Skeletal muscle hypoplasia represents the only significant lesion in peripheral organs of ruminants infected with Schmallenberg virus during gestation.

Schmallenberg virus (SBV), an arbovirus within the family Bunyaviridae, represents a ruminant pathogen that has caused epidemic abortion and birth of malformed or stillborn animals in many European countries since August 2011. Histological and immunohistochemical analysis of peripheral tissues of SBV-infected animals, including lymphoid tissues, endocrine organs and tissues of the gastrointestinal, urogenital and respiratory system, were analyzed in order to elucidate the occurrence of SBV-associated changes and the presence of viral antigens and RNA. Twenty calves and 12 lambs as well as age-matched controls were included in this study. Significant muscular hypoplasia with fatty replacement was noted in affected calves and lambs. In addition, hepatocellular degeneration with lymphohistiocytic inflammation, interstitial fibrosis and biliary hyperplasia was detected in calves. All animals lacked SBV-positive cells in the peripheral organs. These observations resemble those found in Akabane virus- and Cache Valley virus-infected animals and support the occurrence of few residual lesions in peripheral organs following SBV infection.

Lack of schmallenberg virus in ruminant brain tissues archived from 1961 to 2010 in Germany.

Schmallenberg virus (SBV) is an orthobunyavirus of the family Bunyaviridae that is associated with stillbirth and malformations in ruminants. The infection has been identified in many European countries since August 2011. The present study investigated retrospectively the occurrence of SBV infection in ruminants using immunohistochemistry and in-situ hybridization in brain tissues archived between 1961 and 2010 (112 cattle, 57 sheep, 16 goats and 27 wild ruminants). Eighty-five animals with inflammatory brain lesions and 47 animals with malformations were included. Due to the lack of SBV protein and RNA detection, SBV appears to have been introduced recently into Northern parts of Europe from tropical or subtropical regions.

Cellular differentiation and proliferation in the ovine lung during gestation and early postnatal development.

This study investigates epithelial cell differentiation and proliferation in specific anatomical regions of the ovine lung during prenatal and postnatal development. Immunohistochemistry was used to identify ciliated epithelial cells, Clara cells, neuroepithelial bodies and type II pneumocytes in the lungs of preterm (67, 127 and 140 days of gestation), full-term (147 days) and postnatal (9, 16 and 91 days old) lambs. Differentiation of ciliated epithelial cells was seen at 67 days of gestation and at term for Clara cells. Neuroepithelial bodies were first detected at 127 days of gestation. From 16 to 91 days of age there was a significant (P <0.05) increase in beta-tubulin (present in ciliated epithelial cells) and Clara cell protein (present in Clara cells) in multiple regions of the lung. Detection of Ki67, a marker of proliferation, in preterm lambs showed a reduction in proliferation index in multiple anatomical regions of the lung between 70 days of gestation and term. Cell proliferation increased following parturition, and then decreased between 16 and 91 days of age, with the largest reduction occurring in the alveolar compartment. Knowledge of which cells are present at specific times of lung development provides valuable information on the anatomy of the ovine lung, improving its use as a model for ovine and human neonatal disease. In addition, the antibodies used here will be valuable for future studies requiring the identification and quantification of respiratory epithelial cell phenotypes in the sheep lung.

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.

Coevolution of endogenous betaretroviruses of sheep and their host.

Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. Jaagsiekte sheep retrovirus (JSRV) is a pathogenic exogenous retrovirus and the causative agent of ovine pulmonary adenocarcinoma. The sheep genome contains at least 27 copies of endogenous retroviruses (enJSRVs) highly related to JSRV. enJSRVs have played several roles in the evolution of the domestic sheep as they are able to block the JSRV replication cycle and play a critical role in sheep conceptus development and placental morphogenesis. Available data strongly suggest that some dominant negative enJSRV proviruses (i.e. able to block JSRV replication) have been positively selected during evolution. Interestingly, viruses escaping the transdominant enJSRV loci have recently emerged (less than 200 years ago). Thus, endogenization of these retroviruses may still be occurring today. Therefore, sheep provide an exciting and unique system to study retrovirus-host coevolution. (Part of a multi-author review).

Requirements for veterinary research and the recruitment of veterinary surgeons into research.

