Detection of Jaagsiekte sheep retrovirus in the peripheral blood during the pre-clinical period of ovine pulmonary adenomatosis.

The envelope protein (Env) of the Jaagsiekte sheep retrovirus (JSRV) is known to be a unique oncoprotein responsible for inducing ovine pulmonary adenocarcinoma (OPA). The objective of this study was to prepare a specific monoclonal antibody (mAb) against the JSRV Env protein using bioinformatic analysis. According to the structure and epitope prediction results of JSRV Env, the JSRV-Env572-615 antigen was prepared via peptide synthesis (amino acid sequence 572-615, denoted as JSRV-Env572-615). BALB/c mice were immunized to prepare the anti-JSRV-Env572-615 mAb. Spleen cells were fused with SP2/0 myeloma cells after being screened by indirect ELISA and cloned by limiting dilution. The specificity of mAb was evaluated by western blot analysis and immunohistochemistry assays. Western blot results showed that the JSRV Env protein was able to bind to mAb with high specificity. Immunohistochemistry assays demonstrated that the mAb was able to recognize JSRV Env in adenomatous hyperplasia of the lung. Furthermore, JSRV was detected in peripheral blood leukocytes during the pre-clinical period of OPA in 2 of the 25 sheep using this newly synthesized mAb. Therefore, this mAb may be a useful tool for the detection of JSRV in sheep.

The sheep tetherin paralog oBST2B blocks envelope glycoprotein incorporation into nascent retroviral virions.

UNLABELLED: Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A. IMPORTANCE: BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species.

IFITM proteins restrict viral membrane hemifusion.

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

Interplay between ovine bone marrow stromal cell antigen 2/tetherin and endogenous retroviruses.

Endogenous betaretroviruses (enJSRVs) of sheep are expressed abundantly in the female reproductive tract and play a crucial role in conceptus development and placental morphogenesis. Interestingly, the colonization of the sheep genome by enJSRVs is likely still ongoing. During early pregnancy, enJSRV expression correlates with the production of tau interferon (IFNT), a type I IFN, by the developing conceptus. IFNT is the pregnancy recognition signal in ruminants and possesses potent antiviral activity. In this study, we show that IFNT induces the expression of bone marrow stromal cell antigen 2 (BST2) (also termed CD317/tetherin) both in vitro and in vivo. The BST2 gene is duplicated in ruminants. Transfection assays found that ovine BST2 proteins (oBST2A and oBST2B) block release of viral particles produced by intact enJSRV loci and of related exogenous and pathogenic jaagsiekte sheep retrovirus (JSRV). Ovine BST2A appears to restrict enJSRVs more efficiently than oBST2B. In vivo, the expression of BST2A/B and enJSRVs in the endometrium increases after day 12 and remains high between days 14 and 20 of pregnancy. In situ hybridization analyses found that oBST2A is expressed mainly in the endometrial stromal cells but not in the luminal and glandular epithelial cells, in which enJSRVs are highly expressed. In conclusion, enJSRVs may have coevolved in the presence of oBST2A/B by being expressed in different cellular compartments of the same organ. Viral expression in cells unable to express BST2 may be one of the mechanisms used by retroviruses to escape restriction.

Jaagsiekte sheep retrovirus-specific immune responses induced by vaccination: a comparison of immunisation strategies.

Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). No JSRV-specific immunological responses have been detected in clinical cases of OPA or in experimentally infected lambs. The aim of the present study was to induce immune responses in sheep against JSRV proteins using several immunisation strategies. The vaccines were administered subcutaneously and intradermally, or intranasally, in adjuvant. Antibodies were measured by ELISA and immunoblotting, and T cell responses by lymphoproliferation assay. Antibodies specific for JSRV-capsid protein were induced by inoculation of recombinant proteins in adjuvant, and transient JSRV-specific T cell responses by intranasal inoculation with inactivated virus. These results will help in the design of a protective vaccine against JSRV infection and the development of OPA.

Sheep retrovirus structural protein induces lung tumours.

Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats, with significant animal health and economic consequences. The host range of JSRV is in part limited by species-specific differences in the virus entry receptor, hyaluronidase 2 (Hyal2), which is not functional as a receptor in mice but is functional in humans. Sheep are immunotolerant of JSRV because of the expression of closely related endogenous retroviruses, which are not present in humans and most other species, and this may facilitate oncogenesis. Here we show that expression of the JSRV envelope (Env) protein alone in lungs of mice, by using a replication-incompetent adeno-associated virus vector, results in tumours with a bronchiolo-alveolar localization like those seen in sheep. Whereas lethal disease was observed in immunodeficient mice, tumour development was almost entirely blocked in immunocompetent mice. Our results provide a rare example of an oncogenic viral structural protein, show that interaction of the viral Env protein with the virus entry receptor Hyal2 is not required for tumorigenesis, and indicate that immune recognition of Env can protect against JSRV tumorigenesis.

A history of ovine pulmonary adenocarcinoma (jaagsiekte) and experiments leading to the deduction of the JSRV nucleotide sequence.

Jaagsiekte (JS), a contagious cancer affecting the lungs of sheep has been called many names over the years. At a recent workshop in Missilac, France it was agreed that the disease would be called ovine pulmonary adenocarcinoma (OPA). The disease is caused by an infectious retrovirus called jaagsiekte sheep retrovirus (JSRV). This chapter focuses on the early research that led up to the isolation, cloning and sequencing of the exogenous infectious form of JSRV and the demonstration that it has an endogenous counter part that is present in all sheep. As there was no in vitro production source of the virus much of the early research focused on the in vivo production and purification of the virus to obtain sufficient material to use to identify the viral proteins and purify the viral genetic material. Typically, new born lambs were inoculated intra-tracheally with concentrated lung lavage from previously infected sheep lungs. The optimal purification involved the concentration of lung lavage of freshly slaughtered sheep, an extraction with organic solvent, and final purification by both rate zonal and isopycnic centrifugation. Monoclonal and polyclonal antibodies were made against the purified fractions. The polyclonal antibodies were not very specific and the monoclonal antibodies proved to be against antigens expressed in high concentrations in response to any lung pathology. The genomic RNA of the virus was isolated from ex vivo purified materials, and cloned as a collection of cDNAs. The full length sequence was assembled by walking through the cDNA clones. The genome of the exogenous virus is 7462 bases and has the classical gag, pol, env genome arrangement and is flanked by a long terminal repeat (LTR) on each end. An additional open reading frame (ORF) was observed in the viral genome and has been called orfX. A function has not been determined for this ORF. JSRV is classified as a betaretrovirus, with gag and pol closely related to D type retrovirus, whereas env is related to the B type viruses such as the human endogenous retrovirus HERV-K. An interesting finding was that the exogenous infectious virus had an endogenous counter part which is present in the genomes of all sheep and goats. It is estimated that there are between 15 and 20 endogenous loci per sheep genome. No circulating antibodies have been found in OPA-affected sheep. It is suggested that the endogenous JSRV transcripts are expressed at an early age and are cause for the clonal elimination of JSRV specific T cells during T-cell ontogeny. Histopathologically the sheep disease resembles human bronchiolar alveolar carcinoma and has been identified as a natural out bred animal model that could be used to study the human disease.

Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma.

Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). To monitor changes in cellular immune function during JSRV infection, lymphoproliferation in response to various mitogens was measured in the blood of conventionally housed and specific-pathogen-free lambs experimentally infected with JSRV until the development of OPA and compared with uninfected control lambs. In addition, blood samples collected from adult field cases in the terminal stages of OPA and control adult sheep were compared. No difference in the proliferative response to phytohaemagglutinin and pokeweed mitogen between the animal groups was detected. In contrast, reduced responses to concanavalin A stimulation were demonstrated in the JSRV-inoculated lambs, prior to the onset of clinical disease, and also in the terminally ill adult sheep. Peripheral blood leukocytes were monitored to identify phenotypic frequency alterations. The CD4 lymphocytopaenia and neutrophilia reported previously in adult OPA cases were demonstrated but similar phenotypic changes were not identified during experimental infection.