Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Cells infected with Jaagsiekte sheep retrovirus are detected in the bone marrow of asymptomatic sheep

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer caused by Jaggsiekte sheep retrovirus (JSRV). It is difficult to identify animals infected with JSRV but are clinically healthy. The virus does not induce a specific antibody response and, although proviral DNA sequences of JSRV can be found in mononuclear blood cells, the detection is inconsistent. The aim of this study was to investigate the presence of JSRV in the bone marrow of infected sheep and develop a more consistent screening method. Immunohistochemical examination of bone marrow samples from 8 asymptomatic JSRV-infected sheep revealed the presence of positively labelled cells. However, JSRV could not be detected by a highly sensitive polymerase chain reaction (PCR) in bone marrow aspirates periodically collected from these animals. Results suggest that JSRV-infected cells may be present in the bone marrow of symptomless animals, but the number is below the detectable level for PCR. Therefore, this technique does not seem to be helpful for preclinical diagnosis of OPA.     

Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures

Jaagsiekte sheep retrovirus (JSRV) causes a fatal lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). OPA is a significant disease in many sheep-rearing countries and there is no effective method of control. A unique feature of OPA is the overproduction of fluid in the lung of affected animals. This lung fluid contains JSRV and provides a means of transmission through the inhalation of virus. In this study we demonstrated that lung fluid from different OPA cases contained between 10⁷ and 10¹⁰ copies of JSRV RNA per ml. Examination of JSRV RNA survival under conditions that mimic natural conditions suggested that intact JSRV virions may persist for several weeks in the environment. These are the first quantitative data on JSRV in lung fluid and provide valuable information for implementing appropriate biosecurity measures to control the spread of JSRV in the field.     

The morphology and morphogenesis of jaagsiekte retrovirus (JSRV)

Jaagsiekte retrovirus ( JSRV ) was recently shown to be the aetiological agent of jaasiekte (ovine pulmonary adenomatosis). The morphogenesis of JSRV was studied in jaagsiekte tumour tissue. Intracytoplasmic particles, often associated with centrioles, were found in tumour cells. JSRV budded from tumour cells with a complete core which appeared to mature during the budding process. Extracellular particles were found in the alveolar lumen. Immature extracellular particles were rare. Mature extracellular JSRV was membrane-bound and had a slightly eccentric nucleoid with an electron-dense perinucleoidal space. In negatively stained preparations of JSRV the envelope was covered with spikes. JSRV is morphologically distinct from all known retroviruses.     

Immunohistochemical and histopathological findings of ovine pulmonary adenocarcinoma (Jaagsiekte) in Egyptian sheep

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced transmissible lung cancer in sheep. Lungs and associated (bronchial and mediastinal) lymph nodes of seven sheep with OPA were examined. Lungs had few multifocal consolidated slightly elevated gray to white masses ranging from 0.5 to 3 cm in diameter. Histopathologically, these masses appeared as well-differentiated acinar adenocarcinoma with little evidence of anaplasia. The acini composed of well-differentiated cuboidal to low columnar epithelium with clear or vacuolated cytoplasm and low mitotic index. No metastases were observed in the bronchial and mediastinal lymph nodes of any animal. The presence of Jaagsiekte sheep retrovirus (JSRV) was demonstrated in the lungs by immunohistochemistry. JSRV protein was detected in all tumor epithelial cells, histologically normal alveolar type II cells, and few bronchiolar epithelial cells, alveolar macrophages, lymphocytes, and plasma cells. This study is the first to confirm the presence of natural OPA in Egypt.     

稳定表达绵羊肺腺瘤病毒表面蛋白A549细胞系的建立

为研究绵羊肺腺瘤病病毒(JSRV)表面蛋白(SU)的致瘤机制,本研究构建稳定表达SU的羊肺细胞A549细胞系.采用PCR方法从含su基因的pGEX-4T-1-SU重组质粒中扩增SU编码序列,将其克隆至真核表达载体pcDNA3.1(+)中,转染A549细胞.通过G418筛选,对转染阳性细胞进行纯化,获得稳定表达JSRV SU蛋白的A549细胞系.以间接免疫荧光及western blot鉴定SU的表达状况,并运用共聚焦显微镜确认SU蛋白的亚细胞定位.结果表明,重组蛋白SU在A549细胞中有效表达,而且主要分布于细胞质中.该细胞系的建立为SU生物学功能的体外研究提供了平台.     