Veterinary science plays a crucial role in society and it is important that veterinarians are involved. However, there are concerns about a growing gap in the quality of veterinary science compared with other biomedical sciences, and about falling recruitment of veterinarians into research. Andrea Nolan and Massimo Palmarini, on behalf of the rcvs Research Subcommittee, discuss these concerns and the steps being taken to encourage more veterinary graduates into a career in research.

Ovine endogenous betaretroviruses (enJSRVs) and placental morphogenesis.

Endogenous retroviruses (ERVs) account for a substantial portion of the genetic pool of every animal species (e.g. approximately 8% of the human genome). Despite their overwhelming abundance in nature, many questions on the basic biology of ERVs are unanswered. The most important question derives from the observations in many animal species, including humans, of abundant ERVs expressed in the female genital tract. Sheep harbor approximately 20 copies of endogenous betaretroviruses (enJSRVs), which are related to an exogenous oncogenic virus, Jaagsiekte sheep retrovirus (JSRV). enJSRVs are abundantly expressed in the ovine placenta and uterine endometrium throughout gestation. Hyaluronidase 2 (HYAL2), which can serve as a cellular receptor for JSRV and enJSRVs envelope (Env), is expressed by the trophoblast giant binucleate cells and multinucleated syncytia of the placenta. Little is known about the cellular and molecular mechanisms that regulate trophoblast differentiation and syncytia formation during synepitheliochorial placentation in sheep. The temporal and spatial alterations in enJSRVs expression in the ovine uterus and placenta support the hypothesis that trophoblast growth and differentiation into binucleate cells and formation of multinucleated syncytiotrophoblast involves enJSRVs Env and possibly their cellular receptor, HYAL2.

Molecular biology of jaagsiekte sheep retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. Until recently, research on JSRV/OPA was hampered by the lack of a tissue culture system for the propagation of the virus. Historically, pathological samples (lung fluid) collected from sheep affected by OPA were the only source of infectious JSRV. Thus studies on the JSRV/OPA system were conducted only where field isolates of OPA cases were readily available. In the past 10 years, the deduction of the JSRV sequence (York et al. 1991; York 1992), the isolation of an infectious and oncogenic JSRV molecular clone (JSRV21) (Palmarini et al. 1999a) and the establishment of a rapid method to produce infectious virus in vitro (Palmarini et al. 1999a) sparked many studies at the molecular level that strengthened past observations and revealed new properties of this unique virus. Here, we will review the data accumulated so far on the molecular biology of JSRV using the infectious and oncogenic JSRV21 molecular clone as virus of reference.

Endogenous retroviruses related to jaagsiekte sheep retrovirus.

Ovine betaretroviruses consist of exogenous viruses [jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus, (ENTV)] associated with neoplastic diseases of the respiratory tract and 15-20 endogenous viruses (enJSRV) stably integrated in the ovine and caprine genome. Phylogenetic analysis of this group of retroviruses suggests that the enJSRV can be considered as 'modern' endogenous retroviruses with active, exogenous counterparts. Sequence analysis of JSRV, ENTV and enJSRV suggests that enJSRV do not directly contribute to the pathogenesis of ovine pulmonary adenocarcinoma (OPA) or enzootic nasal tumor through large-scale recombination events, but small-scale recombination or complementation of gene function cannot be excluded; experiments involving enJSRV-free sheep, which have not been found, would be needed to investigate this possibility. Evidence of expression of enJSRV structural proteins in tissues of the reproductive tract and lung implies that they do not have a primary role in disease. However, experimental exploitation of exogenous/endogenous retrovirus sequence differences by producing chimeras has been useful in establishing the determinants of JSRV Env-induced transformation. Even if enJSRV do not have a direct role in OPA, their expression during ontogeny or in neonatal life may impact the likelihood of exogenous JSRV infection and disease outcome via the induction of immunological tolerance. Aside from any role in disease, enJSRV loci may serve as useful genetic markers in the sheep and their frequent expression in the reproductive tract of the ewe may portend an important physiologic role in sheep.

