Porcine circoviruses are significant pathogens that affect swine populations worldwide, with implications for animal health and productivity. While PCV2 is well-documented, particularly due to widespread vaccination programs, PCV3 is less understood, and its epidemiological impact is still under investigation. This study screened for PCV2 and PCV3 in pigs and wild boars across Portugal to assess their prevalence. Also, nucleotide sequence determination was performed to evaluate the genetic diversity of these viruses. Stool samples from 160 pigs belonging to different groups (quarantine, nursery, fattening and adult pigs), as well as organ samples from 120 hunted wild boars, were analyzed. Samples were collected from twelve of the eighteen mainland Portuguese districts with positive cases being detected in nine of them. Pigs had a lower prevalence of PCV2 (1.9%) than PCV3 (11.2%), but the opposite was true in wild boars (76.7% for PCV2 and 55.0% for PCV3). The lower PCV2 prevalence in pigs can be attributed to the PCV2 vaccination program implemented. Additionally, these viruses were significantly more prevalent in wild boars (90.8% were infected with at least one of the viruses) than in domestic pigs (only 12.5%). This significant difference highlights the impact of the controlled environment in pig farms on disease prevention in contrast to the higher exposure risks faced by wild boars in their natural habitat. Compared to a previous study from 2023, we observed a slight decrease in the percentage of positive cases for both PCV2 and PCV3. Phylogenetic analysis of sequences obtained by Sanger sequencing allowed us to conclude that the samples from domestic pigs belong to the PCV2a and PCV3c clades, in contrast to the PCV2-positive cases detected in domestic pigs in 2023 that were classified in the PCV2d genotype. Conversely, samples from wild boars belong to the PCV2d and PCV3a clades. These results reveal genotype differences between wild and domestic pigs and shifts from 2023 to 2024. Our findings provide some information about the circulation of these viruses and emphasize the importance of vaccination and continued monitoring for a deeper understanding of their epidemiology to mitigate potential risks to swine health and production.

Porcine circoviruses are significant pathogens that affect swine populations worldwide, with implications for animal health and productivity. While PCV2 is well-documented, particularly due to widespread vaccination programs, PCV3 is less understood, and its epidemiological impact is still under investigation. This study broadly screened for PCV2 and PCV3 in pigs and wild boars across Portugal to assess their prevalence and genetic diversity. Stool samples from 160 pigs belonging to different groups (Quarantine, Nursery, Fattening, Breeding Stalls, Gestation, and Maternity), as well as organ samples from 120 wild boars, were analyzed. Samples were collected from 12 of the 18 mainland Portuguese districts with positive cases being detected in nine of them. Pigs had a lower prevalence of PCV2 (1.9%) than PCV3 (11.2%), but the opposite was true in wild boars (76.7% for PCV2 and 55.0% for PCV3). The lower PCV2 prevalence in pigs can be attributed to the PCV2 vaccination program implemented. Additionally, these viruses were significantly more prevalent in wild boars (90.8% were infected with at least one of the viruses) than in domestic pigs (only 12.5%). This significant difference highlights the impact of the controlled environment in pig farms on disease prevention in opposition to the higher exposure risks faced by wild boars in their natural habitat. Compared to our previous study from 2023, we observed a slight decrease in the percentage of positive cases for both PCV2 and PCV3. Furthermore, the positive cases detected in pigs in 2023 belonged to the PCV2d and PCV3c clades. In contrast, the samples from pigs in the present study belong to the PCV2a and PCV3c clades. Conversely, samples from wild boars belonged to the PCV2d and PCV3a clades. These results reveal genotype differences between wild and domestic pigs and shifts from 2023 to 2024. Our findings provide new insights into the circulation of these viruses and emphasize the importance of vaccination and continued monitoring for a deeper understanding of their epidemiology to mitigate potential risks to swine health and production.

