Effect of Myxoma Virus Species Jump on Iberian Hare Populations.

The myxoma virus species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis) has raised concerns. We assess the decline suffered by Iberian hare populations on the Iberian Peninsula and discuss the association between the effect of myxomatosis and the average abundance index, which we estimated by using hunting bags.

Occurrence of Coxiella burnetii in wild lagomorphs and their ticks in Spanish Mediterranean ecosystems.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, is a zoonotic multi-host vector-borne pathogen of major public health importance. Although the European Food Safety Authority has recently made the monitoring of this bacterium in wildlife a priority, the role of wild lagomorphs in the transmission and maintenance of C. burnetii is poorly understood. AIMS: The aims of this study were to determine the prevalence and risk factors associated with C. burnetii circulation in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) and to assess the presence of this pathogen in ticks that feed on them in Mediterranean ecosystems in Spain, the country with the highest number of reported cases of Q fever in Europe. METHODS: A total of 574 spleen samples were collected from 453 wild rabbits and 121 Iberian hares, and 513 ticks (processed in 120 pools) between the 2017/2018 and 2021/2022 hunting seasons. RESULTS: C. burnetii DNA was detected in 103 (17.9%; 95% CI: 14.8-21.1) of the 574 wild lagomorphs tested. By species, prevalence was 16.3% (74/453; 95% CI: 12.9-19.7) in the European wild rabbit and 24.0% (29/121; 95% CI: 16.4-31.6) in the Iberian hare. At least one positive lagomorph was found on 47.9% of the 96 hunting estates sampled and in every hunting season since 2018/2019. Two risk factors associated with C. burnetii infection were as follows: outbreak of myxomatosis on the hunting estate in the month prior to sampling and high tick abundance observed by gamekeepers on the hunting estate. C. burnetii DNA was also found in 33 of the 120 (27.5%; 95% CI: 19.5-35.5) tick pools tested. The pathogen was detected in 66.7% (4/6), 29.2% (26/89) and 21.4% (3/14) of Haemaphysalis hispanica, Rhipicephalus pusillus and Hyalomma lusitanicum pools respectively. CONCLUSIONS: This study provides new epidemiological data on C. burnetii in European wild rabbits and is the first survey on this zoonotic pathogen performed in Iberian hares. Our results indicate widespread endemic circulation of C. burnetii and highlight the importance of both wild lagomorph species as natural reservoirs of this zoonotic bacterium in Mediterranean ecosystems in southern Spain, which may be of public and animal health concern. The high prevalence and wide diversity of positive tick species suggest the possible role of ticks in the epidemiological cycle of C. burnetii, with the potential risk of transmission to sympatric species, including humans.

Identification and Characterisation of a Myxoma Virus Detected in the Italian Hare (Lepus corsicanus).

Myxoma virus (MYXV) is a Leporipoxvirus (genus) belonging to the family Poxviridae; it is characterised by a genome of approximately 161 kb dsDNA encoding for several proteins that play an essential role in both host spectrum determination and immunomodulation. The healthy reservoir of the virus is Sylvilagus spp. At the same time, in wild and domestic European rabbits (Oryctolagus cuniculus), MYXV is the etiologic agent of myxomatosis, a disease with an extremely high mortality rate. In 2014, an interspecies jump of MYXV was reported in Lepus europaeus in the UK. In 2018, myxomatosis induced by a new recombinant strain called MYXV-To was identified during a large outbreak in Iberian hares (Lepus granatensis) in Spain. Here, we describe the case of myxomatosis in another hare species: an adult male Italian hare (Lepus corsicanus) found dead in 2018 in Sicily with lesions suggestive of myxomatosis and treponema infection. Laboratory tests, e.g., end-point PCR and negative staining electron microscopy, confirmed the presence of both pathogens. MYXV was then isolated from tissue samples in permissive cells and sequenced using NGS technology. Main genomic differences concerning known MYXV strains are discussed.

Estrogen regulated transcription of the non-estrogen-regulated hamster uteroglobin gene in MCF-7 cells.

