></tbody></table>

<table><caption>The batch is also positive against a second swIAV lineage if HI titers are:</caption><thead><tr><th>When a batch of sera is positive against a 1<sup>st</sup> antigen of HA lineage:</th><th>H1<sub>av</sub><br>(clade 1C.2.1)</th><th>H1<sub>av</sub><br>(clade 1C.2.4)</th><th>H1pdm<br>(clade 1A.3.3.2)</th><th>H1<sub>hu</sub><br>(clade 1B.1.2.3)</th><th>H3<br>(1970.1)</th></tr></thead><tbody><tr><td>H1<sub>av</sub> (clade 1C.2.1)</td><td>-</td><td>≥ 80</td><td>≥ 40</td><td>≥ 40</td><td>≥ 20</td></tr><tr><td>H1<sub>av</sub> (clade 1C.2.4)</td><td>≥ 40</td><td>-</td><td>≥ 20</td><td>≥ 20</td><td>≥ 20</td></tr><tr><td>H1<sub>pdm</sub> (clade 1A.3.3.2)</td><td>≥ 40</td><td>≥ 40</td><td>-</td><td>≥ 40</td><td>≥ 20</td></tr><tr><td>H1<sub>hu</sub> (clade 1B.1.2.3)</td><td>≥ 20</td><td>≥ 40</td><td>≥ 40</td><td>-</td><td>≥ 40</td></tr><tr><td>H3 (1970.1)</td><td>≥ 20</td><td>≥ 80</td><td>≥ 20</td><td>≥ 20</td><td>-</td></tr></tbody></table>

The thresholds were determined according to known antigen-antibody cross-reactions.

## Calculation of seroprevalence values and statistical analyses

The design of the surveys (cluster sampling for surveys A and C, unequal weighting of observations among sampled counties when compared to the actual population) was taken into account to obtain population-based estimates of the seroprevalence values. An adjustment of the seroprevalence results was applied for each county based on the real number of pig farms with fattening pigs per county in the study area, using the official database (BDPORC 2012, 2018 or 2022 for surveys A, B and C, respectively). As the database for the French pig production in 2008 was no longer available at the time of the
analyses, the 2012 database was used to survey A. As for survey B farm characteristics were available
because of individual-based sampling from the national database, hence the real distribution of the
different farm types (farrow-to-finish, wean-to-finish or Finishing farm) was also used as a parameter of
adjustment in the estimations. The analyses were carried out with the R Survey package version 4.4 (24).
The regional seroprevalence values were established accounting for the herd location in one of the four
areas previously defined, i.e., North East, North West, South East or South West (Figure 1).

Moreover, for national survey B conducted in 2018, a logistic regression was applied to the adjusted
sample (function svyglm using the R Survey package) to calculate the odds of a herd being seropositive
given several variables (herd type, region and age of sampled pigs).

For comparisons of quantitative data of ELISA titers, Wilcoxon Rank-Sum test was applied and
significant differences were defined if p-value was <0.05 (R version 4.1.2).

Results

High swIAV seroprevalence levels were sustained over time

The national seroprevalence value was estimated to be 91% [83-96]<sub>C195</sub> in 2008 (survey A) and 87% [81-92]<sub>C195</sub> in 2018 (survey B) (table 4). The within-herd positivity distributions ranged from 10% to 100% and were not considered statistically different in both surveys (χ2 p-value = 0.13, figure 2). Most positive farms exhibited more than half-positive pigs per batch, revealing a high within-herd positivity. Thus, at least 6/10 positive pigs were observed in 94% and 89% of positive herds in 2008 and 2018, respectively (figure 2).

At the regional level, the highest seroprevalence value was detected in the North West in both 2008 and 2018 periods (94% and 88%, respectively), at similar rates than that found in this region in 2022 (91%) (table 4). In the North East, an important evolution occurred between 2008 and 2018, rising from 52% to 82%. A similar increase, from 28% in 2008 to 68% in 2018, was measured in the South East, whereas the seroprevalence in the South West remained stable around 49% of positive herds whatever the period.

