Exogenous Jaagsiekte Sheep Retrovirus type 2 (exJSRV2) related to ovine pulmonary adenocarcinoma (OPA) in Romania: prevalence, anatomical forms, pathological description, immunophenotyping and virus identification.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. RESULTS: Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). CONCLUSIONS: In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.

Old origin of a protective endogenous retrovirus (enJSRV) in the Ovis genus.

Sheep, the Jaagsiekte sheep retrovirus (JSRV) and its endogenous forms (enJSRVs) are a good model to study long-time relationships between retroviruses and their hosts. Taking advantage of 76 whole genome resequencing data of wild and domestic Ovis, we investigated the evolution of this relationship. An innovative analysis of re-sequencing data allowed characterizing 462 enJSRVs insertion sites (including 435 newly described insertions) in the Ovis genus. We focused our study on endogenous copies inserted in the q13 locus of chromosome 6 (6q13). Those copies are known to confer resistance against exogenous JSRV thanks to alleles bearing a mutation in the gag gene. We characterized (i) the distribution of protective and non-protective alleles across Ovis species and (ii) the copy number variation of the 6q13 locus. Our results challenged the previous hypothesis of fixation and amplification of the protective copies in relation with domestication, and allowed building a new model for the evolution of the 6q13 locus. JSRV would have integrated the 6q13 locus after the Ovis-Capra divergence (5-11 MYA) and before the Ovis diversification (2.4-5 MYA). The protective mutation in the enJSRV 6q13 copy appeared shortly after its insertion and was followed by genomic amplifications, after the divergence between Pachyform lineage on one side and the Argaliform and moufloniform lineages on the other (2.4-5 MYA). Considering the potential selective advantage of the protective mutation, its fixation in both sheep and its closest wild relative Ovis orientalis may be due to natural selection before domestication from O. orientalis populations.

Evidence against a role for jaagsiekte sheep retrovirus in human lung cancer.

BACKGROUND: Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats that can be transmitted by aerosols produced by infected animals. Virus entry into cells is initiated by binding of the viral envelope (Env) protein to a specific cell-surface receptor, Hyal2. Unlike almost all other retroviruses, the JSRV Env protein is also a potent oncoprotein and is responsible for lung cancer in animals. Of concern, Hyal2 is a functional receptor for JSRV in humans. RESULTS: We show here that JSRV is fully capable of infecting human cells, as measured by its reverse transcription and persistence in the DNA of cultured human cells. Several studies have indicated a role for JSRV in human lung cancer while other studies dispute these results. To further investigate the role of JSRV in human lung cancer, we used highly-specific mouse monoclonal antibodies and a rabbit polyclonal antiserum against JSRV Env to test for JSRV expression in human lung cancer. JSRV Env expression was undetectable in lung cancers from 128 human subjects, including 73 cases of bronchioalveolar carcinoma (BAC; currently reclassified as lung invasive adenocarcinoma with a predominant lepidic component), a lung cancer with histology similar to that found in JSRV-infected sheep. The BAC samples included 8 JSRV DNA-positive samples from subjects residing in Sardinia, Italy, where sheep farming is prevalent and JSRV is present. We also tested for neutralizing antibodies in sera from 138 Peruvians living in an area where sheep farming is prevalent and JSRV is present, 24 of whom were directly exposed to sheep, and found none. CONCLUSIONS: We conclude that while JSRV can infect human cells, JSRV plays little if any role in human lung cancer.

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.

Diagnosis and phylogenetic analysis of ovine pulmonary adenocarcinoma in China.

Ovine pulmonary adenocarcinoma (OPA) is a lung tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is common in sheep, and it is most commonly observed in China. Without preventative vaccines and serological diagnostic tools for assay of OPA, identification of JSRV based on reverse transcription polymerase chain reaction (RT-PCR) is very important for prevention and control measures for OPA in practice management. In this study, the diagnosis of OPA was made from analysis of clinical signs, pathological observations, JSRV-like particle discovery, and RT-PCR of the target env gene. The phylogenetic analysis showed that the China Shandong (SD) strain studied in this article belonged to exogenous JSRV, and it was very similar to 92k3, which was isolated from sheep in the Kenya (Y18305). The current study reported a severe outbreak of OPA in Shandong Province, China. The observations could offer a comparative view of the env gene of JSRV.