表达绵羊肺腺瘤病毒跨膜蛋白HepG2细胞系的建立

为深入研究绵羊肺腺瘤病毒(JSRV)与绵羊肺腺瘤病(OPA)发病关系,本研究采用PCR方法从pGEX-4T-1-TM重组质粒中扩增编码JSRV跨膜蛋白(TM)的基因序列,并引入标签多肽HA序列和限制性内切酶位点,将其重组至真核表达载体pcDNA3.1(+)中,构建了重组质粒pcDNA-TM-HA.通过转染HepG2细胞并用G418筛选,对稳定表达TM的阳性细胞进行纯化,获得了稳定表达JSRV tm基因的HepG2细胞系.间接免疫荧光及western blot检测结果表明,重组蛋白TM-HA在HepG2细胞中得到正确表达.JSRV TM蛋白稳定表达细胞系的建立为进一步研究该蛋白与JSRV诱导OPA发病关系提供了重要的实验平台.     

Colostrum and milk can transmit jaagsiekte retrovirus to lambs

Ovine pulmonary adenocarcinoma (OPA) is a contagious disease caused by jaagsiekte sheep retrovirus (JSRV). In the three studies performed, we have obtained data of the importance of colostrum/milk (C/M) in the transmission of JSRV. In the first study, a group of sheep from a flock with a long history of OPA, samples from colostrum and peripheral blood leucocytes (PBLs) were collected. Two specific PCRs (U3-LTR and env of the JSRV) were carried out. Using U3PCR 8/34 sheep were positive in colostrum whereas with envPCR 7/34 were positive. From these animals only one was positive with U3PCR in the PBLs. Evidence of the transmission of JSRV infection by C/M was obtained in two more separate studies. In the second study, PBLs from five lambs from JSRV+ ewes and two from JSRV-ewes were tested by the U3PCR. They were fed C/M by their mothers during 3 months and slaughtered 7 months after birth. Three out of five lambs from the JSRV+ sheep become PBL positive at 3-4 months old and the other two were also positive at 4-6 months of age. One lamb of the JSRV-sheep became also PBL positive at an age of 3 months. In the third study, a group of lambs from JSRV negative mothers were fed with C/M from JSRV+ sheep and housed in separate unit. For comparison, another group of the same origin and maintained in another different unit, were fed with C/M containing a JSRV virus preparation. All lambs were blood sampled monthly and JSRV infection was detected as early as 15 days and several times onwards in both groups. Control groups fed with C/M from JSRV free flock and JSRV blood test negative sheep were always negative. Together these results indicate that suckling is an important natural transmission route for JSRV.     

Bronchioloalveolar carcinoma not related to jaagsiekte sheep retrovirus in a goat

A spontaneous lung tumor in a 5-year-old goat of the Murciano-Granadina breed is described in this paper. Clinical signs of cachexia and tachypnoea were evident, and a considerable amount of white mucous foamy fluid was discharged from the nostrils when the animal's head was lowered. A lung tumor with the characteristics of bronchioloalveolar carcinoma was detected during histopathologic examination. The tumor cells were positive for surfactant proteins C and B, confirming that alveolar type II cells were the origin of the neoplasia. Tumor samples were tested by polymerase chain reaction, immunoblotting, and immunohistochemistry for the presence of Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), another retrovirus very closely related to JSRV, but all tests were negative. Therefore, this is the first reported case of spontaneous bronchioloalveolar carcinoma not related to JSRV or ENTV infection in a goat.     