Table 4: National and regional estimated seroprevalence values of swIAV infections in France in 2008, 2018 and 2022.
<table><thead><tr><th>Study area</th><th>2008</th><th>2018</th><th>2022</th></tr></thead><tbody><tr><td></td><td>Survey A<br>N=186</td><td>Survey B<br>N=487</td><td>Survey C<br>N=116</td></tr><tr><td>Hexagonal France</td><td><strong>91%</strong> [83-96] CI95%<br>n<sub>pos</sub>=127</td><td><strong>87%</strong> [81-92] CI95%<br>n<sub>pos</sub>=364</td><td></td></tr><tr><td>North East</td><td><strong>52%</strong> [18-84] CI95%<br>n<sub>pos</sub>=8</td><td><strong>82%</strong> [63-93] CI95%<br>n<sub>pos</sub>=33</td><td>-</td></tr><tr><td>North West</td><td><strong>94%</strong> [90-97] CI95%<br>n<sub>pos</sub>=105</td><td><strong>88%</strong> [82-92] CI95%<br>n<sub>pos</sub>=295</td><td><strong>91%</strong> [73-97] CI95%<br>n<sub>pos</sub>=92</td></tr><tr><td>South East</td><td><strong>28%</strong> [6-70] CI95%<br>n<sub>pos</sub>=5</td><td><strong>68%</strong> [39-87] CI95%<br>n<sub>pos</sub>=14</td><td>-</td></tr><tr><td>South West</td><td><strong>49%</strong> [31-68] CI95%<br>n<sub>pos</sub>=9</td><td><strong>49%</strong> [42-56] CI95%<br>n<sub>pos</sub>=22</td><td>-</td></tr></tbody></table>

N=number of tested herds. Confidence interval (CI) at 95% is given into brackets; n pos =number of positive herds by ELISA.

*Estimated seroprevalence values of the different swIAV lineages changed over time*

The frequencies of batches found infected by only one virus subtype were estimated below 50% for both national surveys, and anti-HA antibodies against two or three different antigens were detected in around a quarter of cases (table 5). The presence of anti-HA antibodies directed towards only one antigen was shown in 50% [40-59] CI95 of the farms tested in the North West area in 2022 while 30% [23-37] CI95 showed several swIAV lineage exposures. Whatever the period, the mean ELISA titer of positive herds increased when multiple lineages exposures were revealed by HI tests (table 5 and figure 3). Statistical differences in ELISA titers were obtained for batches showing a multiple swIAV exposure, a single swIAV lineage exposure or an unidentified swIAV lineage exposure with the same pattern at each period (p-value <0.05). The mean ELISA titers were significantly lower for positive herds, which belonged to "unidentified swIAV lineage exposure" group compared to those obtained for herds belonging to single or multiple exposure groups (p-value<0.05).

Table 5: Estimated frequencies of single, multiple or unidentified swIAV lineage exposure in 2008, 2018 and 2022.
<table><thead><tr><th rowspan="2">swlAV lineage exposure</th><th>2008</th><th>2018</th><th>2022</th></tr><tr><th>Survey A</th><th>Survey B</th><th>Survey C</th></tr></thead><tbody><tr><td><strong>Multiple swlAV lineage exposure</strong></td><td><strong>21% [16-29]<sub>CI95</sub></strong><br>n<sub>pos</sub>=20<br><strong>20862</strong></td><td><strong>26% [22-29]<sub>CI95</sub></strong><br>n<sub>pos</sub>=87<br><strong>15168</strong></td><td><strong>30% [23-37]<sub>CI95</sub></strong><br>n<sub>pos</sub>=26<br><strong>12403</strong></td></tr><tr><td><strong>Single swlAV lineage exposure</strong></td><td><strong>47% [37-58]<sub>CI95</sub></strong><br>n<sub>pos</sub>=65<br><strong>16210</strong></td><td><strong>49% [45-53]<sub>CI95</sub></strong><br>n<sub>pos</sub>=211<br><strong>8516</strong></td><td><strong>50% [40-59]<sub>CI95</sub></strong><br>n<sub>pos</sub>=52<br><strong>5675</strong></td></tr><tr><td><strong>Unidentified swlAV lineage exposure</strong></td><td><strong>23% [16-32]<sub>CI95</sub></strong><br>n<sub>pos</sub>=42<br><strong>9519</strong></td><td><strong>13% [12-14]<sub>CI95</sub></strong><br>n<sub>pos</sub>=66<br><strong>4836</strong></td><td><strong>11% [7-18]<sub>CI95</sub></strong><br>n<sub>pos</sub>=14<br><strong>1902</strong></td></tr></tbody></table>

Confidence interval (CI) at 95% is given into brackets; n<sub>pos</sub>=number of positive batches by ELISA; Mean ELISA titers of positive batches are given in bold in the third line.