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.

Endogenous retroviruses of sheep: a model system for understanding physiological adaptation to an evolving ruminant genome.

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. The sheep genome contains 27 endogenous betaretroviruses (enJSRVs) related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. The enJSRVs can protect their host against JSRV infection by blocking early and late steps of the JSRV replication cycle. In the female reproductive tract, enJSRVs are specifically expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophectoderm growth and development. Collectively, available evidence in sheep and other mammals indicate that ERVs coevolved with their hosts for millions of years and were positively selected for biological roles in genome plasticity and evolution, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental development.

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model.

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.

[Enzootic nasal adenocarcinoma in a dairy sheep flock]. / Enzootische nasale Adenokarzinome in einem Milchschafbestand.

In a herd of dairy sheep several losses occurred due to a respiratory syndrome in combination with progressive wasting. Clinical and pathomorphological diagnostics of 3 sheep revealed the presence of cancerous masses in the nasal cavities. These neoplasms were identified as adenocarcinomas originating from the nasal mucosa. Etiologically, they were attributed to JRSV (Jaagsiekte Sheep Retrovirus) by detection of capsid protein 24 in western blot. The significance of the disease in Switzerland is discussed, also in the context of lung adenomatosis.

Jaagsiekte sheep retrovirus is not detected in human lung adenocarcinomas expressing antigens related to the Gag polyprotein of betaretroviruses.

A proportion of human lung adenocarcinomas (hLACs) express an antigen related to the major capsid protein (CA) of Jaagsiekte sheep retrovirus (JSRV), a Betaretrovirus that causes a transmissible lung cancer in sheep. In this study, we have investigated whether JSRV or related betaretroviruses are expressed in hLACs. Results obtained indicate that JSRV is not associated with human lung adenocarcinomas. However, a proportion of hLACs reacted positively in immunohistochemistry with antibodies specific towards different domains of the JSRV Gag suggesting that a bona fide retrovirus antigen could be expressed in these tumours. Further studies will be necessary to ascertain whether the detection of antigens cross-reacting with betaretrovirus Gag antisera in some hLACs is due to expression of a human endogenous retrovirus or, more unlikely, of an uncharacterized exogenous retrovirus.

PCR examination of bronchoalveolar lavage samples is a useful tool in pre-clinical diagnosis of ovine pulmonary adenocarcinoma (Jaagsiekte).

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The disease is a particular problem in flocks in many parts of the world. The aim of the study was to assess screening methods for individual animals as a prelude to future eradication trials. Results of histological examination were used as the standard to evaluate the relative sensitivity and specificity of an established heminested polymerase chain reaction (PCR) test for JSRV proviral DNA from blood and bronchoalveolar lavage (BAL) samples. PCR results from tissue samples are included as control data. PCR testing of blood samples was found to have an estimated sensitivity of only 10% (95% confidence interval (CI) 3-20) while the sensitivity of the PCR test on BAL samples was 89% (CI 79-96) in comparison to the results of histological examination. We conclude that PCR testing of BAL samples is an effective confirmatory test for sheep with suspected clinical OPA. It is also a useful tool for the pre-clinical identification of individual infected sheep within an infected flock and therefore may prove beneficial in future control or eradication programmes.

Eradication of ovine pulmonary adenocarcinoma by motherless rearing of lambs.

The principles of maedi-visna eradication programmes were applied to a field trial for the eradication of ovine pulmonary adenocarcinoma (OPA). In two maternal flocks the prevalence of gross and histological lesions in slaughtered animals was 18.3 per cent and 29.8 per cent, respectively. The lambing period was supervised for three consecutive years from 1999 to 2001, during which the lambs were taken away from their mothers at birth, deprived of maternal colostrum, and hand-reared away from other sheep. Over the three-year period, 322 hand-reared animals, mainly male lambs between 10 and 14 months old, were slaughtered; their lungs were examined grossly, 52.5 per cent of them were examined histologically, and 105 samples of caudal mediastinal lymph nodes were examined by PCR. No OPA tumours were detected in the slaughter specimens from the derived flock, but one lamb had histological lesions in one lung location; intrauterine transmission was ruled out in this case. No clinical OPA has subsequently been observed in the hand-reared flock. Bronchoalveolar lavage samples from the breeding stock were examined by PCR in order to rule out further subclinical cases of OPA. No Jaagsiekte retrovirus was detected in any of the 488 samples.

