Domestic sheep and bighorn sheep carry distinct gammaherpesviruses belonging to the genus Macavirus.

The genus Macavirus of the subfamily Gammaherpesvirinae comprises two genetically distinct lineages of lymphotropic viruses. One of these lineages includes viruses that can cause malignant catarrhal fever (MCF), which are known as MCF viruses (MCFV). All MCFVs are genetically and antigenically related but carried by different hosts. In this study, we report the recognition of new MCFV carried by bighorn sheep. The virus was first identified in a bighorn sheep from Banff National Park, Alberta, Canada. Analysis of a conserved region of the viral DNA polymerase gene of the virus carried by this bighorn sheep showed 85.88% nucleotide identity to the MCFV carried by domestic sheep, ovine herpesvirus 2 (OvHV-2). Further investigation of bighorn samples obtained from animals in the US and Canada showed 98.87-100% identity to the DNA polymerase sequence of the first bighorn in the study. Phylogenetic analysis indicated that the MCFV carried by bighorn sheep is closely related but distinct from OvHV-2. Epidemiological and virulence features of the newly recognized MCFV are still unknown and warrant further investigation. Considering the current nomenclature for MCFVs, we suggest a tentative designation of ovine herpesvirus-3 (OvHV-3) for this newly identified bighorn sheep MCFV.

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.

SNPs in candidate genes MX dynamin-like GTPase and chemokine (C-C motif) receptor-5 are associated with ovine pulmonary adenocarcinoma progression in Latxa sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer in sheep caused by Jaagsiekte sheep retrovirus (JSRV). OPA is present in many sheep-rearing countries causing economic and welfare issues, as currently no efficient vaccines or treatments are available. Breed differences suggest a host genetic component may influence the pathogenesis of OPA, but so far few genes have been identified. In this work, a genetic association study was carried out in Latxa dairy sheep which were classified as cases/controls based on the presence/absence of OPA lung tumours. Candidate genes included cytokines and a receptor and innate immunity genes. After SNPs in the candidate genes were identified, the distribution of alleles in cases and controls was compared by means of logistic regression analyses at the allelic, genotypic and haplotypic levels. The association analysis showed that several candidate genes were significantly associated with resistance or susceptibility to OPA; two of the candidates, CCR5 and MX1, remained significantly associated with resistance and susceptibility respectively, even after Bonferroni correction.

The use of endogenous retroviruses as markers to describe the genetic relationships among local Swedish sheep breeds.

The aim of this study was to describe the genetic relationships among five Swedish sheep breeds using insertional polymorphisms of six endogenous Jaagsiekte retroviruses of sheep. Although the Swedish breeds were found to have genomes of 'primitive' origin, there also are indications of the presence of more recently derived sheep breeds within the ancestries of three of the breeds.

"Ménage à Trois": the evolutionary interplay between JSRV, enJSRVs and domestic sheep.

Sheep betaretroviruses represent a fascinating model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with a variety of highly related endogenous JSRVs, referred to as enJSRVs. During evolution, some of them were co-opted by the host as they fulfilled important biological functions, including placental development and protection against related exogenous retroviruses. In particular, two enJSRV loci, enJS56A1 and enJSRV-20, were positively selected during sheep domestication due to their ability to interfere with the replication of related competent retroviruses. Interestingly, viruses escaping these transdominant enJSRVs have recently emerged, probably less than 200 years ago. Overall, these findings suggest that in sheep the process of endogenization is still ongoing and, therefore, the evolutionary interplay between endogenous and exogenous sheep betaretroviruses and their host has not yet reached an equilibrium.

Detection and molecular characterization of naturally transmitted sheep associated malignant catarrhal fever in cattle in India.

Malignant catarrhal fever (MCF) is a fatal herpesvirus infection of domestic and wild ruminants, with a short and dramatic clinical course characterized primarily by high fever, severe depression, swollen lymph nodes, salivation, diarrhea, dermatitis, neurological disorders, and ocular lesions often leading to blindness. In the present study, fatal clinical cases of sheep associated malignant catarrhal fever (SA-MCF) were identified in cattle in the state of Karnataka. These cases were initially presented with symptoms of diarrhea, respiratory distress, conjunctivitis, and nasal discharges. Laboratory diagnosis confirmed the detection of ovine herpesvirus-2 (OvHV-2) genome in the peripheral blood samples of two ailing animals. The blood samples collected subsequently from sheep of the neighboring areas also showed presence of OvHV-2 genome indicating a nidus of infection in the region. The positive test results were further confirmed by nucleotide sequencing of the OIE approved portion of tegument gene as well as complete ORF8 region of the OvHV-2 genome. Phylogenetic analysis based on the sequence of the latter region indicated close genetic relationship with other OvHV-2 reported elsewhere in the world.

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.

Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.

Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep.

Bluetongue virus, a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue, which is a non-contagious Culicoides mediated blood-borne disease. The present study characterizes the pathogenicity of a Taiwan prototype BTV2/KM/2003 in Corriedale sheep inoculated subcutaneously into the ear pinna. Histologically, multifocal petechiated hemorrhage, with mild to moderate inflammation and edema, were present in the contralateral ear pinna, tongue, and facial skin, without remarkable lesions in lymphoid organs. By days post-infection (DPI) 7, viral VP7 antigen, detected by immunohistochemistry, presented in the spleen, chiefly located in the outer rim of <3 cell thickness of marginal zone macrophages bordering the marginal zone and red pulp, and T lymphocytes of the red pulp. By DPI 11, viral signals shifted from the marginal zone to macrophages and small lymphocytes within follicles of the spleen. In situ hybridization with VP7 gene probe detected strong signals in the spleen, chiefly spanning the whole width of 5-10 cell thickness of the marginal zone, including the marginal zone macrophages and marginal zone B cells, as well as macrophages of sheathed capillaries in the red pulp. This study demonstrates molecular as well as morphologic evidence of the presence of bluetongue virus in the marginal zone of the spleen, most likely associated with viremia in acute infection, as previously demonstrated by the authors.