绵羊肺腺瘤病毒检测方法的研究进展

绵羊肺腺瘤（OPA）是由β-反转录病毒属绵羊肺腺瘤病毒（JSRV）引起的一种肿瘤性传染病,该病潜伏期长,经呼吸道传播,冬季圈养种羊发病率高,目前无治疗措施,病死率为100%。OPA的持续存在对种羊生产形成了潜在威胁并造成较大经济损失,严重危害养羊业健康发展。所以,对OPA的早期精确诊断是防制本病的前提,尤其是在出入境检验检疫过程中对进出口羊的JSRV检测尤为重要。随着分子生物学技术的不断发展,JSRV分子生物学检测方法也得到不断的创新和改进。作者就近年来针对检测JSRV的聚合酶链式反应（PCR）、核酸探针杂交技术、酶联免疫吸附试验（ELISA）、环介导等温扩增技术（LAMP）等方法的研究概况作一综述,为寻找快速准确并适用于出入境检疫的JSRV检测方法和进一步发展研究新型JSRV检测方法提供参考。     

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.     

猪瘟病毒囊膜结构(糖)蛋白E~(rns)和E2的生物学特性研究

猪瘟病毒(CSFV)、牛病毒性腹泻病毒(BVDV)、边界病病毒(BDV)和长颈广鹿瘟病毒(Giraffe pestvirus)为黄病毒科瘟病毒属成员,其病毒结构、抗原性和遗传特性密切相关。CSFV囊膜结构(糖)蛋白E2(gp55)是诱导机体产生中和抗体及激发保护性免疫应答的主要抗原蛋白。囊膜结构(糖)蛋白Erns/E0(gp48)具有RNA酶活性,在病毒增殖及中和病毒感染中发挥重要作用,是诱导机体产生保护性免疫应答的第二抗原蛋白。E2和Ens与细胞表面受体的相互作用介导CSFV对细胞的感染过程。本文综述CSFV囊膜结构(糖)蛋白Erns和E2的生物学特性研究进展。     

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.     

Characterization of viral polypeptides from avian rotavirus

The synthesis of avian rotavirus (AvRV-1) polypeptides in MA 104 cells was investigated. Extracts of cells labeled with either [35S]methionine or [3H]mannose were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was detected first at 6 hours postinfection. Ten major viral polypeptides were detected at this time. The presence of five viral structural proteins (100K, 90K, 88K, 45K, and 37K) were demonstrated in AvRV-1-infected cells by immunoprecipitation analysis. One structural polypeptide (37K) was identified as a glycoprotein. Two viral polypeptides (30K and 28K) were identified as nonstructural glycoproteins. By tunicamycin inhibition of glycosylation, a 32K polypeptide was identified. This 32K polypeptide later was proven to be the precursor of 37K structural glycoprotein by immunoprecipitation analysis, beta-N-acetylglucosaminidase H (Endo H) treatment, and peptide mapping analysis. Avian rotavirus contained high-mannose oligosaccharide content in the glycoproteins similar to the glycoproteins of mammalian rotaviruses.     

An influx of macrophages is the predominant local immune response in ovine pulmonary adenocarcinoma

Infection with a retrovirus, Jaagsiekte sheep retrovirus (JSRV), causes ovine pulmonary adenocarcinoma (OPA). The excess production of surfactant proteins by alveolar tumour cells results in increased production of pulmonary fluid, which is characteristically expelled through the nostrils of affected sheep. The immune response to JSRV and the tumour is poorly understood: no JSRV-specific circulating antibodies or T cells have been detected to date. The aim of the present study was to obtain phenotypic evidence for a local immune response in OPA lungs. Specific-pathogen free lambs were infected intratracheally with JSRV. When clinical signs of OPA were apparent, the lungs were removed at necropsy and immunohistochemistry (IHC) was performed on lung sections using a panel of mouse anti-sheep mAbs. No influx of dendritic cells, B cells, CD4, CD8 or gammadelta T cells was seen in the neoplastic nodules or in their periphery. MHC Class II-positive cells were found intratumourally, peritumourally and in the surrounding alveolar lumina. In the tumours, many of these cells were shown to be fibroblasts and the remainder were likely to be mature macrophages. In the alveolar lumen, the MHC Class II-positive cells were CD14-positive and expressed high levels of IFN-gamma. They appeared to be immature monocytes or macrophages which then differentiated to become CD14-negative as they reached the periphery of the tumours. A high level of MHC Class I expression was detected on a range of cells in the OPA lungs but the tumour nodules themselves contained no MHC Class I-positive cells. On the basis of these findings, it is proposed that the lack of an effective immune response in OPA could result from a mechanism of peripheral tolerance in which the activity of the invading macrophages is suppressed by the local environment, possibly as a consequence of the inhibitory properties of the surfactant proteins.     