At the national level, the major swlAV lineage infecting pigs in production herds in 2008 was the H1<sub>hu</sub>N2, with an estimated seroprevalence value of 54% towards H1<sub>hu</sub> clade 1B.1.2.3 (figure 4). The HI antibody titers varied from 20 to 2560. The second most seroprevalent swlAV was the H1<sub>av</sub>N1 lineage as the seropositivity against H1<sub>av</sub> clade 1C.2.1 antigen reached 38% (figure 4). The HI antibody titers varied from 20 to 640. In 2018, trends have reversed, with 56% of seropositive batches towards H1<sub>av</sub>N1 presenting the same HI antibody titers range than in 2008, and 36% towards H1<sub>hu</sub>N2 (titers range [20-640]). The seropositivity against H1N1<sub>pdm</sub> was tested in 2018 and 15% of batches were positive towards H1<sub>pdm</sub> clade 1A.3.3.2 (titers range [20-640]). The seroprevalence of H3N2 was very low in both surveys, with only 1.5% and 0.6% of positive batches in 2008 and 2018, respectively. The HI antibody titers ranges for the H3 lineage were [80] in 2008 and [20-40] in 2018.

When estimating the seroprevalence values at the regional level, contrasted situations were observed depending on the area. In the North East area, the three swlAV lineages tested in 2008 were detected: the highest seroprevalence value was towards H1<sub>av</sub>N1 (29%) whereas the prevalence values of H1<sub>hu</sub>N2 and H3N2 lineages were similar (19% and 18%, respectively) (figure 4). In the North West area, the highest seroprevalence value was reached by the H1<sub>hu</sub>N2 lineage (57%), followed by H1<sub>av</sub>N1 that reached 40%, whereas the prevalence of the H3N2 lineage was estimated to be very low (1%). In the South East, antibodies were detected against only two swlAV lineages, H1<sub>av</sub>N1 and H1<sub>hu</sub>N2, with similar prevalence values (11% and 12%, respectively). In the South West, only antibodies against H1<sub>av</sub>N1 lineage were detected, at a seroprevalence level of 14%.
In 2018, the situation evolved as infections due to the H1N1pdm virus were evidenced in the four areas of the territory. The North East area remained a region where all tested swIAV lineages were detected, with seroprevalence values in decreasing order as follows: H1N1pdm (34%), H1avN1 (30%), H1huN2 (21%) and H3N2 (7%) (figure 4). In the North West area, the seroprevalences of the lineages H1avN1 (57%) and H1huN2 (37%) prevailed over those of H1N1pdm (15%) and H3N2 (0.5%). In the South East and South West areas, the pigs were only exposed to H1avN1 and H1N1pdm. However, their seroprevalence levels were similar (38% and 37%, respectively) in South East, but different (21% and 7.6%, respectively) in South West.

The survey conducted in the North West area in 2022 showed that the high seroprevalence value towards H1avN1 (HA-clade 1C.2.1) was maintained over time (53%; titer range [20-2560]) while the seroprevalence towards H1huN2 strongly diminished (10%; titer range [20-160]) (figure 4). In contrast, the seroprevalence of H1avN2 (HA-clade 1C.2.4) was estimated at 36%, with a titer range from 20 to 5120. The seroprevalence of H1N1pdm counted for 19% (titer range [20-320]).

As mentioned before, several cases of unidentified lineage exposure were revealed regardless of the survey (table 5). The proportion of cases where the swIAV subtype responsible for the presence of antibodies in sera remained undetermined, reached 23% and 13% at the national level in 2008 and 2018, respectively. In both surveys, the frequency of unidentified subtype varied depending on the region. In 2008 it was equal to 35% [15-61]Cl95 in South West, 23% [15-34]Cl95 in North West, 11% [2-39]Cl95 in South East and 5% [0.8-25]Cl95 in North East (figure 4). In 2018, the highest frequency of unidentified swIAV subtype was observed again in South West (22% [16-30]Cl95) but the lowest was found in South East (5.5% [1-26]Cl95) (figure 4). It decreased by 10 points in North West and increased by 14 points in North East. In 2022, the frequency of unidentified positive cases (11%) remained similar to that calculated in 2018 (13%).

*Impact of geographic area, pig production type and age of pig on swIAV seroprevalence in 2018*

In 2018, herds in South West were evaluated to be 5 times (OR=0.21 [0.08-0.55]Cl95) less affected by swIAV infection than those in North East, which was considered as the reference in the statistical model (table 6). The risk of influenza infection in North West and North East, as well as in South East and North East, were not statistically different (p-value >0.05).

Herds located in North West were 3 times more exposed to the risk of H1avN1 virus infection than those from North East (OR=3.11 [2.24-4.31]Cl95) and twice more exposed to the risk of H1huN2 virus infection (OR=2.18 [1.49-3.20]Cl95) (table 6). However, they were 3 times less impacted by H1N1pdm infection (OR=0.33 [0.24-0.46]Cl95). Also, pigs seemed to be less impacted by H1N1pdm infection in South West than in North East (OR=0.16 [0.07-0.34]Cl95).
At the national level, the post-weaning-finishing type of pig production was estimated 3 times less infected than farrow-to-finish type (OR = 0,33 [0,23-0,46]$_{CI95}$) (table 6). This seemed to be especially the case in North West area (OR = 0,32 [0,20-0,51]$_{CI95}$), but in North East and South East, the finishing production type was more infected compared to the farrow-to-finish herds (OR = 6,80 [1,72-26,88]$_{CI95}$ and 4,35 [2,31-8,19]$_{CI95}$ respectively).