Transformation and oncogenesis by jaagsiekte sheep retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma (OPA). JSRV is a potent carcinogen in the experimental setting, inducing end-stage tumors at around 6 weeks of age when newborn lambs are inoculated intratracheally. Despite this rapid oncogenesis, inspection of the JSRV genome sequence does not reveal any obvious viral oncogenes. In this review, recent advances in studies of JSRV oncogenic transformation are described. Molecular cloning of an infectious and oncogenic JSRV provirus was instrumental in the studies. DNA transfection of JSRV proviral DNA into mouse NIH3T3 cells results in morphological transformation, indicating that the JSRV genome carries an oncogene. Further experiments identified the JSRV envelope protein as the transforming gene, and a PI3 kinase docking site in the cytoplasmic tail of the transmembrane (TM) protein was shown to be necessary for transformation. Avian DF-1 cells infected with an avian retroviral vector (RCAS) expressing the JSRV envelope protein also undergo tumorigenic transformation. Possible mechanisms of transformation are discussed, and a cooperating role for insertional activation of proto-oncogenes in tumorigenesis is also considered. The transforming potential of the JSRV envelope protein may be necessary for JSRV infection and replication in vivo.

Expression of endogenous betaretroviruses in the ovine uterus: effects of neonatal age, estrous cycle, pregnancy, and progesterone.

The ovine genome contains 15 to 20 copies of endogenous retroviruses (enJSRVs) highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus. enJSRVs are highly expressed in the endometrial lumenal epithelia (LE) and glandular epithelia (GE) of the ovine uterus. The effects of neonatal age, estrous cycle, pregnancy, and progesterone on expression of enJSRVs in the ovine uterus were determined. Expression of enJSRV RNAs was absent from the uterus of ewes at birth, but enJSRV RNAs were expressed specifically in the LE and developing GE from postnatal day (PND) 7 to PND 56. In adult ewes, enJSRV RNAs were detected only in the epithelia of the uterine endometrium, as well as epithelia of the oviduct, cervix, and vagina. In cyclic ewes, endometrial enJSRV RNA abundance was lowest on day 1, increased 12-fold between days 1 and 13, and then decreased to day 15. In pregnant ewes, levels of endometrial enJSRV RNAs were high on day 11, increased to day 13, and then decreased to day 19. In day 17 and 19 conceptuses, enJSRV RNAs were also detected in binucleate cells of the trophectoderm. Immunoreactive JSRV capsid and envelope proteins were detected in the endometrial LE and GE, as well as in the binucleate cells of the conceptus. In transfection assays utilizing ovine endometrial LE cells, progesterone increased transcriptional activity of several enJSRV long terminal repeats. Collectively, these results indicate that transcription of enJSRVs in the endometrial epithelia of the ovine uterus is increased by progesterone and might support a role for enJSRVs in conceptus-endometrium interactions during the peri-implantation period and early placental morphogenesis.

Retrovirus-induced ovine pulmonary adenocarcinoma, an animal model for lung cancer.

Studies on the molecular mechanisms of transformation of retrovirus-induced neoplasms in domestic and laboratory animal species have provided insights into the genetic basis of cancer. Ovine pulmonary adenocarcinoma (OPA) is a retrovirus-induced spontaneous lung tumor of sheep that has striking analogies to some forms of human adenocarcinoma. The etiologic agent of OPA, jaagsiekte sheep retrovirus (JSRV), is unique among retroviruses for having a specific tropism for the differentiated epithelial cells of the lung, and it is the only virus known to cause a naturally occurring lung adenocarcinoma. Expression of the JSRV envelope protein is sufficient to induce cell transformation in vitro, possibly via the activation of the phosphatidylinositol 3-kinase/Akt-signaling pathway mediated by the cytoplasmic tail of the transmembrane protein. The aim of this review is to draw the attention of basic and clinical scientists engaged in lung cancer research to this unique animal model, to explore the possible use of OPA as a tool to investigate the mechanisms of pulmonary carcinogenesis, and to underline the similarities between OPA and some forms of human lung adenocarcinoma. The possibility of a viral etiology for the latter will be evaluated in this review.

Association of jaagsiekte sheep retrovirus with pulmonary carcinoma in Sardinian moufflon (Ovis musimon).

