Influenza A in Wild Boars: Viral Circulation in the Emilia-Romagna Region (Northern Italy) between 2017 and 2022

Simple Summary Wild boars and feral pigs are underinvestigated hosts for influenza A viruses (IAVs). This study confirmed and evaluated viral circulation in the Emilia-Romagna wild boar population between 2017 and 2022. Samples were collected at post mortems and screened for IAVs; 0.37% of the tested animals provided positive results. Positive samples were subtyped, isolated, and genotyped via full-genome sequencing. The results highlight the co-circulation of the same viral genotypes in overlapping years in both pigs and wild boars in the same geographical area. Considering the role of domestic and wild Sus scrofa species in the IAVs’ ecology, surveillance against these viruses in the wild boar population needs to be implemented. Abstract A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars’ IAVs detected in the Emilia-Romagna region (2017–2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife–livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains’ complete genome analysis showed an evolving and interesting situation that deserves further investigation.

IAV infection in a pig population usually leads to a highly contagious respiratory disease, which is mainly characterized by high morbidity (up to 100%) and low mortality rates [16][17][18]. Although the knowledge of IAV infection dynamics in pig hosts is constantly evolving worldwide, viral circulation in wild boars and feral pigs is poorly investigated, despite the fact that the three of them belong to the Sus scrofa species [19], and only a few reports are available about the Italian situation [20,21]. European epidemiological surveys on IAV circulation in the Eurasian wild boar population demonstrated a seroprevalence ranging between 1.4% and 25.9% [3,[19][20][21][22][23][24][25][26] and a virological prevalence between 0.8% and 3.4% [3,20,21]. In addition, the wild boar ecological niche enables the possibility of IAV spillover events from wild birds to wild Sus scrofa species [19], as has been reported with serological evidence in the US feral swine population [27].
Wild boars have dramatically increased in number throughout Italy in recent years, resulting in significant agricultural damage and representing a burden for viral infectious disease spread [28]. The latest virological Italian data, published in 2012, reported an H1avN1 subtype in the wild boar population of the Emilia-Romagna region which was a predominant strain co-circulating in the domestic swine population in the same period [20].
The main aim of this study was to investigate the current IAV viral circulation in the Emilia-Romagna wild boar population, taking into account the increased genetic variability of the circulating strains among the domestic swine population [29]. For this purpose, the identified IAVs were subtyped and genotyped, and the evaluation of the results in a high-density commercial swine production geographical area was performed.

Study Design and Sampling
Virological surveillance was performed in the Emilia-Romagna region (Northern Italy) between 2017 and 2022 via an active and passive surveillance program, using the samples collected during the Regional Wildlife Control Plan. Sampling was organized with the collaboration of the Regional Agriculture Office, Fishing and Hunting Offices, and local veterinary services; the surveillance was carried out in the whole regional territory, covering 8 out of the 9 provinces of the region (Bologna, Forlì-Cesena, Modena, Parma, Piacenza, Ravenna, Reggio Emilia, Rimini). Wild boars' carcasses or viscera were collected and delivered, refrigerated, to the IZSLER laboratories by rangers, hunters, and local veterinary authorities.

SwIAVs Detection, Subtyping and Viral Isolation
Lung specimens conferred to the IZSLER laboratories were homogenized in 1:10 in phosphate-buffered saline (PBS); total RNA was extracted from 100 µL of the homogenized sample according to the One for All vet kit (Indical Bioscience GmbH, Leipzig, Germany) manufacturer's instruction.
All of the positive samples were identified at the subtype level using a nested multiplex end-point RT-PCR, as previously described [29], which allowed viral differentiation into the subtypes H1avN1, H1huN2, H3N2, and H1pdm09N1 according to the amplicon product length (for the HA gene the primers set: Simultaneously, viral isolation from positive samples was attempted, inoculating both 11-day-old SPF embryonated chicken eggs and susceptible cell lines (MDCK and Caco-2) [17,31].