To study the estrogen regulated transcription of the uteroglobin (UG) gene, the founding member of the secretoglobin family widely expressed in many different mammalian species, we re-created functional estrogen response elements (EREs) in the UG gene promoter from a species where UG expression is not regulated by estrogens: the hamster Mesocricetus auratus (Ma), to ascertain if the lack of functional EREs is the real cause of its estrogen insensitivity. Functional EREs in the hamster promoter, including the consensus ERE (cERE), failed to respond to an appropriate estrogen stimulus compared with its estrogen regulated ortholog from the brown hare Lepus capensis (Lc). As the nucleotide sequence is the only difference between genetic constructs from these two species, we suspected that the UG promoter from the hamster probably contains cis-acting genetic elements that negatively impairs the estrogen-regulated transcription mediated by the functional ERE. Accordingly, we prepared chimeric DNA constructs which eventually allowed to identify a region located 29 base pairs (bp) downstream of the ERE as responsible for the lack of estrogen-responsiveness of the Ma-UG gene in the breast cancer cell line MCF-7. This region contains the sequence ACACCCC which has been identified as the core sequence of the Sp/ Krüppel-like factor (KLF) family of transcription factors. This finding is relevant, not only due to the observation on a novel mechanism that control estrogen-induced transcription, but also because it may encourage further investigation for better defining specific genes with an ERE that do not respond to estrogen signaling in MCF-7 cells, a cell line widely employed as an in vitro model in breast cancer research.

Characterization of the Tongue Worm, Linguatula serrata (Pentastomida), Identified from Hares (Lepus europaeus) in Romania.

Linguatula serrata (Frölich, 1789) is a widespread parasite known as the tongue worm belonging to the family Linguatulidae. The adult form of the parasite is usually located in the upper respiratory tract of domestic and wild carnivores while the larval forms are located in the visceral organs of intermediate hosts (various herbivorous mammals). Twenty-four European brown hares (Lepus europaeus) were examined in this study, of which two were positive with L. serrata nymphs. The collected nymphs were examined morphologically using electron-microscopic analysis and molecularly by amplification of 18S rRNA and COX1 genes. Lung tissue samples were also collected and histopathological examination was performed. Histopathological examination revealed the following lesions: generalized inflammatory oedema, granulomas with necrosis, calcification and fibrosis in the bronchial tree. The results of molecular sequencing for L. serrata specimens collected from the European brown hares are deposited in GenBank. This study presents the first report on Linguatula serrata nymphs collected from L. europaeus in Romania, using molecular and morphological characterization simultaneously.

Genetic Characteristics and Phylogeographic Dynamics of Lagoviruses, 1988-2021.

Rabbit haemorrhagic disease virus (RHDV), European brown hare syndrome virus (EBHSV), rabbit calicivirus (RCV), and hare calicivirus (HaCV) belong to the genus Lagovirus of the Caliciviridae family that causes severe diseases in rabbits and several hare (Lepus) species. Previously, Lagoviruses were classified into two genogroups, e.g., GI (RHDVs and RCVs) and GII (EBHSV and HaCV) based on partial genomes, e.g., VP60 coding sequences. Herein, we provide a robust phylogenetic classification of all the Lagovirus strains based on full-length genomes, grouping all the available 240 strains identified between 1988 and 2021 into four distinct clades, e.g., GI.1 (classical RHDV), GI.2 (RHDV2), HaCV/EBHSV, and RCV, where the GI.1 clade is further classified into four (GI.1a-d) and GI.2 into six sub-clades (GI.2a-f). Moreover, the phylogeographic analysis revealed that the EBHSV and HaCV strains share their ancestor with the GI.1, while the RCV shares with the GI.2. In addition, all 2020-2021 RHDV2 outbreak strains in the USA are connected to the strains from Canada and Germany, while RHDV strains isolated in Australia are connected with the USA-Germany haplotype RHDV strain. Furthermore, we identified six recombination events in the VP60, VP10, and RNA-dependent RNA polymerase (RdRp) coding regions using the full-length genomes. The amino acid variability analysis showed that the variability index exceeded the threshold of 1.00 in the ORF1-encoded polyprotein and ORF2-encoded VP10 protein, respectively, indicating significant amino acid drift with the emergence of new strains. The current study is an update of the phylogenetic and phylogeographic information of Lagoviruses that may be used to map the evolutionary history and provide hints for the genetic basis of their emergence and re-emergence.

Factors affecting populations of the endemic Iberian hare (Lepus granatensis) after the first myxomatosis outbreaks in Central Spain.