Surveillance of Jaagsiekte sheep retrovirus in sheep in Hokkaido, the northern island of Japan

Surveillance of jaagsiekte sheep retrovirus (JSRV) infection was performed by polymerase chain reaction (PCR) of blood DNA samples collected from 40 sheep and goats in 10 different flocks in Hokkaido, the northern island of Japan. No exogenous (oncogenic) JSRV sequence was detected by PCR in these samples, while the ovine endogenous retrovirus sequence was successfully amplified in all samples. Our paper is the first demonstration of JSRV surveillance in Japan and shows no evidence of oncogenic JSRV infection in sheep and goats in Hokkaido.     

Immunogenic and antigenic properties of recombinant soluble glycoprotein of rabies virus

Rabies virus glycoprotein is a type I transmembrane protein exposed on the surface on the mature virus particle that induces virus neutralizing antibodies. In the present study, 60 amino acid C-terminal hydrophobic anchor (transmembrane) and cytoplasmic domains of glycoprotein were deleted from full-length glycoprotein and fused with polyhistidine tag. The N-terminal viral signal peptide was also replaced with CD33 signal peptide for efficient secretion in mammalian cells. Following transfection of Madin Darby bovine kidney (MDBK) cells with plasmid encoding this soluble form of glycoprotein, polyclonal populations of stably transfected resistant cells were obtained after G418 selection. The protein was expressed as a glycosylated protein and secreted outside the cells utilizing N-terminal CD33 signal peptide. The secreted soluble glycoprotein was purified from cell culture supernatant by Ni--agarose affinity chromatography utilizing C-terminal polyhistidine tag. Like full-length glycoprotein, the expressed recombinant soluble glycoprotein was found to be immunogenic when injected in rabbits. In this study, we have assessed the potential of recombinant soluble glycoprotein as diagnostic antigen in ELISA and found that this recombinant protein can be used as diagnostic antigen in ELISA for detecting anti-glycoprotein antibodies in immunized host.     

Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology

The viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine development strategies against both viruses. Subunit, DNA and vectored vaccine candidates have been developed using this glycoprotein as the primary antigen, demonstrating that gD has the capacity to induce robust virus neutralizing antibodies and strong cell-mediated immune responses, as well as protection from clinical symptoms, in target species. This review highlights the structural and functional characteristics of BoHV-1, BoHV-5 and where appropriate, Human herpesvirus gD, as well as its role in viral entry and interactions with host cell receptors. Furthermore, the interactions of gD with the host immune system are discussed. Finally, the application of this glycoprotein in new vaccine design is reviewed, taking its structural and functional characteristics into consideration.     

Approaches for genetic purity testing of live recombinant viral vaccines using a human adenovirus:rabies model.

A two part purity testing regimen for genetically engineered live viral vaccines is described using a human adenovirus 5: rabies glycoprotein gene recombinant as a model vaccine. Initially, restriction endonuclease analysis of the recombinant viral genome verified the integrity of the recombinant construct and identified the vector genome. The second stage employed the polymerase chain reaction to facilitate a more detailed study of the target rabies glycoprotein cassette. The size of the target region was predicted from known nucleic acid sequence information and compared to that obtained after electrophoresis with molecular weight standards. Digestion of the polymerase chain reaction product with a second restriction endonuclease cleaved the target into a number of small fragments. Resolution of the fragments by gel electrophoresis allowed analysis of the target region alone, verifying its identity and integrity.