Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I.

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I-expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I-overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2-an enzyme without a counterpart in mammals-as a candidate target for leishmanicidal drugs.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Biological Evaluation and Mechanistic Studies of Quinolin-(1H)-Imines as a New Chemotype against Leishmaniasis.

Leishmaniasis is one of the most challenging neglected tropical diseases and remains a global threat to public health. Currently available therapies for leishmaniases present significant drawbacks and are rendered increasingly inefficient due to parasite resistance, making the need for more effective, safer, and less expensive drugs an urgent one. In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, we have screened in-house antiplasmodial libraries against axenic and intracellular forms of Leishmania infantum, Leishmania amazonensis, and Leishmania major. Several of the screened compounds showed half-maximal inhibitory concentrations (IC50s) against intracellular L. infantum parasites in the submicromolar range (compounds 1h, IC50 = 0.9 µM, and 1n, IC50 = 0.7 µM) and selectivity indexes of 11 and 9.7, respectively. Compounds also displayed activity against L. amazonensis and L. major parasites, albeit in the low micromolar range. Mechanistic studies revealed that compound 1n efficiently inhibits oxygen consumption and significantly decreases the mitochondrial membrane potential in L. infantum axenic amastigotes, suggesting that this chemotype acts, at least in part, by interfering with mitochondrial function. Structure-activity analysis suggests that compound 1n is a promising antileishmanial lead and emphasizes the potential of the quinoline-(1H)-imine chemotype for the future development of new antileishmanial agents.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum (LiNDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae, two ubiquinone-binding sites (UQI and UQII) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of LiNDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the LiNDH2 ubiquinone-binding site 1-UQI. Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum. The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016-2017).

We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.

AVIAN POXVIRUS INFECTION IN A FLAMINGO (PHOENICOPTERUS RUBER) OF THE LISBON ZOO.

Avian poxviruses (APV) are very large viruses spread worldwide in a variety of hosts. They are responsible for a disease usually referred to as pox, mainly characterized by nodular lesions on feather-free regions of the body. On May 2010, a young American flamingo (Phoenicopterus ruber) of the Lisbon Zoo (Portugal) developed a nodular lesion suggestive of poxvirus infection on its right foot. Avipoxvirus was isolated from the lesion and a fragment of the P4b-encoding gene was amplified by polymerase chain reaction. The nucleotide sequence of the amplicon was determined and analyzed. A close relationship (100% identity) was observed between the flamingo poxvirus and isolates from great bustard (Hungary 2005), house sparrow (Morocco 2009), MacQueen's bustard (Morocco 2011), and Houbara bustard (Morocco 2010 and 2011), suggesting interspecies transmission as a possible source of infection. To strengthen the investigation, the 5' and 3' ends of genes cnpv186 and cnpv 187, respectively, were also analyzed. The cnpv186-187 fragment exhibited 100% identity with MacQueen's bustard and Houbara bustard isolates, both from Morocco 2011. Phylogenetic analyses based in both fragments grouped the flamingo isolate consistently within clade B2 of canarypox. However, the phylogenetic relationships among the different representatives of avian poxviruses were more comprehensive in the tree based on the concatenated coding sequences of the cnpv186-187 fragment, rather than on the P4b-coding gene. The clearer displacement and distribution of the isolates regarding their host species in this last tree suggests the potential usefulness of this genomic region to refine avian poxvirus classification.

The mitochondrial complex I of trypanosomatids--an overview of current knowledge.

The contribution of trypanosomatid mitochondrial complex I for energy transduction has long been debated. Herein, we summarize current knowledge on the composition and relevance of this enzyme. Bioinformatic and proteomic analyses allowed the identification of many conserved and trypanosomatid-specific subunits of NADH:ubiquinone oxidoreductase, revealing a multifunctional enzyme capable of performing bioenergetic activities and possibly, also of functioning in fatty acid metabolism. A multimeric structure organized in 5 domains of more than 2 MDa is predicted, in contrast to the 1 MDa described for mammalian complex I. The relevance of mitochondrial complex I within the Trypanosomatidae family is quite diverse with its NADH oxidation activity being dispensable for both procyclic and bloodstream Trypanosoma brucei, whereas in Phytomonas serpens the enzyme is the only respiratory complex able to sustain membrane potential. Aside from complex I, trypanosomatid mitochondria contain a type II NADH dehydrogenase and a NADH-dependent fumarate reductase as alternative electron entry points into the respiratory chain and thus, some trypanosomatids may have bypassed the need for complex I. The involvement of each of these enzymes in the maintenance of the mitochondrial redox balance in trypanosomatids is still an open question and requires further investigation.

First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

Rabbit hemorrhagic disease virus 2, 2010-2023: a review of global detections and affected species.

Rabbit hemorrhagic disease virus 2/genotype GI.2 (RHDV2/GI.2; Caliciviridae, Lagovirus) causes a highly contagious disease with hepatic necrosis and disseminated intravascular coagulation in several Leporidae species. RHDV2 was first detected in European rabbits (Oryctolagus cuniculus) in France in 2010 and has since spread widely. We gather here data on viral detections reported in various countries and affected species, and discuss pathology, genetic differences, and novel diagnostic aspects. RHDV2 has been detected almost globally, with cases reported in Europe, Africa, Oceania, Asia, and North America as of 2023. Since 2020, large scale outbreaks have occurred in the United States and Mexico and, at the same time, cases have been reported for the first time in previously unaffected countries, such as China, Japan, Singapore, and South Africa, among others. Detections have been notified in domestic and wild European rabbits, hares and jackrabbits (Lepus spp.), several species of cottontail and brush rabbits (Sylvilagus spp.), pygmy rabbits (Brachylagus idahoensis), and red rock rabbits (Pronolagus spp.). RHDV2 has also been detected in a few non-lagomorph species. Detection of RHDV2 causing RHD in Sylvilagus spp. and Leporidae species other than those in the genera Oryctolagus and Lepus is very novel. The global spread of this fast-evolving RNA virus into previously unexploited geographic areas increases the likelihood of host range expansion as new species are exposed; animals may also be infected by nonpathogenic caliciviruses that are disseminated by almost all species, and with which genetic recombination may occur.