Genetic Characterization of the Isolated Viruses
SwIAVs isolates were further genetically and antigenically analyzed using nextgeneration sequencing (NGS) with Illumina technology (MiSeq Sequencing System-Illumina Inc., San Diego, CA, USA), as recently reported [29]. Gene sequences of the eight IAV genome segments, such as gene combinations, were compared with SwIAVs circulating in domestic pigs in the same geographical area [29] and with sequences retrieved from the GenBank Influenza virus resource database (https://www.ncbi.nlm.nih.gov/genomes/ FLU/Database/nph-select.cgi?go=database; accessed on 26 April 2022). The sequences of SwIAV HA gene segments were then aligned with ClustalW using MEGAX [32], and maximum-likelihood (ML) phylogenetic tree analysis was performed using the IQ-TREE-2 software [33][34][35]. The phylogenetic tree robustness was statically evaluated by bootstrap analysis with 1000 bootstrap replicates. The phylogenetic tree was visualized using Figtree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 26 April 2022), and the origin of each segment was determined by its clustering with reference strains (Supplementary S1-dataset of sequences used in this study). Finally, wild boar H1 sequences were analyzed and named using the swine H1 influenza classification tool (http://www.fludb.org, accessed on 26 April 2022) [14].

SwIAV Subtypes Circulation (Years 2017-2022)
From 1 January 2017 to 4 April 2022, 4605 wild boar lung samples were analyzed in 2414 SwIAV one-step real-time RT-PCR tests. The lungs were pooled according to date and location of sampling, with a maximum of five individuals per pool. The sampling abundance evaluated for province distribution is reported in Figure 1.

SwIAV Genotypes and Phylogenetic Analysis
Three out of the seventeen (17.65%) positive samples were isolated and fully genetically characterized via an NGS approach. The isolated strains were from wild boar samples collected in different years and from disparate geographical areas: (i) a 2018 strain from Ravenna province (GenBank accession number MW136322-MW136329); (ii) a 2020 strain from Parma province (GenBank accession number MW621858-MW621865); and (iii) a 2021 strain from Forlì-Cesena province (GenBank accession number ON468439-ON468446).
Whole-genome phylogenetic analysis of the isolates was performed, and the eight genome segments were aligned and analyzed in order to assign each virus to a specific lineage, genotype, and origin. Two of them were subtyped as H1N1 but belonged to different genotypes: the 2018 isolate was an HA-1C-N1av (H Clade 1C.2.1) in genotype U, while the 2020 isolate was an HA-1C-N1av (H Clade 1C.2.1) in genotype A (Harder's nomenclature [36]). Lastly, the 2020 isolate was an H1pdm09N1-derived subtype (HA-1A-N1av-Clade 1A.3.3.2) in genotype 31 ( Table 2). Those results were also compared with the SwIAVs circulating in domestic pig hosts and are reported in Table 2. Table 2. Data analysis of the genotypes among the H1N1 subtype detected in wild boars; strains were assigned to the corresponding genotype, as previously described [13,15,37]. The wild boars' SwIAVs were compared to the strains circulating in swine farms (data from [29]

Discussion
Swine have a well-known crucial role in IAVs' ecology and in viral interspecif mission, being susceptible to both avian and human IVs and thus acting as an IA ing vessel". This capability leads to the development of multiple reassortant (avian/human, human/swine, or human/avian/swine reassortants), which are a spillover infections and are crucial in viral epidemiology [4,6,39,40]. Even thou boars and feral pigs both belong to the Sus scrofa species and can be infected w avian and swine IAVs in the same way as domestic pigs [19,27], these hosts and th in the SwIAVs' ecology are underinvestigated. To date, several reports about Sw roprevalence in European countries are available [3,[19][20][21][24][25][26]41,42], but only them are about virological circulation [19][20][21] and the genotyping of the viral isola During this study, a total of 4605 wild boar biological samples were investig SwIAVs to evaluate the active viral circulation; of these, 17 tested positive (0.37 relatively low prevalence detected could have two explanations: a comparatively demic circulation in the wild boar hosts or a not completely representative sampl positivity level observed in this study could support the evidence of the occur epidemic outbreaks in the wild hosts. At the same time, convenience sampling pe during the Regional Wildlife Control Plan might not be truly representative of t circulation in the wild boar population. To support this hypothesis, further invest are certainly needed, such as the use of different samples or sampling procedu

