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Importance: Ovine pulmonary adenomatosis (OPA) and maedi-visna disease (MVD) are chronic and progressive infectious diseases in sheep caused by Jaagsiekte sheep retrovirus (JSRV) and maedi-visna virus (MVV), respectively., Objective: To investigate the pathological changes and conduct viral gene analysis of OPA and MVD co-occurrence in Inner Mongolia, China., Methods: Using gross pathology, histopathology, immunohistochemistry, ultrastructural pathology, PCR, and sequence analysis, we investigated the concurrent infection of JSRV and MVV in 319 Dorper rams slaughtered in a private slaughterhouse in Inner Mongolia, in 2022., Results: Of the 319 rams included, 3 showed concurrent JSRV and MVV infection. Gross lung pathology showed diffuse enlargement, consolidation, and greyish-white miliary nodules on the lung surface; the trachea was filled with a white foamy fluid; hilar and mediastinal lymph nodes were significantly enlarged. Histopathology results revealed typical OPA and MVD lesions in the lung tissue. Immunohistochemical results were positive for JSRV envelope protein (Env) in the tumor cells and MVV CA in alveolar macrophages. Transmission electron microscopy showed several virions and autophagosomes in the lung tissue, severely damaged mitochondria, and the induced mitophagy. Nucleotide sequences obtained for JSRV env and MVV gag showed the highest homology with the Inner Mongolian strains of JSRV env (JQ837489) and MVV gag (MW248464)., Conclusions and Relevance: Our study confirmed that OPA and MVD co-occurrence and identified the pathological changes in Inner Mongolia, China, thereby providing references for the identification of concurrent JSRV and MVV infections., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)

Background: In order to characterize the complete range of lesions, especially minimal, affecting mammary gland and viral antigen distribution and target cells using immunohistochemistry in naturally Visna/maedi (VM) 84 infected sheep were studied, forty-four from flocks with clinical cases (A) and 35 randomly sampled from two abattoirs (B) together with five negative controls (C). An immunocytochemistry technique was developed and further milk samples (n = 39) were used to study viral excretion, carrier cells and the role of milk and colostrum in the transmission of the disease., Results: All sheep from group C and three sheep from group B were negative to VM in tissue sections by histopathology, immunohistochemistry and PCR, and also in serum using ELISA. Several degrees of CD3 + lymphocytic interstitial mastitis were observed in groups A and B: minimal (+) n = 26 sheep; moderate (++), n = 32 and severe (+++), n = 12. No differences in lesion distribution were observed between groups A and B. Viral presence was confirmed by immunohistochemistry using two different antibodies and/or PCR in every tissue with lesions while serology was negative in six sheep with lesions. Two milk samples taken from milk tanks from two flocks from group A and fourteen milk samples from 29 infected sheep from group B were positive to VM (most of them from animals with moderate and severe lesions). Positivity was only found in macrophages, even in focal and minimal lesions, while no positivity was observed in epithelial or any other cells in either tissue and milk samples., Conclusions: This new observation of the minimal lesions described in this work increased the prevalence of VM lesions in mammary gland up to 90.9% and VM should be considered as a differential diagnosis when minimal interstitial lesions are detected. A high prevalence of VM was observed in intensive milk-producing sheep, ELISA serology did not detect as positivity all infected animals, while histology, IHC or PCR showed higher sensitivity. The cytological technique developed was very useful in milk-cell studies using hematoxylin and eosin and immunocytochemistry. Viral detection in milk samples (16/39) confirms a potential but limited role of milk/colostrum in viral transmission.

Maedi-visna, a disease caused by small ruminant lentiviruses (SRLVs), is present in sheep from many countries, also including Germany. An amino acid substitution (E/K) at position 35 of the transmembrane protein 154 (TMEM154) as well as a deletion in the chemokine (C-C motif) receptor type 5 gene (CCR5) were reported to be associated with the serological MV status and/or the SRLV provirus concentration in North American sheep populations. The aim of this study was to test if those two gene variants might be useful markers for MV susceptibility in Germany. For this purpose, more than 500 sheep from 17 serologically MV positive German sheep flocks with different breed backgrounds were genotyped applying PCR-based methods. Both, crosstab and non-parametric analyses showed significant associations of the amino acid substitution at position 35 of TMEM154 with the serological MV status (cut-off-based classification) and the median MV ELISA S/P value in all samples and in two of the four analyzed breed subsets. The deletion in the CCR5 promoter did not show a consistent association with serological MV status or median ELISA S/P value. It can be concluded that the amino acid substitution at position 35 of TMEM154 is a promising marker for breeding towards a lower number of serologically MV positive sheep in German flocks, at least in flocks of the Texel breed, while this remains questionable for the deletion in the CCR5 promoter. The findings of this study still need to be verified in additional sheep breeds.

