Inês Caetano

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.

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.

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. with small rodents serving as the main reservoir. In Croatia, the serogroup Pomona has been detected most frequently, but its genomic diversity remains insufficiently characterized. This study presents the first whole genome sequencing analysis of 48 Croatian Leptospira spp. isolates collected from small rodents over a 14-year period. Serological typing confirmed that all the isolates belonged to the serogroup Pomona. Genomic analysis assigned them to L. kirschneri based on high genomic similarity using average nucleotide identity (ANI). The isolates were assigned to ST-98 using traditional multilocus sequence typing (MLST), while cgMLST identified seven genotype clusters, many of which showed geographic structuring. Phylogenetic analyses based on single nucleotide polymorphisms (SNPs) supported this structure and revealed a monophyletic clade of Croatian isolates distinct from other global L. kirschneri strains. Serological typing, MLST, and phylogenetic clustering support classification of the isolates as L. kirschneri, serogroup Pomona, most likely serovar Mozdok, although serovar Tsaratsovo cannot be excluded. These results indicate the existence of a geographically restricted and potentially host-adapted lineage of L. kirschneri in Croatia. The integration of ecological, serological, and genomic data in this study emphasizes the value of whole genome sequencing for understanding the population biology of Leptospira spp. serogroup Pomona. Moreover, it supports targeted, country-specific surveillance and control strategies for leptospirosis through the identification of circulating serovars and species in reservoir hosts, in line with a One Health approach.

To date, five herpesviruses have been identified in Leporidae (LeHV-1, LeHV-2, LeHV-3, LeHV-4, and LeHV-5). Two of these have been shown to infect the European rabbit (Oryctolagus cuniculus), causing either asymptomatic infection (LeHV-2, a gammaherpesvirus) or virulent disease (LeHV-4, an alphaherpesvirus). Unfortunately, apart from LeHV-4, for which complete genome sequences are available, molecular data on leporid herpesviruses are extremely limited, with no sequences available in public databases for LeHV-1 and LeHV-3, and only a few short sequences for LeHV-2 and LeHV-5. In this study, we investigated the presence of herpesviruses in biological samples from wild rabbits (n = 34) found dead in the field during 2024. A pan-herpesvirus nested PCR directed to the herpesviral DNA polymerase gene was used for screening. Positive samples (n = 14, 41.17%) were further investigated by sequencing analysis of a longer region of the DNA polymerase gene, as well as the glycoprotein B gene and the terminase gene. Blastn analysis of the amplicons revealed the highest similarity to gammaherpesvirus. Phylogenetic analyses based on glycoprotein B, DNA polymerase, and concatenated amino acid sequences consistently placed the newly identified LeHV-6 in close proximity to LeHV-5. Both viruses form a well-supported clade within the Gammaherpesvirinae, clustering with rodent-associated herpesviruses, such as Murine herpesvirus, MuHV-4, and A. sylvaticus rhadinovirus 1. Considering the species susceptibility and the nucleotide similarities with the five previously described leporid herpesviruses, we conclude that a new rabbit gammaherpesvirus has been identified, which we propose to name LeHV-6.

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.

The first official case of bluetongue virus serotype 3 (BTV-3) in Portugal was confirmed in sheep from the district of Évora in September 2024. Notably, mortality was observed in pregnant sheepdogs within the affected sheep flocks. This study presents four cases of pregnant dogs infected with BTV-3 in mid-September 2024, all of which aborted prior to death. BTV-3 was identified by RT-qPCR following initial positive results from pan-BTV RT-qPCR. The virus was subsequently isolated from the blood of one of the dogs in BHK-21 cells, and a partial sequence of the vp2 gene was obtained. This sequence showed 100% similarity to sheep BTV3/3234/PT2024, identified in Portugal in September 2024, as well as to BTV-3/NET2023, first reported in the Netherlands in 2023. These findings suggest that the viruses may be related or share a common origin. Co-infection with common canine viruses and pathogenic bacteria was ruled out, confirming that the fatalities were due to BTV-3 infection, probably by ingestion of sheep placenta after lambing. Our results confirm the potential for the transmission of BTV-3 to non-ruminant species, particularly carnivores, and, therefore, the wider ecological implications of this virus. In addition, the identification of transplacental transmission of BTV-3 in one of the dogs provides new evidence highlighting the complexity of the virus’ transmission mechanisms.

In September 2024, bluetongue virus serotype 3 (BTV-3) was first identified in Portugal, specifically in the Alentejo region (Évora District), using molecular diagnostic methods. The initial case involved a sheep exhibiting severe clinical symptoms, including head oedema, prostration, nasal discharge, and significant respiratory distress. A subsequent case was documented in another sheep from a different farm within the same district, which presented with high fever (41.5 °C), nasal discharge, and arthritis, ultimately resulting in mortality. Within one month of these initial detections, additional cases in both sheep and cattle were reported in neighbouring districts, indicating the virus rapid spread within the region. In response to this emerging threat, extensive monitoring efforts are being undertaken to delineate the distribution of BTV-3, and vaccination campaigns targeting this serotype have been initiated. These measures aim to mitigate the impact of the virus on livestock health and prevent further transmission.

In September 2024, bluetongue virus serotype 3 (BTV-3) was first identified in Portugal, specifi-cally in the Alentejo region (Évora District), using molecular diagnostic methods. The initial case involved a sheep exhibiting severe clinical symptoms, including head oedema, prostration, nasal discharge, and significant respiratory distress. A subsequent case was documented in another sheep from a different farm within the same district, which presented with high fever (41.5°C), nasal discharge, and arthritis, ultimately resulting in mortality. Within one month of these initial detections, additional cases in both sheep and cattle were reported in neighboring districts, indi-cating the virus rapid spread within the region. In response to this emerging threat, extensive monitoring efforts are being undertaken to delineate the distribution of BTV-3, and vaccination campaigns targeting this serotype have been initiated. These measures aim to mitigate the impact of the virus on livestock health and prevent further transmission.