The Iberian hare (Lepus granatensis) is an endemic species distributed in Spain and Portugal. Myxomatosis outbreaks affecting this species were detected in 2018 in Central and Southern Spain, spreading afterward. Aiming to evaluate factors affecting the status of hare population after the arrival of myxomatosis, we conducted 108 nocturnal hare counts in Central Spain during two study periods (winter/spring and summer/autumn) in 54 different hunting grounds, covering 1071 km and observing 884 individuals. The mean density in winter/spring was 7.66 hares/100 ha, (range 6.14-9.54/100 ha), while in summer/autumn, it was 3.4 hares/100 ha (range 2.6-4.4/100 ha). Densities of hares were not affected by the dominant habitat and the presence/absence of myxomatosis outbreaks. Hares were more abundant at hunting grounds at a higher altitude and in those conducting targeted management, while detection of myxomatosis was related to lower altitude and higher levels of game management. A MaxEnt model used to generate a risk map for myxomatosis occurrence showed that the temperature annual range was the most important predictor, which suggests that environmental factors affecting myxomatosis vectors (mosquitoes, fleas, and ticks) could play a key role in disease transmission. As myxomatosis in hares is becoming endemic, hare densities may be improved by game management and the monitoring and surveillance of this emerging disease. These surveillance programs could be the basis of effective collaborations between hunters, researchers, and environmental managers.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Pathological changes and viral antigen distribution in tissues of Iberian hare (Lepus granatensis) naturally infected with the emerging recombinant myxoma virus (ha-MYXV).

BACKGROUND: A cross-species jump was confirmed in 2018, when a novel recombinant myxoma virus (MYXV) (ha-MYXV) caused high mortality in Iberian hare (Lepus granatensis) in the Iberian Peninsula. METHOD: The aim of this study was to evaluate the main lesions, tissular distribution and target cells of ha-MYXV in Iberian hare. Gross postmortem examinations and histological and immunohistochemical studies to detect ha-MYXV were carried out in 28 animals that were confirmed as ha-MYXV positive by PCR. RESULTS: The main macroscopic lesions were bilateral blepharoconjunctivitis, epistaxis, intense congestion and oedema in several organs and some internal haemorrhages. Visible myxomas were not found. Histopathological examination revealed hyperplastic epidermis with predominant hyperkeratosis and myxoid matrix in the dermis. ha-MYXV-positive keratinocytes showed hydropic degeneration and cytoplasmic inclusion bodies. Alveolar oedema, interstitial pneumonia, dramatic lymphoid depletion in the spleen and necrosis in the liver and testis were observed. ha-MYXV was mainly detected in epithelial and myxoma cells in the skin, and also in macrophages, lymphocytes, fibroblasts and endothelial cells in several organs, as well as in hepatocytes and Leydig cells. LIMITATIONS: A non-homogeneous number of samples were included in all the animals. Future experimental studies with controlled variables are necessary. CONCLUSION: These findings correspond to an unusual form of myxomatosis, characterised by an acute or hyperacute presentation.

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.

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.

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.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Endoparasites of the European brown hare (Lepus europaeus Pallas, 1778 L.) (Lagomorpha: Leporidae) from Bulgaria.

In recent decades in Europe there has been a decline in the population of European brown hare (Lepus europaeus L.). Diseases, including parasitoses, are one of the factors considered to be the cause for the phenomenon. In this connection the aim of the present work was to update the data on the composition and distribution of endoparasites of this small mammal in Bulgaria against the background of the situation in Europe. Thirty sets of abdominal organs and forty seven faecal samples of hares from different regions of the country were investigated for presence of parasites. The faeces were analyzed with the coproscopical methods of Fulleborn, serial sedimentations, and the modified Baermann technique. Helminthological necropsies of the abdominal organs were carried out through the common technique. Parasites of the following taxa were established: Trematoda (Dicrocoelium dendriticum), Cestoda (Cysticercus pisiformis, Mosgovoyia pectinata), Nematoda (Trichuris sylvilagi, Strongylidae, Strongyloides sp., Protostrongylus spp.), Protozoa (Eimeria spp.) and Arthropoda (Pentastomum dentatum). Strongyloides sp. is reported for the first time as a part of the parasite fauna of the European brown hare in Bulgaria. Trichuris sylvilagi was the most prevalent parasite (63.3%), followed by gastrointestinal strongylids (57.43%), Eimeria spp. (55.3%), Protostrongylus spp. and Strongyloides sp. (31.9%), D. dendriticum (14.3%), C. pisiformis (10%), and M. pectinata (6.67%). The studies showed that eimeriid coccidia, gastrointestinal strongylids, and trichurids are the most common endoparasites among the population of the European brown hare both in Bulgaria and the European continent.