Bronchiolo-alveolar carcinoma has been described in man and in several animal species, including cattle, dogs, opossums, goats and sheep. In sheep, a bronchiolo-alveolar carcinoma, known as ovine pulmonary carcinoma (OPC), is caused by jaagsiekte sheep retrovirus (JSRV), an exogenous type D retrovirus. In the mid-1980s, a severe outbreak of a disease resembling OPC was described in captive Sardinian moufflon (Ovis musimon). In the present study, the use of polymerase chain reaction (PCR) amplification of nucleic acids extracted from archival material established that JSRV was associated with OPC in affected moufflon. JSRV was detected in the lungs and mediastinal lymph nodes. Immunohistochemical and in-situ PCR demonstrated that in the lungs, JSRV proviral DNA was localized in transformed and untransformed type II pneumocytes and in the alveolar macrophages. In the mediastinal lymph nodes, JSRV DNA was mainly located in the cortical follicles and paracortex. These data suggest that JSRV is the cause of OPC in Sardinian moufflon, as it is in Sardinian sheep.

A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G(1)-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.

Clinically relevant cognitive impairment after cardiac surgery: a 6-month follow-up study.

BACKGROUND AND PURPOSE: The majority of studies on neuropsychological complications after cardiac surgery used the raw variation of selective tests scores to define the occurrence of cognitive decline. We prospectively estimated the frequency of cognitive impairment after cardiac surgery, with a particular emphasis on persistent and clinically relevant cognitive decline. Possible baseline and operative predictors were also evaluated. METHODS: An extensive neuropsychological battery was administered to 110 patients (mean age 64.1+/-9.4 years; 70.9% males) undergoing cardiac surgery before and 6 months after the operation. After evaluating the variations in the cognitive performances, two independent neuropsychologists ranked the patients as unchanged-improved, mildly-moderately deteriorated, or severely deteriorated, using a global and functionally oriented judgement. The degree of the impairment was determined in relation to its impact on everyday life activities. RESULTS: Ten patients (9.1%) were ranked as severely deteriorated, 22 (20%) as mildly-moderately deteriorated, and 78 (70.9%) as unchanged-improved. Cognitively impaired patients were older (p=0.031), more often females (p=0.005), with a low education level (p=0.013). At multivariate analysis, female gender (odds ratio (OR) 6.14, 95% confidence interval (95% CI) 2.16-17.50), baseline use of beta-blockers (OR 4.55, 95% CI 1.30-15.92), and PaO2 at arrival in intensive care unit (OR for 1 mm Hg increment 1.012, 95% CI 1.004-1.020) were significant predictors of cognitive impairment of any degree. Positive predictors of severe cognitive impairment were history of hypertension (OR 5.33, 95% CI 1.03-27.64) and PaO2 at arrival intensive care unit (OR for 1 mm Hg increment 1.020, 95% CI 1.006-1.035), while education was protective (OR per year of increment 0.53, 95% CI 0.31-0.90). CONCLUSIONS: A considerable proportion of cardiac surgery patients may undergo clinically relevant cognitive impairment. The knowledge of variables influencing cognitive outcome is essential for the adoption of preventive measures.

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2-3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21DeltaGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

Evidence for a protein related immunologically to the jaagsiekte sheep retrovirus in some human lung tumours.

Human bronchioloalveolar carcinoma (BAC) is a lung cancer, morphologically similar to an endemic contagious lung neoplasm of sheep called sheep pulmonary adenomatosis (SPA) or jaagsiekte. SPA is caused by an exogenous type B/D retrovirus (jaagsiekte sheep retrovirus (JSRV)), which prompted the present study to obtain evidence of a retrovirus in BAC. A panel of 249 human lung tumours, 21 nontumour lung lesions, four normal lung tissues, 23 adenocarcinomas from other organs and a cell line expressing a human endogenous retrovirus protein was examined immunohistochemically using a rabbit antiserum directed against the JSRV capsid protein. Specific staining was detected only in the cytoplasm of recognizably neoplastic cells in the pulmonary alveoli of 39 of 129 (30%) BACs, 17 of 65 (26%) lung adenocarcinomas and two of seven large cell carcinomas. The remaining samples were negative. These results support the hypothesis that some human pulmonary tumours may be associated with a jaagsiekte sheep retrovirus-related retrovirus, warranting further studies.

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus.

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.