Small ruminant lentiviruses (SRLVs), which comprise caprine arthritis-encephalitis virus (CAEV) and maedi-visna virus (MVV), are prevalent in goats and sheep worldwide, including in Japan. However, little is known about the molecular characteristics of goat lentiviruses in Japan. In this study, a molecular and phylogenetic analysis of the long gag region was performed. The phylogenic tree demonstrated that all samples belonged to SRLV subtype B1. Two clusters were identified, with one cluster distinct from previously reported strains of subtype B1. In addition, several alterations in the amino acid sequence were detected in immunodominant epitopes of the gag region. To gain a deeper understanding of the genetic diversity of SRLVs in Japan, it will be necessary to increase the sample size and conduct a broader survey. The present report is important for establishing baseline information on the prevalence of SRLV in Japan and providing data to develop a new, more sensitive diagnostic test for effective control of SRLV.

Production and excretion of small ruminant lentiviruses (SRLVs) varies with the stage of the host reproductive cycle, suggesting hormonal involvement in this variation. Stress may also affect viral expression. To determine if hormones affect SRLV transcriptional activity, the expression of green fluorescent protein (GFP) driven by the promoters in the U3-cap region of the long terminal repeats (LTRs) of different strains of SRLV was assessed in cell culture. High concentrations of steroids (progesterone, cortisol and dehydroepiandrosterone) inhibited expression of GFP driven by SRLV promoters. This effect decreased in a dose-dependent manner with decreasing concentrations of steroids. In some strains, physiological concentrations of cortisol or dehydroepiandrosterone (DHEA) induced the expression of GFP above the baseline. There was strain variation in sensitivity to hormones, but this differed for different hormones. The presence of deletions and a 43 base repeat in the U3 region upstream of the TATA box of the LTR made strain EV1 less sensitive to DHEA. However, no clear tendencies or patterns were observed when comparing strains of different genotypes and/or subtypes, or those triggering different forms of disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A control system for Visna/maedi virus (VMV) infection based on serologic segregation and management strategies was applied in an infected Spanish dairy Manchega breed sheep flock (n=670) that was affected by a severe respiratory process associated to VMV. The control started in 2004 and consisted on the serological study of animals, segregation in two different flocks (seropositive and seronegative), separate management of flocks, selection of young female lambs for replacement only from seronegative ewes offspring, immediate removal of seropositive animals detected in the seronegative flock and a management tending toward the reduction and final culling of the seropositive flock. The serological control was repeated yearly or twice a year, approximately. Initial VMV seroprevalence of the undivided flock was 66.4% (January 2004) that descended to 47.3%, 12.8%, 2.2% and 0.2% between July 2004 and May 2006. Residual seroprevalence fluctuated slightly thereafter with a peak of 2.2% in April 2008. After segregation, number of animals in the seronegative flock was 378 that descended to 323 in October 2005. Since then, this number has increased steadily reaching 650 sheep in December 2011. The seropositive flock was progressively reduced by culling until its total disappearance in June 2010. This work presents the detailed results obtained in the control strategy against VMV in a single dairy sheep flock by implementing a segregation system based on serologic testing. The system is highly successful, as it reduces to residual levels VMV infection in about two years without the need of culling a high number of animals, as required by other methods. Moreover, the original size flock was been recovered within 8 years and has led to a subjective improvement of animal health and welfare in the flock. The residual seroprevalence could be eliminated at this stage by applying more sensitive molecular or other serological techniques to reach eradication., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi-visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.

The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.

Maedi-visna is an important virus infection of sheep having prolonged incubation period (slow disease) and reflecting two distinct forms clinically and pathologically. In this study, the presence of MVV was investigated serologically in 58 Amasya Herik sheep breed and 525 Karayaka sheep breed. Seropositivity rates in Amasya Herik sheep breed and Karayaka sheep breed were detected as 69.0% and 18.5%, respectively. MVV antibodies were found in 137 of 583 serum samples (23.5%). Positivity rates for the provinces varied and were as follows: Samsun 19.4%, Sinop 15.4%, Ordu 25.8%, Trabzon 26.7%, Rize 36.7%, Amasya 69.0% and Tokat 35.0%, however no antibody response was detected in all of the sheep in Giresun province.