Finally, two ranges of age ([10;15] and [≥16] weeks, respectively) were compared and it appeared that the oldest pigs had twice more risk than the youngest ones to be seropositive (OR = 2,43 [1,07-5,55]$_{CI95}$) (table 6).

Table 6: Impact of geographic area, pig production type and age of sampled pigs on swIAV seroprevalence.
Epidemiological factors

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td colspan="5"><em>Geographic area on swlAV positivity</em></td></tr><tr><td>North East</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>North West</td><td>1,60</td><td>0,56</td><td>4,55</td><td>NS</td></tr><tr><td>South East</td><td>0,45</td><td>0,11</td><td>1,89</td><td>NS</td></tr><tr><td>South West</td><td>0,21</td><td>0,08</td><td>0,55</td><td>0,01</td></tr></tbody></table>

Geographic area on H1<sub>av</sub>N1 positivity

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td>North East</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>North West</td><td>3,11</td><td>2,24</td><td>4,31</td><td>&lt; 0,0001</td></tr><tr><td>South East</td><td>1,44</td><td>0,26</td><td>8,02</td><td>NS</td></tr><tr><td>South West</td><td>0,62</td><td>0,36</td><td>1,07</td><td>NS</td></tr></tbody></table>

Geographic area on H1<sub>hu</sub>N2 positivity

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td>North East</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>North West</td><td>2,18</td><td>1,49</td><td>3,20</td><td>&lt; 0,0001</td></tr><tr><td>South East</td><td>0</td><td>0</td><td>0</td><td>&lt; 0,0001</td></tr><tr><td>South West</td><td>0</td><td>0</td><td>0</td><td>&lt; 0,0001</td></tr></tbody></table>

Geographic area on H1N1<sub>pdm</sub> positivity

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td>North East</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>North West</td><td>0,33</td><td>0,24</td><td>0,46</td><td>&lt; 0,0001</td></tr><tr><td>South East</td><td>1,12</td><td>0,46</td><td>2,75</td><td>NS</td></tr><tr><td>South West</td><td>0,16</td><td>0,07</td><td>0,34</td><td>&lt; 0,0001</td></tr></tbody></table>

Production type on swlAV positivity

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td>Farrow-to-finish</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Finishing</td><td>0,63</td><td>0,38</td><td>1,04</td><td>NS</td></tr><tr><td>Post-weaning-finishing</td><td>0,33</td><td>0,23</td><td>0,46</td><td>&lt; 0,0001</td></tr></tbody></table>

Age of pigs on swlAV positivity

<table><thead><tr><th></th><th>OR</th><th>2,5% OR</th><th>97,5% OR</th><th>p-value</th></tr></thead><tbody><tr><td>&lt; 16 WOA</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>≥ 16 WOA</td><td>2,43</td><td>1,07</td><td>5,55</td><td>0,035</td></tr></tbody></table>
Odd Ratio (OR), 95% confidence interval and p-value were calculated by logistic regression using data from survey B conducted in 2018. NS: not significant, WOA: week of age

Discussion

National surveys conducted in 2008 and 2018 revealed that swIAV infections were largely affecting the pig population in hexagonal France at both period. The swIAV seroprevalence in 2008, reaching 91%, was similar to that obtained in Spain at the same time (22). Indeed, a cross-sectional survey organized in 2008–2009 by testing 10 finishing pigs in 98 farms located in ten regions across Spain, resulted to a seroprevalence of 93.9% by anti-NP ELISA (25). The sampling strategy is important to measure a seropositivity against swIAV that is actually related to the circulation of the viruses in herds. That is the reason why the target population was pigs over 10 weeks of age and not sows nor piglets. Thanks to this sampling frame, the three surveys focused on serological responses to swIAV infection and discarded the problem of interference linked to breeding herd vaccination or transmission of maternally derived antibodies. In the 2018 survey, we observed a higher risk of antibodies detection in oldest pigs (≥ 16 weeks). Logically, there is an increasing probability of swIAV exposure linked to the age, as the oldest animals could have been infected several times with different swIAV subtypes successively. It is in line with a study conducted in 2019–2023 in commercial farrow-to-finish pig farms in Greece that estimated variable seroprevalences according to the age groups of swine (26). We also observed an impact of herd type with a higher risk of antibodies detection in farrow-to-finish herds compared to the post-weaning-finishing herds at the national scale. The self-sustaining forms of the infection that may depend on production systems and intensive herd management could explain this observation. Amongst factors, sows herds could play a role in the within-herd virus dynamics and in the swIAV persistence at the herd level (4, 6, 15). The within-herd frequency of seropositive pigs observed in both national surveys was high. It was not surprising since swine influenza is a very contagious disease, as supported by the high reproduction number estimated in field condition (4) and as we previously calculated in experimental conditions (6).