Defective valyl-tRNA synthetase hampers the mitochondrial respiratory chain in Neurospora crassa.

Respiratory chain deficiency can result from alterations in mitochondrial and/or cytosolic protein synthesis due to the dual genetic origin of mitochondrial oxidative phosphorylation. In the present paper we report a point mutation (D750G) in the bifunctional VARS (valyl-tRNA synthetase) of the fungus Neurospora crassa, associated with a temperature-sensitive phenotype. Analysis of the mutant strain revealed decreased steady-state levels of VARS and a clear reduction in the rate of mitochondrial protein synthesis. We observed a robust induction of the mitochondrial alternative oxidase with a concomitant decrease in the canonical respiratory pathway, namely in cytochrome b and aa3 content. Furthermore, the mutant strain accumulates the peripheral arm of complex I and depicts decreased levels of complexes III and IV, consistent with severe impairment of the mitochondrial respiratory chain. The phenotypic alterations of the mutant strain are observed at the permissive growth temperature and exacerbated upon increase of the temperature. Surprisingly, glucose-6-phosphate dehydrogenase activities were similar in the wild-type and mutant strains, whereas mitochondrial activities for succinate dehydrogenase and alternative NADH dehydrogenases were increased in the mutant strain, suggesting that the VARSD-G mutation does not affect overall cytosolic protein synthesis. Expression of the wild-type vars gene rescues all of the mutant phenotypes, indicating that the VARSD-G mutation is a loss-of-function mutation that results in a combined respiratory chain deficiency.

Snapshot of the Phylogenetic Relationships among Avian Poxviruses Circulating in Portugal between 2017 and 2023.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing avian pox disease, characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosystem. Several diagnostic methods are available, and bird vaccination can be an effective preventive measure. Ten APV-positive samples were analyzed to update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023. A P4b gene fragment was amplified using a PCR, and the nucleotide sequence of the amplicons was determined using Sanger sequencing. The sequences obtained were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on Avipoxviruses in Portugal.

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

Does cultural background influence the dissemination and severity of the COVID-19 pandemic?

The COVID-19 pandemic has spread throughout the globe affecting countries worldwide. However, several differences have been observed in the number of daily new cases, the COVID-19 reproduction rate, and the severity of the disease in different countries. Previous studies have mostly highlighted government restriction policies to mitigate the pandemic effects as reasons for such differences. This study focuses on 101 countries and proposes that each country's cultural background is also accountable for such differences. We considered the six Hofstede's cultural dimensions (power distance, individualism, masculinity, uncertainty avoidance, long term orientation, and indulgence) and statistically analyzed their correlation with several COVID-19 impact metrics in comparison to several restriction policies. Our results support our claim that national culture influences both acceptance and subsequent adoption of restriction policies and the implementation by each government of those policies. We highlight that the attitudes towards and trust in political institutions, policies and governance is influenced by the cultural background, which is reflected in the pandemic numbers. As a main takeaway from this study, we conclude that data-driven models which aim at predicting the pandemic impact evolution at a global scale should also include variables that reflect the cultural background of each nation.

Co-infection by classic MYXV and ha-MYXV in Iberian hare (Lepus granatensis) and European wild rabbit (Oryctolagus cuniculus algirus).

Myxomatosis is an emergent disease in the Iberian hare (Lepus granatensis). In this species, the disease is caused by a natural recombinant virus (ha-myxoma virus [MYXV]) identified for the first time in 2018 and has since been responsible for a large number of outbreaks in Spain and Portugal. The ha-MYXV, which harbours a 2.8 Kb insert-disrupting gene M009L, can also infect and cause disease in wild and domestic rabbits, despite being less frequently identified in rabbits. During the laboratory investigations of wild leporids found dead in Portugal carried out within the scope of a Nacional Surveillance Plan (Dispatch 4757/17, MAFDR), co-infection events by classic (MYXV) and naturally recombinant (ha-MYXV) strains were detected in both one Iberian hare and one European wild rabbit (Oryctolagus cuniculus algirus). These two cases were initially detected by a multiplex qPCR detection of MYXV and ha-MYXV and subsequently confirmed by conventional PCR and sequencing of the M009L gene, which contains an ha-MYXV-specific insertion. To our knowledge, this is the first documented report of co-infection by classic MYXV and ha-MYXV strains either in Iberian hare or in European wild rabbit. It is also the first report of infection of an Iberian hare by a classic MYXV strain. These findings highlight the continuous evolution of the MYXV and the frequent host range changes that justify the nonstop monitoring of the sanitary condition of wild Leporidae populations in the Iberian Peninsula.

Evaluation of Commercial Myxomatosis Vaccines against Recombinant Myxoma Virus (ha-MYXV) in Iberian Hare and Wild Rabbit.

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains.

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

Spillover events of rabbit haemorrhagic disease virus 2 (recombinant GI.4P-GI.2) from Lagomorpha to Eurasian badger.

Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In these animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P-GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data show the presence of calicivirus compatible virions in the nucleus of hepatocytes, which constitutes a paradigm shift for caliciviruses' replication cycle.