Multiclonal pattern of Jaagsiekte sheep retrovirus integration sites in ovine pulmonary adenocarcinoma.

Insertional mutagenesis and envelope (Env)-mediated oncogenesis are hypothesized mechanisms by which Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA). Twenty-eight JSRV integration sites in lung tumors (LTs) from four sheep with OPA were cloned and sequenced by a multiple step gene walking technique. Using nested PCR, clonal expansion of these integration sites could be detected, if at all, only in the localized regions of LT from which the integration sites were derived. One sheep had a viral integration site in a sequence with 85 and 81% identity, respectively, over 100 bp to exon 2 of the human and mouse receptor protein tyrosine phosphatase gamma genes. Clonal integration of Jaagsiekte sheep retrovirus in this gene was demonstrated by nested PCR and Southern blot hybridization in the DNA sample from which the integration site was cloned, but not in other LT or kidney DNA samples from the same sheep. OPA may develop from multiple independent oncogenic events and a role for insertional mutagenesis cannot be ruled out.

Natural history of JSRV in sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep and, rarely, goats that arises from two types of secretory epithelial cell that retain their luxury function of surfactant synthesis and secretion. It is classified as a low-grade adenocarcinoma and is viewed as a good model for epithelial neoplasia because of its morphological resemblance to the human lung tumour, bronchioloalveolar adenocarcinoma. OPA is present in most of the sheep rearing areas of the globe and, in affected flocks, tumours are present in a high proportion of sheep. OPA is associated with the ovine retrovirus, jaagsiekte sheep retrovirus (JSRV), and is transmissible only with inocula that contain JSRV. All sheep contain JSRV-related endogenous viruses, but JSRV is an exogenous virus that is associated exclusively with OPA. JSRV is detected consistently in the lung fluid, tumour and lymphoid tissues of sheep affected by both natural and experimental OPA or unaffected in-contact flockmates and never in sheep from unaffected flocks with no history of the tumour. JSRV replicates principally in the epithelial tumour cells, but also establishes a disseminated infection of several lymphoid cell types, including peripheral blood leukocytes (PBLs). Longitudinal studies in flocks with endemic OPA have revealed JSRV in PBLs before the onset of clinical OPA and even in the absence of discernible lung tumour. The prevalence of JSRV infection is 40%-80%, although only 30% of sheep appear to develop OPA lesions. A unique feature of OPA is the absence of a specific humoral immune response to JSRV, despite the highly productive infection in the lungs and the disseminated lymphoid infection. This feature is associated with reduced responsiveness to some mitogens, although the phenotypic profile of the peripheral blood remains unaltered. The reduced response is an early and sustained event during infection and may indicate that the failure of infected sheep to produce specific antibodies to JSRV is a direct consequence of infection.

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.

Identification of Hyal2 as the cell-surface receptor for jaagsiekte sheep retrovirus and ovine nasal adenocarcinoma virus.

Jaagsiekte sheep retrovirus (JSRV) and ovine nasal adenocarcinoma virus (ONAV) replicate in the airway and cause epithelial cell tumors through the activity of their envelope (Env) proteins. Identification of the receptor(s) that mediate cell entry by these viruses is crucial to understanding the oncogenic activity of Env and for the development of gene therapy vectors based on these viruses that are capable of targeting airway cells. To identify the viral receptor(s) and to further study the biology of JSRV and ONAV, we developed retroviral vectors containing Moloney murine leukemia virus components and the Env proteins of JSRV or ONAV. We used a new technique involving positional cloning by phenotypic mapping in radiation hybrid cells to identify and clone the human receptor for JSRV, Hyal2, which also serves as the receptor for ONAV. Hyal2 is a glycosylphosphatidylinositol-anchored cell-surface protein that has low hyaluronidase activity and is a member of a large family that includes sperm hyaluronidase (Spam) and serum hyaluronidase (Hyal1). Hyal2 is located in a region of human chromosome 3p21.3 that is often deleted in lung cancer, suggesting that it may be a tumor suppressor. However, its role in JSRV or ONAV tumorigenesis, if any, is still unclear. JSRV vectors are capable of transducing various human cells, and are being further evaluated for gene therapy purposes.