In both our national surveys, the sampling was appropriate to estimate the swIAV seroprevalence and its
evolution over time, with more accurate results obtained with the 2018 sampling as shown by smaller
confidence intervals in the 2018 survey. The seroprevalence value was already high (91%) in 2008 and
remained as such in 2018 (87%). We reported an increase of seroprevalence between 2008 and 2018
only regionally, in North East and South East, which could be linked to the emergence and spread of the
new lineage H1N1pdm within the pig population after the pandemic flu in 2009. The H1N1pdm virus has
rapidly disseminated in the pig population worldwide, especially in countries previously free of swIAV.
For example in Norway, active serosurveillance had highlighted for years a naive pig population for all
swIAV, but after 2009 herds had been infected by the H1N1pdm virus and seroprevalence had raised in a
short time (27, 28). Moreover, the authors pointed that the seroprevalence was higher in the most
densely populated pig areas. We also observed the highest swIAV seroprevalence in the most intensive production region, i.e., the North West of France, in our both national surveys.

The analytical approach was consistent across all study period: first ELISA was used to detect antibodies against the swIAV nucleoprotein providing insights into disease prevalence, followed by HI tests, which are more time-consuming and challenging to interpret. Selecting the appropriate antigens is a critical step in conducting HI tests, as it ensures accurate detection of antibodies with a good sensitivity, which can offer valuable information on prevalence of the different virus lineages. At each period, the antigen selection was consistent with knowledge of circulating viruses at the European and/or French levels (10–16, 18). To accurately set positivity thresholds and interpret the HI titers, it is crucial to control the antigenic distances between reference viruses and the level of cross-reactions in the HI tests. For study A in 2009, we followed the guidelines established by the European consortium ESNIP (23), but adjustments were necessary when additional reference antigens were incorporated for study B and C in 2018 and 2022. Moreover, it seemed informative to run HI test beginning at 1:10 serum dilution and consider HI titer equal to 10 possibly, to gain in sensitivity. Concerning the survey A from 2009, considering doubtful sera would have reduced the number of undetermined lineage exposure batches to reach a similar proportion of that was observed in survey B in 2018 (data not shown). Therefore, we encourage using a pre-treatment method of sera which allows HI test starting with a serum dilution range from 1:10.

In 2008–2009, passive surveillance of swIAV infections was not organized at the national level in France and the collected data were limited to the North West (11). Consequently, national survey A has well completed knowledge on viruses in circulation and pointed some regional specificities. It showed the presence of H3 lineage in the North East. This information was supported by data from several other European countries, including Belgium, where the H3N2 virus was prevalent at this time (23). In France, while no more H3N2 virus was detected since the end of the 90s, one farm located in a high pig density area, around ten kilometres from the Belgian border, was found infected by an H3N2 virus in 2011, which confirmed sporadic cases of infection by this lineage in this region (29), in line with results from both surveys A and B. In North West, the very low seroprevalence of this lineage is consistent with the fact that this virus has no longer been detected by passive surveillance. Another regional specificity was the absence of H1<sub>hu</sub>N2 in the South West, the only region with a single lineage exposure until 2009. However, it can be pointed out the highest frequency of unidentified lineage exposure in this region in both surveys A and B. Even so the lack of identification of anti-HA antibodies was probably due to a weaker sensitivity of HI tests compared to ELISA, as revealed by the lowest mean ELISA titers obtained on these batches, it cannot be excluded that the absence of a reaction is linked to the choice of tested antigens, that was based on virological data. We performed a HI test with an additional H1<sub>av</sub>N2 antigen harbouring an HA-1C.2.4 and punctually isolated in 2015 in this region (30). This supplementary investigation of all batches obtained in the South West in survey B in 2018 did not show the presence of anti-HA-1C.2.4 antibodies (data not shown). We also tested a sub-sample of 18 undetermined serotype batches from the four regions of the survey B to test a human H3N2 antigen, but no positive results were obtained
(data not shown). It could be informative to test other IAV, particularly of avian origin, which might have crossed the species barrier from poultry to swine. In any case, these results recall the importance to set up virological surveillance, especially through the RESAVIP network, in particular in South West.