Porcine circovirus types 2 (PCV2) and 3 (PCV3) are major pathogens affecting swine health and productivity, yet important gaps remain in understanding their evolution and circulation in Europe, particularly within wild boar populations that may serve as reservoirs. This study examined the genetic diversity and evolutionary dynamics of PCV2 and PCV3 in Portugal, drawing on viral genomes obtained from domestic pigs and wild boars to explore transmission patterns, spillover events and the contribution of recombination to viral emergence. We identified two PCV2 genotypes (PCV2a and PCV2d) and two PCV3 genotypes (PCV3-2a and PCV3-3g) circulating in Portuguese swine. Phylogeographic reconstruction revealed multiple introductions of both PCV2 and PCV3 from China into Europe, followed by regional diversification and subsequent spread within European wild boar populations. Evidence of bidirectional viral exchange between domestic pigs and wild boars was also observed. Recombination played a notable role in PCV2 evolution, with consistent signals detected among PCV2a sequences and indications that the PCV2h genotype likely originated from a recombinant event involving a Portuguese PCV2a strain and a Chinese PCV2d strain. By contrast, no recombination was detected in PCV3, suggesting that its evolution is primarily mutation-driven. Overall, these findings highlight the complex evolutionary history of swine circoviruses in Europe and underscore the importance of continuous genomic surveillance in both domestic and wild hosts. The study reinforces the value of a One Health approach for monitoring and controlling emerging circoviruses with implications for animal health and livestock production.

The Hepatitis E virus (HEV) is a zoonotic virus of global concern that circulates in both domestic and wild pig populations. Understanding its presence and dynamics in wildlife reservoirs is crucial for assessing spillover risks and designing One Health surveillance strategies. HEV is classified into eight distinct genotypes, two of which (genotypes 3 and 4) infect both humans and swine, representing a major public health concern in Europe. This study explored the occurrence, genetic diversity, and evolutionary relationships of HEV in wild boars (Sus scrofa scrofa) from mainland Portugal. Organ samples from a total of 120 wild boar were collected from seven different districts in Portugal. HEV RNA was detected in four animals (3.3%), all of which were collected from municipalities in the Évora district, near the land border with Spain. Of the four wild boars that tested positive for HEV in the diagnostic context, it was possible to genotype and characterize one through full sequencing. Phylogenetic analysis based on the complete genome, revealed that this strain clustered with predominantly human-derived HEV-3m sequences from Spain and France, highlighting its zoonotic potential. The inclusion of 14 sequences from domestic swine in a second phylogenetic analysis performed with a small fragment allowed to conclude that sub-genotypes 3e, 3f and 3m are circulating in this population with no spatial or temporal segregation. Phylogeographic analysis suggests that there have been several transmission events between Spain and France and estimated that this HEV strain is most likely to have been introduced from Spain. The fact that these four positive samples from wild boar in south-eastern Portugal all have a common spatial origin, together with the absence of detections in the other six districts tested and the presence of known HEV hotspots in south-western Spain, supports the hypothesis that this strain in Portugal is most likely from HEV circulation in Spanish wild boar, facilitated by cross-border movement of wild boar along the south-eastern Portuguese border. Although recombination events were identified in several HEV-3 strains, none were detected in the sequence obtained in this study or in other HEV-3m strains. This study provides the first molecular evidence of HEV-3m circulation in wild boars in Portugal, offering valuable insight into the HEV strain circulation in European wildlife populations. The zoonotic potential of HEV and the likelihood of interspecies transmission highlight the need for coordinated cross-border surveillance and integrated One Health strategies.

Hepatitis E virus (HEV) is a zoonotic pathogen of global concern that circulates in both domestic and wild swine populations. Understanding its presence and dynamics in wildlife reservoirs is crucial for assessing spillover risks and designing One Health surveillance strategies. This study investigated the occurrence, genetic diversity, and evolutionary relationships of HEV in wild boars from mainland Portugal. A total of 120 animals from seven districts were tested, with HEV RNA detected in four cases (3.3%), all from the Évora district near the Spanish border. One positive sample was successfully sequenced, and phylogenetic analysis based on the complete genome classified it within the HEV-3m subtype, clustering with predominantly human-derived sequences from Spain and France, which highlights its zoonotic potential. A second phylogenetic analysis based on a partial genomic fragment, including sequences from domestic pigs in Portugal, revealed the co-circulation of subtypes 3e, 3f, and 3m without clear spatial or temporal patterns. Phylogeographic analysis suggested that the identified strain was most likely introduced from Spain, supporting the hypothesis of cross-border transmission through wild boar movement. No recombination events were detected in the sequence obtained in this study. These findings provide the first molecular evidence of HEV-3m circulation in wild boars in Portugal, offering valuable insight into the HEV strain circulation in European wildlife populations. The zoonotic potential of HEV and the likelihood of interspecies transmission highlight the need for coordinated cross-border surveillance and integrated One Health strategies.