Toxicity of chlortetracycline and oxytetracycline on Vallisneria natans (Lour.) Hare.

Tetracyclines are frequently detected in water bodies due to their widespread use in aquaculture and animal husbandry. A hydroponic experiment was conducted to explore the phytotoxic effects of Vallisneria natans (Lour.) Hare exposed to various concentrations of chlortetracycline (CTC) and oxytetracycline (OTC) (0, 0.1, 1, 10, 30, 50, and 100 mg/L) for 7 days (7 D) and 14 days (14 D), respectively. The results showed that similar to OTC treatment for 7 D, the relative growth rates (RGR) and catalase (CAT) activity of V. natans, after 7 D of CTC exposure, decreased significantly at 10 mg/L and 30 mg/L, respectively. The content of soluble protein notably decreased when CTC ≥ 10 mg/L and OTC ≥ 30 mg/L. The hydrogen peroxide (H2O2) content was significantly stimulated when OTC ≥ 10 mg/L, while it hardly changed when exposed to CTC. After 14 D, the malondialdehyde (MDA) and H2O2 contents of V. natans were significantly higher than those of the control group under a high concentration of OTC (≥ 30 mg/L), but they did not change significantly under a high concentration of CTC. The activity of polyphenol oxidase (PPO), under CTC treatment after 14 D, showed first a significant increase then decreases; the maximum value (125% of the control) was noticed at 10 mg/L CTC, while it remained unchanged when exposed to OTC. The soluble protein content significantly decreased at 10 mg/L CTC and 0.1 mg/L OTC, respectively. The RGR, CAT, and peroxidase (POD) activities, similar to OTC treatment after 14 D, decreased evidently when CTC was 10 mg/L, 30 mg/L, and 0.1 mg/L, respectively. CTC and OTC harm the chlorophyll content of V. natans after 14 D, and the reductions of chlorophyll a and carotenoid were more pronounced than chlorophyll b. The results suggest that CTC and OTC both have a negative effect on the growth of V. natans, and OTC can cause oxidative damage in V. natans but CTC harms the metabolism process without inducing oxidative damage. Overall, the toxicity of OTC to V. natans is stronger than that of CTC.

A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies.

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated.

Monitoring of emerging myxoma virus epidemics in Iberian hares (Lepus granatensis) in Spain, 2018-2020.

Myxomatosis is an infectious disease caused by the myxoma virus (MYXV), which has very high mortality rates in European wild rabbits (Oryctolagus cuniculus). While sporadic cases of myxomatosis have also been reported in some hare species, these lagomorphs are considered to have a low susceptibility to MYXV infection. In the present study, we describe the spatiotemporal evolution and main epidemiological findings of novel hare MYXV (ha-MYXV or MYXV-Tol) epidemics in Iberian hares (Lepus granatensis) in Spain. In the period 2018-2020, a total of 487 hares from 372 affected areas were confirmed to be MYXV-infected by PCR. ha-MYXV outbreaks were detected in most of the Spanish regions where the Iberian hare is present. The spatial distribution was not homogeneous, with most outbreaks concentrated in the southern and central parts of Spain. Consecutive outbreaks reported in the last two years suggest endemic circulation in Spain of this emerging virus. A retrospective study carried out just after the first epidemic period (2018-2019) revealed that the virus could have been circulating since June 2018. The number of outbreaks started to rise in July, peaked during the first half of August and October and then decreased sharply until January 2019. The apparent mean mortality rate was 55.4% (median: 70%). The results indicated high susceptibility of the Iberian hare to ha-MYXV infection, but apparent resistance in the sympatric hare species present in Spain and less infectivity in European rabbits. The novel ha-MYXV has had significant consequences on the health status of Iberian hare populations in Spain, which is of animal health and conservation concern. The present study contributes to a better understanding of ha-MYXV emergence and will provide valuable information for the development of control strategies. Further research is warranted to assess the impact of this emerging virus on wild lagomorph populations and to elucidate its ecological implications for Iberian Mediterranean ecosystems.