By extrapolation and for simplicity, the detection of anti-HA antibodies was here associated to the circulation of main known virus lineages. For instance, reactivity towards the H1<sub>av</sub>N1 (HA-1C.1.2.1) antigen followed infection by the H1<sub>av</sub>N1 (HA-1C.1.2.1) virus but might also result from infection by a H1<sub>av</sub>N2 (HA-1C.1.2.1) virus. Thus, infections with reassortant viruses could not be distinguished here. For example, in 2006–2008, the virological surveillance conducted in France identified sporadic H1<sub>hu</sub>N1 (HA-1B.1.2.3) and H1<sub>av</sub>N2 (HA-1C.1.2.1) virus strains in low proportion (11). Nonetheless, whatever their frequencies, these infections could not be distinguished by HI test and were here confounded with H1<sub>hu</sub>N2 (HA-1B.1.2.3) and H1<sub>av</sub>N1 (HA-1C.1.2.1) infections, respectively, but this potential misclassification was considered as negligible according to our knowledge of the detected swIAV.

While overall seroprevalence remained unchanged between 2008 and 2018, the seroprevalences of the various viral lineages changed significantly, due in particular to the introduction of the H1N1<sub>pdm</sub> virus. The H1N1<sub>pdm</sub> was present in all four regions and was even the major lineage in North East in 2018, confirming the enzootic circulation of this virus in the pig population in France, which has led to an increase in viral diversity. Globally, the relative frequencies of the different anti-HA antibodies detected in 2018 at the national scale reflected a distribution of virus lineages that was qualitatively similar to that of the lineages identified in 2017–2018 through the event-based surveillance (18).

In 2022, the seroprevalences of viral lineages in North West of France have evolved as compared to 2018, in line with the emergence of the H1<sub>av</sub>N2 (HA-1C.2.4) lineage in Brittany in 2020 (17, 18), a new lineage whose prevalence has been estimated at 36%, slightly lower than that of the H1<sub>av</sub>N1 virus. By contrast, the event-based surveillance carried out in 2021 showed a higher proportion of influenza cases due to the H1<sub>av</sub>N2 (1C.2.4) virus than to the H1<sub>av</sub>N1 virus (18). The H1<sub>av</sub>N2 (1C.2.4) virus, which is genetically and antigenically distant from the other H1<sub>av</sub> (1C.2) viruses detected in France, was responsible for an epizootic, probably because it was able to escape pre-existing population immunity (31). Thus, this emerging virus was responsible for many clinical cases, but serological data from survey C revealed that the oldest H1<sub>av</sub>N1 might have continue to circulate in the population subclinically or with less marked symptoms than the new genotype, leading to fewer diagnostic investigations from practitioners.

Previous studies had already highlighted the simultaneous presence of anti-HA antibodies directed against different subtypes (10, 23). In 2006–2008, another study conducted within a European serosurvey studying the influenza dynamics in finishing pigs, showed that several subtypes infected on average more than 40% of farms (23). In the three surveys carried out here, exposure to several lineages was found in around a quarter of farms nationwide, and in slightly higher proportions in the North West of France. These results emphasized the consecutive or simultaneous circulation of different swIAV
lineages in herds. These situations favorable to co-infections may generate new viruses through genetic reassortment, some of which are sporadically picked up by event-based surveillance. These reassortant viruses may represent a threat for animal and public health.

## Conclusions

Serological surveys alone would not provide comprehensive information about the swIAV circulating in the country, nor would they allow us to monitor virus evolution by themselves. However, they do provide complementary data to virological detection, which is highly informative in terms of the prevalence of infections, about potentially asymptomatic circulations and uncovering rare events. In view of all these results, and despite the difficulties involved in carrying them out, it would be appropriate to be able to conduct national serological surveys on a regular basis, to support and reinforce the event-based monitoring elsewhere.

## Declarations

### Ethics approval and consent to participate

Not applicable

### Consent for publication

Not applicable

### Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

### Competing interests

The authors declare that they have no competing interests.

### Funding

This project was partially funded by the Interprofession Nationale Porcine (INAPORC) for Survey B.

### Authors' contributions

SH: study design, analysing, interpreting the data and writing the manuscript. NR: conception, study design, statistical analysis. NB, SQ, SG, RF, GP, performing the serological tests. GR: support for statistical analysis. AJ: conception and analysing the data of Survey C. GS: conception, study design, supervision, writing the manuscript. All authors read and approved the final manuscript.

