Identification of a gammaherpesvirus belonging to the malignant catarrhal fever group of viruses in Pyrenean chamois (Rupicapra p. pyrenaica).

High prevalence (46 %) of a gammaherpesvirus was confirmed by molecular detection in the lungs of hunted Pyrenean chamois. The partial glycoprotein B sequence up to the DNA polymerase gene showed 96.6 % nucleotide sequence identity to the Rupicapra rupicapra gammaherpesvirus 1 and 81.5 % to ovine herpesvirus 2. This novel sequence clusters within sequences derived from the malignant catarrhal fever group of viruses, and the corresponding virus is tentatively named Rupicapra pyrenaica gammaherpesvirus 1 (RpHV-1). No specific histological lesions were associated with RpHV-1, nor were any detrimental effects on host health. The epidemiological, phylogenetic and histopathological results suggest that Pyrenean chamois is the natural host of RpHV-1.

PAPILLOMAVIRUS IN HEALTHY SKIN AND MUCOSA OF WILD RUMINANTS IN THE ITALIAN ALPS.

We investigated healthy skin and mucosal specimens of wild ruminants in the Italian Alps. We identified bovine papillomavirus (BPV)-2 DNA in the healthy skin of wild ruminants and documented coinfection of BPV-1 and Cervus elaphus papillomavirus (CePV)-1 in a healthy red deer (Cervus elaphus). We also demonstrated cross-infections of BPVs of the genus Xipapillomavirus, both as single virus infection and also in association with Deltapapillomavirus types 1 and 2, confirming that host tropism of papillomaviruses is not as species-specific as previously thought. Our results suggest that subclinical infections could be linked to the presence of domestic ruminants sharing the same habitat with wild species and that the wildlife may act as a reservoir for papillomaviruses affecting domestic species.

A novel papillomavirus isolated from a nasal neoplasia in an Italian free-ranging chamois (Rupicapra r. rupicapra).

Most amniotes are the hosts of many, distantly related papillomaviruses (PVs). Infection by PVs can be asymptomatic, or lead instead to benign or malignant lesions. However, PVs infecting animals and associated with malignancies are still largely understudied. In the present study, we communicate the complete genome of a novel PV found in a nasal neoplasia of a free-ranging alpine chamois (Rupicapra r. rupicapra) in an Italian national park. Long-PCR and cloning approaches followed for Sanger sequencing were used to identify the first PV found in chamois. The genome of the novel virus - RrupPV1 - of 7256 bp in length, presents the classical PV structure, and lacks the interE2-L2 region that hosts the E5 gene in AlphaPVs and in DeltaPVs. The nucleotide identity percentage of the L1 ORF, places RrupPV1 together with OaPV3 in the same genus. The latter is a PV isolated from a squamous cell carcinoma in sheep in Sardinia. Full-genome phylogenetic reconstructions suggest that these two viruses are sister taxa, and that both of them are very distantly related to any other known PV. Many cetartiodactyl species are infected by non-monophyletic PVs. Our results exemplify further the multiple links between the infection by certain, distantly related PVs and the development of diverse cancers in animals and highlight the need of a systematic search of oncogenic and non-oncogenic animal PVs.

Evolutionary dynamics of endogenous Jaagsiekte sheep retroviruses proliferation in the domestic sheep, mouflon and Pyrenean chamois.

The oncogenic exogenous Jaagsiekte sheep retrovirus (JSRV), responsible for ovine pulmonary adenocarcinoma, has several endogenous counterparts termed enJSRVs. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo long terminal repeat (LTR) formation, several members of enJSRVs have been identified as nearly intact and probably represent recent integration events. To determine the level of enJSRV polymorphism in the sheep population and related species, we have undertaken a study by characterizing enJSRVs copies and independent integration sites in six domestic sheep and two wild species of the sheep lineage. enJSRVs copies were detected by amplifying the env-LTR region by PCR, and for the detection of the insertion sites, we used two approaches: (1) an in silico approach based on the recently published Sheep Reference Genome Assembly (OARv3.0) and (2) an experimental approach based on PCR suppression and inverse PCR techniques. In total, 103 enJSRV sequences were generated across 10 individuals and enJSRV integrations were found on 11 of the 28 sheep chromosomes. These findings suggest that there are still uncharacterized enJSRVs, and that some of the integration sites are variable among the different species, breeds of the same species, subspecies and geographic locations.

Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica).