Discussion
Swine have a well-known crucial role in IAVs' ecology and in viral interspecific transmission, being susceptible to both avian and human IVs and thus acting as an IAV "mixing vessel". This capability leads to the development of multiple reassortant strains (avian/human, human/swine, or human/avian/swine reassortants), which are agents of spillover infections and are crucial in viral epidemiology [4,6,39,40]. Even though wild boars and feral pigs both belong to the Sus scrofa species and can be infected with both avian and swine IAVs in the same way as domestic pigs [19,27], these hosts and their role in the SwIAVs' ecology are underinvestigated. To date, several reports about SwIAV seroprevalence in European countries are available [3,[19][20][21][24][25][26]41,42], but only a few of them are about virological circulation [19][20][21] and the genotyping of the viral isolates [20].
During this study, a total of 4605 wild boar biological samples were investigated for SwIAVs to evaluate the active viral circulation; of these, 17 tested positive (0.37%). The relatively low prevalence detected could have two explanations: a comparatively low endemic circulation in the wild boar hosts or a not completely representative sampling. The positivity level observed in this study could support the evidence of the occurrence of epidemic outbreaks in the wild hosts. At the same time, convenience sampling performed during the Regional Wildlife Control Plan might not be truly representative of the viral circulation in the wild boar population. To support this hypothesis, further investigations are certainly needed, such as the use of different samples or sampling procedures; for instance, Vittecoq et al. [19] reported a 1% SwIAV prevalence (95% confidence) in wild boars' nasal swabs collected in the Camargue area.
At the subtype level, H1N1 was the most frequently detected subtype (five out of seven), identified as H1pdm09N1 (three samples) and H1avN1 (two samples). The identification of the subtype using the nested multiplex end-point RT-PCR is strongly recommended for isolated viruses; however, viral isolation was successful in 3 samples out of the 17 positives. To the authors' knowledge, the explanation of these results is due to the weak positivity (high Ct value) in the M gene RT-PCRs or the carcasses' conservation.
The NGS analysis of the isolated strains showed an evolving and interesting situation that deserves further investigation. The 2018 strain (genotype U) was a rare genotype detected in Italy (1.1%) but was closely related (nucleotide identity 99.01-99.74 for all gene segments) to a SwIAV reported in a swine farm in 2019 in the Veneto region (A/swine/Italy/76625/2019) ( Figure 2); these were the only genotype U reports in Northern Italy and were from non-related geographical areas.
The HA-1C.2.1-N1av strain isolated in 2020 belonged to SwIAV genotype A, which is the dominant H1N1 genotype in the swine farms in Northern Italy, representing 58.3% of all the H1N1 subtypes isolated [29].
The 2021 HA-1A.3.3.2 strain belonged to a new genetic cluster of SwIAVs recently reported in the Italian domestic swine population (sub-cluster a- Figure 3), which is provisionally named genotype 31 [29]. These viruses are reassortants with different combinations of internal genes (pdm09 and avian-origin) ( Table 2), which are antigenically different from the previously identified Italian HA-1A strains and have recently been detected in several pig farms in Northern Italy since 2020 [29].
This study represents an ongoing evaluation of influenza A viral circulation in the Emilia-Romagna wild boar population, despite some results limitations. It represents an assessment of SwIAV dynamics in wild hosts, even though the low percentage of subtyped samples as well as isolated strains affected the evaluation of the viral genotypic diversity. The NGS analysis and the genotyping results highlight the simultaneous circulation of the same viral genotypes in overlapping years in both pigs and wild boars; these results support the hypothesis of spillover events between the domestic and the wild reservoirs, with the continuous introduction of SwIAV viral strains at the wildlife-livestock interface.
Furthermore, the demonstration of the continuous introduction of domestic SwIAVs into the Emilia-Romagna wild boar population represents a biosecurity problem in this geographical area, which has a high density of commercial swine farms. Italian farms' biosecurity needs to be improved and constantly monitored in order to prevent disease transmission via the wildlife-livestock interface, also considering the recent introduction of African swine fever (ASF) in wild boars in the Piedmont region and the Rome province [43,44].

Conclusions
This study confirmed SwIAV viral circulation in wild boars and represents an upgrade on the research into the Italian situation. Considering the Sus scrofa species' role in the development of reassortant strains, which are agents of spillover infections, and livestock's potential role as an epidemiological bridge between wildlife and humans [1,2], the dynamic control of the viral hypervariability through systematic surveillance against SwIAVs in the wild boar population is certainly needed.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12121593/s1. Supplementary File S1: dataset of sequences used in this study.