### Acknowledgements
The authors would like to thank their colleagues from Anses, Ploufragan, F. Eono, V. Dorenlor and E. Eveno for their contributions to the 2008 A survey. They also thank the Association Nationale Sanitaire Porcine (ANSP) for making available the sera collected in 2018. Finally, they would like to thank Labocea 22 for carrying out ELISA and HI tests for Survey C.

## References

1. Janke BH. Influenza A virus infections in swine: pathogenesis and diagnosis. Veterinary pathology. 2014;51(2):410-26.

2. Fablet C, Marois-Créhan C, Simon G, Grasland B, Jestin A, Kobisch M, et al. Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross-sectional study. Veterinary microbiology. 2012;157:152-63.

3. Dykhuis Haden C, Painter T, Fangman T, Holktamp D. Assessing production parameters and economic impact of swine influenza, PRRS and Mycoplasma hyopneumoniae on finishing pigs in a large production system. American Association of Swine Veterinarians. 2012:75-6.

4. Rose N, Hervé S, Eveno E, Barbier N, Eono F, Dorenlor V, et al. Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events. Veterinary research. 2013;44(1):72.

5. Simon G, Thiroux S, Mateu de Antonio EM, Everett H, Chiapponi C, Harder T, et al., editors. Understanding the dynamics and evolution of swine influenza viruses in endemically infected pig herds: coordinated longitudinal studies in six european countries. ESPHM - 14th European Symposium of Porcine Health Management; 2023 May 31 - June 2; Thessaloniki, Greece.

6. Cador C, Hervé S, Andraud M, Gorin S, Paboeuf F, Barbier N, et al. Maternally-derived antibodies do not prevent transmission of swine influenza A virus between pigs. Veterinary research. 2016;47(1):86.

7. Henritzi D, Petric PP, Lewis NS, Graaf A, Pessia A, Starick E, et al. Surveillance of European Domestic Pig Populations Identifies an Emerging Reservoir of Potentially Zoonotic Swine Influenza A Viruses. Cell Host & Microbe. 2020.

8. McLean RK, Graham SP. The pig as an amplifying host for new and emerging zoonotic viruses. One health (Amsterdam, Netherlands). 2022;14:100384.

9. Nelson MI, Worobey M. Origins of the 1918 Pandemic: Revisiting the Swine "Mixing Vessel" Hypothesis. American journal of epidemiology. 2018;187(12):2498-502.

10. Van Reeth K, Brown IH, Durrwald R, Foni E, Labarque G, Lenihan P, et al. Seroprevalence of H1N1, H3N2 and H1N2 influenza viruses in pigs in seven European countries in 2002-2003. Influenza and Other Respiratory Viruses. 2008;2(3):99-105.

11. Kyriakis CS, Brown IH, Foni E, Kuntz-Simon G, Maldonado J, Madec F, et al. Virological Surveillance and Preliminary Antigenic Characterization of Influenza Viruses in Pigs in Five European Countries from 2006 to 2008. Zoonoses and Public Health. 2011;58:93-101.
12. Simon G, Larsen LE, Durrwald R, Foni E, Harder T, Van Reeth K, et al. 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. PloS one. 2014;9(12):e115815.

13. Watson SJ, Langat P, Reid SM, Lam TT, Cotten M, Kelly M, et al. Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013. Journal of virology. 2015;89(19):9920-31.

14. Chastagner A, Hervé S, Quéguiner S, Hirchaud E, Lucas P, Gorin S, et al. 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. Viruses. 2020;12(11).

15. Hervé S, Garin E, Calavas D, Lecarpentier L, Ngwa-Mbot D, Poliak S, et al. Virological and epidemiological patterns of swine influenza A virus infections in France: Cumulative data from the RESAVIP surveillance network, 2011–2018. Veterinary microbiology. 2019;239:108477.

16. Chastagner A, Hervé S, Bonin E, Quéguiner S, Hirchaud E, Henritzi D, et al. Spatio-temporal distribution and evolution of the A/H1N1 2009 pandemic virus in pigs in France from 2009 to 2017: identification of a potential swine-specific lineage. Journal of virology. 2018.

17. Hervé S, Chastagner A, Quéguiner S, Barbier N, Gorin S, Blanchard Y, et al. Dissemination in 2020, in swine herds from north-western France, of a H1avN2 swine influenza virus of a genotype newly introduced in brittan y. Bullet Epidémiol Santé Animale Aliment. 2021;2(92).

18. Richard G, Hervé S, Chastagner A, Quéguiner S, Beven V, Hirchaud E, et al. Major change in swine influenza virus diversity in France owing to emergence and widespread dissemination of a newly introduced H1N2 1C genotype in 2020. Virus Evolution. 2025;veae112.