Cancer mortality in workers employed in cattle, pigs, and sheep slaughtering and processing plants.

OBJECTIVES: We studied mortality in two separate cohorts of workers in abattoirs (N=4996) and meat processing plants (N=3642) belonging to a meatcutters' union, because they were exposed to viruses that cause cancer in food animals, and also to chemical carcinogens at work. METHODS: Standardized mortality ratios (SMRs) and proportional mortality ratios (PMRs) were estimated for each cohort as a whole and in subgroups defined by race and sex, using the US general population mortality rates for comparison. Study subjects were followed up from January 1950 to December 2006, during which time over 60% of them died. RESULTS: An excess of deaths from cancers of the base of the tongue, esophagus, lung, skin, bone and bladder, lymphoid leukemia, and benign tumors of the thyroid and other endocrine glands, and possibly Hodgkin's disease, was observed in abattoir and meat processing workers. Significantly lower SMRs were recorded for cancer of the thymus, mediastinum, pleura, etc., breast cancer, and non-Hodgkin's lymphoma. CONCLUSION: This study confirms the excess occurrence of cancer in workers in abattoirs and meat processing plants, butchers, and meatcutters, previously reported in this cohort and other similar cohorts worldwide. Large nested case-control studies are now needed to examine which specific occupational and non-occupational exposures are responsible for the excess. There is now sufficient evidence for steps to be taken to protect workers from carcinogenic exposures at the workplace. There are also serious implications for the general population which may also be exposed to some of these viruses.

Pathology and pathogenesis of ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte, is a transmissible lung tumour of sheep caused by jaagsiekte sheep retrovirus (JSRV). JSRV induces neoplastic transformation of alveolar and bronchiolar secretory epithelial cells and the resulting tumours can grow to occupy a significant portion of the lung. Tumour growth is frequently accompanied by the overproduction of fluid in the lung, which further compromises normal respiration. The period between infection and the appearance of clinical signs may be several months or years and many JSRV-infected sheep do not exhibit clinical signs at all during their lifespan. This allows the spread of OPA into new flocks through contact with infected but apparently normal animals. OPA was first described in the early 19th century; however, it has still not been possible to devise effective methods for controlling its spread and it remains an important problem in most countries where sheep are farmed. This is due in part to the absence of an immunological response to JSRV in infected animals, which has hindered the development of serological diagnostic tests and vaccines. In addition to its veterinary importance, OPA is regarded as a potential large animal model for human lung adenocarcinoma and this has stimulated research into the pathogenesis of the ovine disease. This work has produced some significant results, including the finding that one of the JSRV structural proteins is directly involved in oncogenesis. The recent advances in understanding JSRV and the pathogenesis of OPA should lead to novel strategies for diagnosis and control of this disease and for its exploitation as a comparative model for human lung cancer.

Revealing the history of sheep domestication using retrovirus integrations.

The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their "retrotype" and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as "primitive" on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.

Development of a management program for a mixed species wildlife park following an occurrence of malignant catarrhal fever.

During late 2001 and early 2002, a mixed species wildlife park in North Carolina experienced an acute outbreak of morbidity and mortality in Pere David's deer (Elaphurus davidianus), axis deer (Axis axis), blackbuck antelope (Antelope cervicapra), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus). Clinical signs varied from fulminant disease, progressing from depression to bloody scours to death in fewer than 4 days in Pere David's deer, to a more protracted form of disease, ranging from 2 wk to 3 mo, in axis deer. In moribund axis deer, high levels of anti-malignant catarrhal fever (MCF) virus antibody by competitive-inhibition enzyme-linked immunosorbent assay were detected. Ovine herpesvirus 2 (OvHV-2) DNA was detected in peripheral blood leukocytes of the affected axis deer. No other MCF viruses were detected. Retrospective examination of frozen tissue samples from the affected Pere David's deer and blackbuck antelope also confirmed the presence of OvHV-2 DNA. Initial control efforts were directed at preventing further deaths of clinically susceptible animals by removing MCF virus reservoir species, particularly ovine species. The most prevalent ovine species in the wildlife park was mouflon sheep (Ovis musimon). All sheep were removed from the park by June 2002, and the last MCF death occurred in October 2002. Since mouflon sheep had been a prominent attraction in the wildlife park, the owner wanted a means to reintroduce this species to the park. Derivation of OvHV-2-uninfected mouflon lambs was undertaken using the previously described program for production of OvHV-2-free sheep (Ovis ovis). The rederived MCF virus-negative mouflon sheep were introduced into the park in approximately January 2004. As of December 2007, no further cases of MCF have occurred since the removal of OvHV-2-positive mouflon sheep and reintroduction of the virus-free lambs. This paper describes the successful management and control of MCF in a densely populated mixed species animal park.

Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node.

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.

Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5' LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.

Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages.

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.