An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: A central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and env encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions--TM amino terminal and SU V4, C4 and V5 segments--in order to assess virus compartmentalization in CNS., Results: Eight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the env sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found., Conclusions: Bayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection., Competing Interests: The authors have declared that no competing interests exist.

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection. Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks. This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos. This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10(3) TCID(50)/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier., (Copyright 2010 Elsevier Inc. All rights reserved.)

Microscopic examination of pneumonic lungs of the Ethiopian highland sheep (n = 35) was made and compared with the pneumonic lungs from ten sheep and 66 goats from the lowlands. Lesions compatible with sheep pulmonary adenomatosis (SPA; 8/35, 22.8%), and maedi-visna (MV; 9/35, 25.7%) were recorded only in sheep from the central highlands. Interstitial pneumonia (43.2%), bronchopneumonia (35.1%), and verminous pneumonia (6.3%) were recorded in both sheep and goats from the high- and the lowlands. SPA was documented for the first time in sheep from Ethiopia in this report. We believe that MV and SPA were introduced into Ethiopia through importation of exotic sheep. These infections should be considered in dealing with the diagnosis of respiratory diseases in all the sheep breeds in the central highlands and in the exotic and the crossbred sheep in the other parts of the country.

Lesions were examined at different levels of the central nervous system (CNS) in 64 sheep with natural maedi-visna (MV) meningoencephalitis. All animals showed lesions in more than one of the CNS locations examined; the lesions in the cranial regions were periventricular, while those in the spinal cord affected the white matter funicles. Lesions were found particularly in the cerebellar peduncles (non-suppurative meningoencephalitis), followed by the corpus callosum, hippocampus and thoracic spinal cord. Vascular, infiltrative and malacic histopathological patterns were recognized. One pattern predominated in each section examined, although mixed forms occurred. Vascular lesions occurred with similar frequency at all CNS levels, but infiltrative and malacic lesions predominated at rostral and caudal levels, respectively. Cells consistent with macrophages and shown immunohistochemically to be associated with MV virus were seen in malacic and infiltrative lesions, at the periphery of damaged areas.

CD8(+) cytotoxic T-lymphocyte (CTL) responses have been shown to be important in the control of human and simian immunodeficiency virus infections. Infection of sheep with visna/maedi virus (VISNA), a related lentivirus, induces specific CD8(+) CTL in vivo, but the specific viral proteins recognized are not known. To determine which VISNA antigens were recognized by sheep CTL, we used recombinant vaccinia viruses expressing the different genes of VISNA: in six sheep (Finnish LandracexDorset crosses, Friesland and Lleyn breeds) all VISNA proteins were recognized except TAT. Two sheep, shown to share major histocompatibility complex (MHC) class I alleles, recognized POL and were used to map the epitope. The pol gene is 3267 bp long encoding 1088 aa. By using recombinant vaccinia viruses a central portion (nt 1609-2176, aa 537-725) was found to contain the CTL epitope and this was mapped with synthetic peptides to a 25 aa region (aa 612-636). When smaller peptides were used, a cluster of epitopes was detected: at least three epitopes were present, at positions 612-623: DSRYAFEFMIRN; 620-631: MIRNWDEEVIKN; and 625-635: EEVIKNPIQAR. A DNA-prime-modified vaccinia virus Ankara (MVA)-boost strategy was employed to immunize four sheep shown to share MHC class I allele(s) with the sheep above. Specific CTL activity developed in all the immunized sheep within 3 weeks of the final MVA boost although half the sheep showed evidence of specific reactivity after the DNA-prime immunizations. This is the first report, to our knowledge, of induction of CTL by a DNA-prime-boost method in VISNA infection.