19. Muzykina L, Barrado-Gil L, Gonzalez-Bulnes A, Crespo-Piazuelo D, Cerón JJ, Alonso C, et al. Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses. Viruses. 2024;16(4).

20. BDPorc. The professional pig database BDPorc [Available from: https://bdporc.com]. Accessed 30 january 2025.

21. Grech-Angelini S, Hervé S, Rose N, Barbier N, Casabianca F, Maestrini O, et al. Serological survey of influenza A viruses in domestic and wild Suidae in Corsica (France), a Mediterranean island environment. Preventive Veterinary Medicine. 2018;157:94-8.

22. WOAH. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2023. In: Health WOfA, editor.2023. p. 1-18.

23. Kyriakis CS, Rose N, Foni E, Maldonado J, Loeffen WL, Madec F, et al. Influenza A virus infection dynamics in swine farms in Belgium, France, Italy and Spain, 2006-2008. Veterinary microbiology. 2013;162(2-4):543-50.

24. Lumley T. Survey: analysis of complex survey samples. R package version 44. 2024.

25. Simon-Grife M, Martin-Valls GE, Vilar MJ, Garcia-Bocanegra I, Mora M, Martin M, et al. Seroprevalence and risk factors of swine influenza in Spain. Veterinary microbiology. 2011;149(1-2):56-63.
26. Papatsiros VG, Papakonstantinou GI, Meletis E, Koutoulis K, Athanasakopoulou Z, Maragkakis G, et al. Seroprevalence of Swine Influenza A Virus (swIAV) Infections in Commercial Farrow-to-Finish Pig Farms in Greece. Veterinary Sciences. 2023;10(10).

27. Hofshagen M, Gjerset B, Er C, Tarpai A, Brun E, Dannevig B, et al. Pandemic influenza A(H1N1)v: human to pig transmission in Norway? Eurosurveillance Europe's journal on infectious disease surveillance, epidemiology, prevention and control. 2009;14(45).

28. Er C, Skjerve E, Brun E, Framstad T, Lium B. Occurrence and spread of influenza A(H1N1)pdm09 virus infection in Norwegian pig herds based on active serosurveillance from 2010 to 2014. Epidemiol Infect. 2016;144(15):3148-65.

29. Hervé S, Quéguiner S, Barbier N, Gorin S, Saulnier A, Simon G. Isolation of a swine influenza virus of H3N2 subtype in a pig herd located in North department. Bulletin épidémiologique, santé animale et alimentation Anses-DGAI, 2012;51:22.

30. Bonin E, Hervé S, Quéguiner S, Barbier N, Gorin S, Garin E, et al. Distinction of several subpopulations of H1avN2 swine influenza viruses in France. Bulletin épidémiologique, santé animale et alimentation. 2016;75:11.

31. Deblanc C, Queguiner S, Gorin S, Richard G, Moro A, Barbier N, et al. Pathogenicity and escape to pre-existing immunity of a new genotype of swine influenza H1N2 virus that emerged in France in 2020. Veterinary research. 2024;55(1):65.

## Figures

<figure><img src="image_2.png" /><figcaption>**Figure 1**</figcaption></figure>

**Counties where the sampled herds are located according to the three surveys and the region.** Survey A-2008 on the left; Survey B-2018 in the middle; Survey C-2022 on the right. Counties have been grouped in four geographic areas of hexagonal France (continental excluding Corsica) and coloured as follows: North West in blue; North East in yellow; South West in purple; South East in green
Blood samples were collected from pigs at slaughterhouses or farm sites and sera were stored at -20°C until analyses.

<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

**Within-herd distribution of positive samples.** The number of positive pig(s) per batch (among 10 tested) is given in relation to the frequencies of ELISA-positive herds in national surveys conducted in 2008 (survey A) and 2018 (survey B).
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

Distribution of ELISA titers according to the type of swIAV lineage exposure in 2008, 2018 and 2022.

Box-plots are represented by year, each plot represents the batch's ELISA titer and is colored in red for the multiple swIAV lineage exposure, in green for the single swIAV lineage exposure and in blue for the unidentified swIAV lineage exposure.
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>

**Estimated seroprevalence values of the swIAV lineages at the national and regional levels in 2008, 2018 and 2022.** The four areas were North East, North West, South East and South West in 2008 (up), 2018 (middle) and only North West in 2022 (down). The swIAV lineages identified were H1av clade 1C.2.1 in pink, H1hu clade 1B.1.2.3 in blue, H1pdm clade 1A.3.3.2 in red, H3 in green and H1av clade 1C.2.4 in
purple. The frequency of unidentified swIAV lineage was in grey. The confidence interval at 95% was identified by the black bar.

**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* Stable.docx