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.

Advances in the study of transmissible respiratory tumours in small ruminants.

Sheep and goats are widely infected by oncogenic retroviruses, namely Jaagsiekte Sheep RetroVirus (JSRV) and Enzootic Nasal Tumour Virus (ENTV). Under field conditions, these viruses induce transformation of differentiated epithelial cells in the lungs for Jaagsiekte Sheep RetroVirus or the nasal cavities for Enzootic Nasal Tumour Virus. As in other vertebrates, a family of endogenous retroviruses named endogenous Jaagsiekte Sheep RetroVirus (enJSRV) and closely related to exogenous Jaagsiekte Sheep RetroVirus is present in domestic and wild small ruminants. Interestingly, Jaagsiekte Sheep RetroVirus and Enzootic Nasal Tumour Virus are able to promote cell transformation, leading to cancer through their envelope glycoproteins. In vitro, it has been demonstrated that the envelope is able to deregulate some of the important signaling pathways that control cell proliferation. The role of the retroviral envelope in cell transformation has attracted considerable attention in the past years, but it appears to be highly dependent of the nature and origin of the cells used. Aside from its health impact in animals, it has been reported for many years that the Jaagsiekte Sheep RetroVirus-induced lung cancer is analogous to a rare, peculiar form of lung adenocarcinoma in humans, namely lepidic pulmonary adenocarcinoma. The implication of a retrovirus related to Jaagsiekte Sheep RetroVirus is still controversial and under investigation, but the identification of an infectious agent associated with the development of lepidic pulmonary adenocarcinomas might help us to understand cancer development. This review explores the mechanisms of induction of respiratory cancers in small ruminants and the possible link between retrovirus and lepidic pulmonary adenocarcinomas in humans.

[Pathological Diagnoses and Whole-genome Sequence Analyses of the Jaagsiekte Sheep Retrovirus in Xinjiang, China].

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.

Absence of jaagsiekte sheep retrovirus DNA and RNA in bronchioloalveolar and conventional human pulmonary adenocarcinoma by PCR and RT-PCR analysis.

Bronchioloalveolar adenocarcinoma (BAC) morphologically resembles sheep pulmonary adenomatosis (SPA), a contagious ovine pulmonary adenocarcinoma caused by the jaagsiekte sheep retrovirus (JSRV). Previously, positivity for JSRV by immunostaining, reverse-transcription polymerase chain reaction (RT-PCR), and Western blot was reported in most nonmucinous BACs. Our objective in this study was to analyze additional BAC subtypes and conventional adenocarcinomas (CA) to further substantiate this association. Tumor tissue was microdissected from unstained paraffin sections of 26 cases of formalin-fixed, paraffin-embedded BAC (7 mucinous, 17 nonmucinous, 2 sclerosing) and 29 cases of CA. Positive controls consisted of 2 separate paraffin blocks of known SPA. Primer sequences were derived that were capable of hybridizing to all reported strain variants of both the DNA (endogenous) and RNA (exogenous) forms of JSRV. Each sample was tested using both PCR (DNA) and RT-PCR (RNA). All BAC and CA cases were negative for JSRV. Positive controls yielded PCR products that were sequenced and precisely matched the published prototype stain of JSRV. To control for negative effects of tissue fixation, dilutions of positive control tissue were added to BAC and CA samples. Detection of JSRV was evident at 1:50 dilution. Although the possibility of a viral association with BAC cannot be excluded, this study shows that the association with JSRV is probably very weak, if present at all.