There are very few previous reports of expression of native full-length maedi visna virus (MVV) Env gp150 protein in the literature. Therefore the use of different plasmid and viral expression vectors to obtain full-length gp150 was investigated. A mammalian expression plasmid, pN3-Env, was constructed containing the MVV env gene encoding the precursor protein gp150 Env. The functionality of the recombinant plasmid was tested for expression in HEK293 cells. A recombinant modified vaccinia Ankara virus, MVA-Env, with expression detected in avian cells was also made. The expression of the MVV gp150 Env precursor protein was shown for the first time upon transfection of the eukaryotic HEK293 cells by the pN3-Env plasmid DNA as demonstrated by Western blot analysis. These plasmid or viral expression vectors are of potential use in MVV vaccines.

Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.

Maedi-visna is a slow virus infection of sheep leading to a progressing lymphoproliferative disease which is invariably fatal. It affects multiple organs, but primarily the lungs where it causes interstitial pneumonia (maedi). Infection of the central nervous system was commonly observed in Icelandic sheep (visna), infection of mammary glands (hard udder) in sheep in Europe and the USA, and infection of the joints in sheep in the USA. The name ovine progressive pneumonia (OPP) is commonly used in the USA and ovine lentivirus (OvLV) infection is also a name used for maedi-visna. A related infection of goats, caprine arthritis-encephalitis (CAE), is common in Europe and the USA. The natural transmission of maedi-visna is mostly by the respiratory route, but also to newborn lambs by colostrum and milk. Intrauterine transmission seems to be rare and venereal transmission is not well documented. Macrophages are the major target cells of maedi-visna virus (MVV), but viral replication is greatly restricted in the animal host, apparently due to a posttranscriptional block. The low-grade viral production in infected tissues can explain the slow course of the disease in sheep. The lesions in maedi-visna consist of infiltrates of lymphocytes, plasma cells, and macrophages, and are detectable shortly after experimental transmission. Several studies indicate that the lesions are immune mediated and that cytotoxic T-lymphocytes may be important effector cells. The persistence of the MVV infection is explained by a reservoir of latently infected blood and bone marrow monocytes, which migrate into the target organs and mature into macrophages with proviral DNA transcription, but limited replication of virus. The MVV particles are morphologically similar to those of other retroviruses and the mode of replication follows the same general pattern. The genome organization and gene regulation resembles that of other lentiviruses. In addition to gag, pol and env, MVV has three auxiliary genes (tat, rev and vif), which seem to have similar functions as in other lentiviruses, with a possible exception of the tat gene. A determination of the 9200 nucleotide sequence of the MVV genome shows a close relationship to CAE virus, but limited sequence homology with other lentiviruses, and only in certain conserved domains of the reverse transcriptase and possibly in the surface protein. MVV infection in sheep and HIV-1 infection in humans have a number of features in common such as a long preclinical period following transmission, and a slow development of multiorgan disease with fatal outcome. A brief early acute phase, which is terminated by the immune response, is also an interesting common feature. Like HIV-1, MVV is macrophage tropic and the early stages of the HIV-1 infection which affect the central nervous system and the lungs are in many ways comparable to maedi-visna. In contrast to HIV-1, MVV does not infect T-lymphocytes and does not cause T-cell depletion and immunodeficiency. This is responsible for the difference in the late stages of the HIV-1 and MVV infections and the final clinical outcome. Despite limited sequence homology, certain proteins of MVV and HIV-1 show structural and functional similarities. Studies of MVV may therefore help in the search for new drugs against lentiviruses, including HIV-1.

Four sheep were infected intratracheally with an attenuated molecular clone of maedi-visna virus (MVV). All four became infected. Ten months later these sheep were challenged intratracheally with a genetically similar but pathogenic clone of MVV. Four unvaccinated sheep were infected simultaneously. All sheep became infected by the challenge virus. The vaccinated sheep were not protected against superinfection with the challenge clone. However, virus was isolated more frequently from the blood of the unvaccinated controls than of the vaccinated animals and ten times more frequently from lungs of unvaccinated sheep than from lungs of vaccinated sheep at sacrifice, indicating partial protection.