Since 2001, severe outbreaks of disease associated with border disease virus (BDV) infection have been reported in Pyrenean chamois. The disease is characterized by variable degrees of cachexia, alopecia and neurological manifestations prior to death. The aim of this study was to investigate this disease under experimental conditions. To assess viral virulence, humoral immune response, dissemination and probable routes of transmission, seven chamois (five seronegative and two seropositive for BDV) were inoculated with a BDV isolated from a naturally infected chamois. A group of three chamois were maintained as uninfected controls. The five seronegative chamois became viraemic from day 2 post-inoculation (p.i.) until their death (three animals) or the end of the experiment (on day 34 p.i.) and developed neutralizing antibodies from day 18 p.i. until the end of the study. Continuous shedding of the virus was detected by RT-PCR in oral, nasal and rectal swabs in viraemic chamois from day 5 p.i. Despite none of the viraemic chamois showing obvious neurological signs, all of them had a non-suppurative meningoencephalitis as seen in naturally infected chamois. The two inoculated BDV-seropositive chamois did not become viraemic. This study confirms that BDV is the primary agent of the disease that has been affecting chamois populations in recent years in the Pyrenees and that previously acquired humoral immunity is protective.

Haematology and serum chemistry of Pyrenean chamois (Rupicapra pyrenaica) naturally infected with a border disease virus.

In 2005 and 2006 an outbreak of disease associated with border disease virus (BDV) infection caused high mortality in the Pyrenean chamois (Rupicapra pyrenaica) in the Catalan Pyrenees (NE Spain). The aim of this study was to determine values for different haematological and serum biochemical analytes in 32 free-ranging Pyrenean chamois affected by the disease and to compare them with those obtained from healthy chamois. In the affected chamois red blood cell counts, haemoglobin concentrations, packed cell volumes, mean corpuscular volumes and lymphocyte counts were all lower, while the neutrophil and platelet counts were higher. Glucose, lactate, triglycerides, creatinine, total protein concentrations and alkaline phosphatase activity were also lower, in contrast to the concentrations of total bilirubin, urea and aspartate aminotransferase activity, which were higher. Most of the observed changes could be associated with cachexia and inflammation in the affected chamois. Lymphopenia could be directly related to the BDV, which would lead to immunosuppression and explain the high rate of secondary infection observed in these animals.

Investigation of the role of Austrian ruminant wildlife in the epidemiology of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is an ubiquitous disease of cattle and other ruminants caused by Ovine herpesvirus 2 (OvHV-2), which is endemic in sheep and transmitted from healthy carriers. Further viruses of the MCF group are also able to induce MCF in ruminants. As alpine pasturing is very common in Austria, possible contact with ruminant wildlife carrying and excreting MCF viruses might be suspected as an infection source. To investigate the epidemiologic role of Austrian deer and chamois, spleen samples were collected from 55 red deer (Cervus elaphus), 72 roe deer (Capreolus capreolus), four fallow deer (Dama dama), and five chamois (Rupicapra rupicapra) during the hunting seasons 2001-2003. Samples were tested by both herpesvirus consensus and OvHV-2-specific polymerase chain reaction. As all spleen samples tested negative, there is no indication that in the region and period investigated, MCF viruses circulated in wild ruminants.

Severe outbreak of disease in the southern chamois (Rupicapra pyrenaica) associated with border disease virus infection.

An outbreak of a previously unreported disease affecting southern chamois (Rupicapra pyrenaica) in the central Pyrenees (NE Spain) was recorded in 2001 and 2002. There was a marked temporal distribution, most animals being found between February and June. After the outbreak, the population was found to have decreased by about 42%, most probably due to the disease. We examined 20 affected chamois. Clinical manifestations included depression, weakness and movement difficulties in all cases. Three chamois presented abnormal behaviour, with absence of flight reaction, and 16 showed different degrees of alopecia with skin hyperpigmentation. At necropsy cachexia was observed in all animals, four chamois had abscesses in different parts of the body, four had pneumonia, one had an extensive subcutaneous infection on the head and neck and one had severe orchitis. Microscopic lesions were found in the brain, mainly edema, gliosis, espongiosis, cariorrexis and neuronal multifocal necrosis. A perivascular mononuclear inflammatory infiltrate was present in three of them. Skin lesions included marked follicular atrophy, mild to moderate epidermal hyperplasia with orthokeratotic hyperkeratosis and follicular hyperkeratosis, and hypermelanosis. In 13 chamois there were haemosiderin deposits in the spleen, and in three individuals kidney "cloissone" was observed. Intraeritrocitic parasites were detected either by direct observation or PCR in 8 of 17 chamois. A pestivirus was isolated and detected by RT-PCR from 12 of 13 affected chamois and antigenic characterized as border disease virus by monoclonal antibodies. This is the first time a border disease virus has been associated with an outbreak of a high-mortality disease in a wild species.