Leucomyelitis was the predominant feature in four North American adult sheep (cases 1-4) with ovine lentivirus (OvLV) infection. All four animals were OvLV-seropositive and a syncytogenic virus consistent with OvLV was isolated from the brain of case 3 and the lungs of case 4. Clinically, the sheep had dyspnoea and neurologic signs of varying severity. Changes in the central nervous system included asymmetrical meningoleucomyelitis with white matter degeneration in all four sheep and scattered foci of leucoencephalitis in periventricular, subependymal and other white matter areas of the brain of the three animals (cases 1, 2 and 4) for which the brain was examined. In the lungs of two sheep (cases 3 and 4), there was lymphoid interstitial pneumonia with marked lymphoid hyperplasia. The viral capsid antigen (p25) was detected by immunohistochemistry (IHC) in sections of lung, brain and spinal cord of the four sheep and OvLV RNA was detected by in-situ hybridization (ISH) in lung and spinal cord samples. The results confirm the usefulness of the IHC and ISH for differential diagnosis of visna.

Frequent recombination occurs during replication in all retroviruses examined. This increases the genetic variation in the retroviral population and may be of importance in the evolution of the virus. Maedi-visna virus (MVV), a retrovirus of sheep, has a highly variable envelope gene. In a previous experiment, 20 sheep were infected with an uncloned strain of MVV and virus was isolated at regular intervals for 7 years. We sequenced the envelope genes of a number of these strains and found evidence for recombination that may have contributed to the observed high frequency of antigenic variants.

Visna-Maedi virus (VMV), an ungulate lentivirus, causes a natural infection in sheep. In vitro, VMV infection and replication lead to strong cytopathic effects with subsequent death of host cells. We investigated, in vitro, the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with VMV, by employing diverse strategies to detect its common end-stage alterations. We demonstrated that VMV-infection in sheep choroid plexus cells (SCPC), is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearence of apoptotic bodies. DNA fragmentation was documented by TUNEL assay. Although the mechanism by which VMV activates this cell suicide program is not known, we examined the activation of caspases, the family of death-inducing proteases that resulted in cleavage of several cellular substrates. To study the role of caspases in VMV-induced apoptosis, we focused on several protease targets: procaspase-3 and procaspase-1. During VMV-infection, SCPC display active caspase-3 and no caspase-1 activity. In conclusion, our results suggest that VMV infection, in vitro, induces cell death of SCPC by a mechanism that can be characterized by many of the properties most closely associated with apoptotic cell death.

Efficient transfer of therapeutic genes into nondividing human cells can be accomplished by inserting the genes into lentiviruses and infecting the cells with the modified viruses. The most developed lentivirus gene transfer systems are based on HIV-1, but because of the widespread HIV epidemic, the use of HIV-based vectors for gene therapy may be associated with a safety risk. In an attempt to find another lentivirus which can transduce human cells and might be safer than HIV-1, we generated gene transfer constructs based on the sheep lentivirus Visna. Molecular analysis of the constructs in a transient production system indicated that Visna produced as many mature virus particles as did HIV-1. Moreover, the virus particles incorporated a heterologous surface protein marker-gene-containing vector RNAs as efficiently as did HIV-1. However, the Visna virus transduced target cells poorly because of defects in reverse transcription and integration of the vector. Further modifications must be made to the Visna gene transfer system if the system is to be used in clinical gene therapy applications., (Copyright 2001 Academic Press.)

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node., (Copyright 1999 Academic Press.)

Highly purified recombinant gag and env proteins derived from Icelandic strain 1514 of maedi-visna virus were used in an indirect enzyme immunoassay (ELISA) to detect antibodies to small ruminant lentiviruses in sheep and goat sera. The recombinant protein-based ELISA performed very well relative to whole maedi-visna virus and whole caprine arthritis-encephalitis-virus-based ELISAs in its ability to detect anti-maedi visna virus and anti-caprine arthritis-encephalitis virus antibodies, despite the antigenic and genomic variability that is known to exist within and between these two small ruminant lentiviruses. The data suggest that these recombinant maedi-visna virus proteins can be reliably used in an ELISA for the routine serodiagnosis of lentiviral infections in sheep and goats.

Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.

The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.

During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.

The lentiviruses of sheep replicate almost exclusively in macrophages and cause chronic interstitial pneumonia, arthritis, and mastitis, but only rarely encephalitis. This study was undertaken to determine whether a non-neurovirulent field strain of ovine lentivirus isolated from joint fluid that replicated productively in lung and joint macrophages could be adapted to enter and replicate in the brain and cause encephalitis. The field isolate was passed seven times sequentially by intracerebral inoculation of sheep. The neuroadapted strain of virus caused severe encephalitis typical of visna in four of four sheep inoculated intracerebrally. The virus replicated to high titers in the brains of these animals and in cultured microglia. The inflammatory response in the brain was characterized by intense infiltrates of macrophages and CD8+ and CD4+ T cells. Many of the perivascular macrophages demonstrated TNF-alpha expression and there was upregulation of MHC Class II antigen expression on both inflammatory cells and endothelium. Inoculation of this neuroadapted virus into the bone marrow of three animals resulted in persistent infection and cell-associated viremia, but not encephalitis. Virus was not detected in brains from these animals, indicating that the virus was not neuroinvasive. These data suggest that neuroinvasiveness and neurovirulence are separate pathogenic determinants, both of which are required for the development of encephalitis during natural infection.

Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host-virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host-U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells., (Copyright 1997 Academic Press.)

The presence in farm sheep of a paramyxovirus closely related to parainfluenza virus type 3 (Pi3) from humans or cattle was confirmed using RT-PCR on RNA samples from lung cells from slaughtered animals. Sequencing and restriction enzyme patterns of the amplified fragment of the F gene confirms the distinctness of the isolate, and suitable PCR primers allow specific detection of the ovine virus. A study of the incidence of ovine Pi3 in samples from sheep with or without distinctive histopathological signs of maedi shows that it is uncommon in aged sheep with overt lentiviral disease, but it occurs at moderate frequency in lambs and may, in the presence of visna-maedi virus, contribute to early lesion formation.

The behavior of two genetically different molecular clones of visna virus KV1772-kv72/67 and LV1-1KS1 was compared in vivo and in vitro. On intracerebral inoculation, clone KV1772-kv72/67 induced a similar response in five sheep as has already been reported with neurovirulent derivates of visna virus. Virus was frequently isolated from blood, cerebrospinal fluid (CSF), and lymphoid organs and induced characteristic central nervous system (CNS) lesions. A strong humoral immune response was detected by ELISA, immunoblotting, and neutralization. Six sheep infected with clone LV1-1KS1 showed a completely different picture. No virus could be isolated from blood or CSF during 6 months of infection. At sacrifice all organs were virus-negative except the CNS of one sheep. None of the six sheep developed significant neutralizing antibodies and only low titer antibodies were detected by ELISA and immunoblotting. Minimal CNS lesions were present in one sheep. The molecular clones were also tested in sheep choroid plexus cells (SCP) and macrophages. In macrophages LV1-1KS1 replicated to a significantly lower titer but induced much more cell fusion than KV1772-kv72/67. The clones replicated equally well in SCP cells. Thus, these molecular clones of visna virus, which differ only by 1% in nucleotide sequence, showed a profound difference in replication and pathogenicity both in vitro and in vivo. These results can be used to map viral genetic determinants important for host-lentivirus interactions.

The core p25 and transmembrane (TM) genes of Maedi-Visna virus (MVV) were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. A preliminary assessment of the diagnostic potential of the recombinant antigens (GST-TM and GST-TM-p25) was made by testing the antigens against 46 seropositive and 46 seronegative sheep and comparing the results with a commercial p25 ELISA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA.

All members of the lentivirus family infect cells of the monocyte-macrophage lineage, although this may be obscured during infection in vivo by the effect of the virus on other cells, such as CD4+ lymphocytes. Macrophages are the major cell type infected by the ruminant lentivirus maedi-visna, making it a valuable model for studying the pathogenesis of lentiviruses in these cells.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.

The nucleotide and deduced primary amino acid sequence of the envelope gene of two virus isolates from the brains of Spanish sheep with encephalomyelitis, were determined and compared with those of other flaviviruses. The amino acid alignments showed that the Spanish viruses shared 95 to 96 per cent homology with the envelope protein of louping ill virus and western European tick-borne encephalitis virus. In comparison, the maximum variation in amino acid identities among strains of louping ill virus from the British Isles is 1.8 per cent. The Spanish isolates were distinguishable from all other known flaviviruses by the presence of a unique tripeptide sequence (AQR) at amino acid positions 232 to 234 in the E protein, the position at which a genetic marker for distinct flavivirus species has been identified. Other genetic markers, viz DSGHD (amino acids 320 to 324) and EHLPTA (amino acids 207 to 212), which identify the tick-borne encephalitis group within the genus Flavivirus, were present in the amino acid sequences of the Spanish virus. It is concluded that the cause of sheep encephalomyelitis in Spain is a distinct species in the tick-borne encephalitis virus group.