Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

Microsatellites in immune-relevant regions and their associations with Maedi-Visna and ovine pulmonary adenocarcinoma viral diseases.

Maedi-Visna (MV) and ovine pulmonary adenocarcinoma (OPA) are two retroviral diseases occurring worldwide that affect adult sheep. Differences in incidence, which may be related to sheep-rearing and housing choices, as well as to genetics, and disease progression have been reported for both diseases. In this work four microsatellites located in immune-relevant regions, the major histocompatibility complex (MHC) region, interferon-γ and interleukin-12p35, were genotyped to determine their association with disease progression. The analysed sample included Latxa sheep with and without OPA and MV-characteristic lesions in their lungs. The microsatellites in the MHC were the most diverse, while the ones located in the cytokines were the less polymorphic. In the case of IFN-γ the results suggested the presence of null alleles. Significant results were detected for several microsatellite alleles in the association analysis carried out by logistic regression. All statistical analyses included a flock effect adjustment to avoid false positives due to genetic structuration. MHC Class I microsatellite alleles OMHC1*205 and OMHC1*193 were associated with disease progression for Maedi and OPA, respectively. Moreover, MHC Class II microsatellite allele DRB2*275 was associated with presence of lesions in Maedi. Furthermore, the MHC microsatellites were combined for a bioinformatic haplotype inference with the PHASE software. In total, 73 haplotypes were detected, 18 of them in more than 6 animals. After standard and weighted logistic regression analysis, two of them were significantly associated with susceptibility: OMHC1*205-DRB2*271 for Maedi and OMHC1*193-DRB2*271 for OPA, both with the Class I microsatellite alleles associated in the marker by marker study. Although more extensive analyses are needed to disentangle the relationship between host genetics and disease, as far as we know this is the first study demonstrating a significant association between sheep MHC Class I microsatellite alleles and susceptibility to Maedi-Visna and OPA viral diseases.

Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus.

The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses.

Patterns of lesion and local host cellular immune response in natural cases of ovine maedi-visna.

This study investigates the nervous form of ovine maedi-visna by histological and immunohistochemical techniques. The aim was to study the lesion types and the local cellular immune response related to each lesion type, and the possible relationship between these parameters. Thirty-four Assaf ewes were studied, 29 of which had shown nervous signs. Microscopical lesion patterns were described according to location, extent and predominance of inflammatory cell type. Immunohistochemical labelling of T cells (CD3(+), CD4(+), CD8(+) and cells expressing the γδ form of the T-cell receptor), B cells and macrophages revealed clear differences between the lesion patterns. Two main lesion types were described. Lymphocytic lesions had areas of mild-moderate injury characterized by a predominance of infiltrating T cells. Histiocytic lesions were more severe and had extensive areas of malacia and dominant infiltration by macrophages and B cells. Each animal had a unique lesion pattern and these differences could be due to individual resistance to the progression of infection. The lymphocytic lesions appear to represent initial or latent phases of slow progression, in which the animal presents some natural resistance to the infection. The histiocytic pattern may reflect a poor immune response or a greater virulence of the viral strain.

Identification of the ovine mannose receptor and its possible role in Visna/Maedi virus infection.

This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression.

MHC class II DRB1 gene polymorphism in the pathogenesis of Maedi-Visna and pulmonary adenocarcinoma viral diseases in sheep.

Ovine pulmonary adenocarcinoma (OPA) and Maedi-Visna (Maedi) are two chronic respiratory diseases of retroviral origin which occur worldwide. It is known that different host genetic factors influence the outcome of viral infections. To determine if variation in the Mhc-DRB1 gene was associated with progression to these ovine diseases, sheep lungs with and without OPA and Maedi lesions were collected. A sequence-based method was applied and 40 different alleles were detected in the sample analysed. In the allele-by-allele association analysis, allele DRB1*0325 had a significant association with susceptibility to Maedi (P = 0.045). For OPA, DRB1*0143 and DRB1*0323 were significantly associated with susceptibility (P = 0.024 and P = 0.029), and allele DRB1*0702 was significantly associated with resistance (P = 0.012). Based on these results, the Mhc-DRB1 alleles were classified by effect in three categories-susceptible (S), resistant (R) and neutral (N)-and animals were reassigned the genotypes as S/S, S/R, S/N, R/R, R/N and N/N. In a second analysis, penalised logistic regression models including a flock effect were run. In Maedi, significant association was detected for the N/S heterozygote (P = 0.0007), but not for the S/S homozygote, probably as a result of the low number of S/S animals. In OPA, association was detected for both the S/S and R/R homozygotes (P = 0.005 and P = 0.047). This allele grouping method may be applied in association studies with highly variable genes. This is the first study demonstrating significant associations between sheep Mhc-DRB1 alleles and susceptibility to OPA and Maedi. Therefore, both diseases are suitable candidates for more comprehensive genetic studies.

Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1.

Previous studies initiated defining the role of host genetics in influencing the outcome of exposure to ovine progressive pneumonia virus. However, specific genes influencing host control of virus replication and disease progression have not been identified. This study, using 383 ewes of the Columbia, Polypay, and Rambouillet breeds, tested the hypothesis that host control of OPPV as measured by provirus levels in the peripheral blood associates with certain breeds and MHC class II Ovis aries (Ovar)-DRB1 expressed alleles. Rambouillet ewes were less likely to have measurable provirus levels as compared to Columbia ewes at ages 5 and 6 (P value < 0.02), and they exhibited lower provirus levels when compared to both Columbia and Polypay ewes of the same ages (P value < 0.05). The presence of DRB1*0403- or DRB1*07012-expressed alleles were significantly associated (P value = 0.019 and 0.0002, respectively) with lower OPP provirus levels but only were only found in 11% of the ewe flock. Analysis of each segregating amino acid in the beta1 domain of DR beta-chain revealed that amino acids Y31, T32, N37, T51, Q60, or N74 significantly associated (P value range = 0.0003-0.018) with lower OPP provirus levels, whereas amino acids H32, A38, or I67 associated (P value range = 0.013-0.043) with higher OPP provirus levels. These results suggest that Ovar-DRB1 contributes as one host genetic factor that controls OPP provirus levels, but does not fully account for the breed-specific OPP proviral differences.

Mutational analysis of a principal neutralization domain of visna/maedi virus envelope glycoprotein.

We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.

Development of a candidate DNA vaccine against Maedi-Visna virus.

DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a beta-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut(24)), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut(24) and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut(24) plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal.

Serum containing ovine IgG2 antibody specific for maedi visna virus envelope glycoprotein mediates antibody dependent cellular cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant GST:SUenv fusion protein in complete Freund's adjuvant produced an antibody response which contained IgG1 and IgG2 antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo may contribute to the persistence of MVV-infected macrophages in vivo.

Maedi-visna virus and its relationship to human immunodeficiency virus.

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.

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

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.

Differential levels of mRNAs for cytokines, the interleukin-2 receptor and class II DR/DQ genes in ovine interstitial pneumonia induced by maedi visna virus infection.

The relative levels of selected cytokine, interleukin-2 receptor, class II DR and DQ RNAs, and maedi visna virus (MVV) RNA were measured by reverse-transcriptase polymerase chain reaction (RT-PCR) in the lungs of sheep with natural maedi visna virus infection (n = 8) and a group of age/sex/breed-matched MVV seronegative sheep (n = 4). These animals were divided into two groups, irrespective of serostatus, according to the severity of lymphocytic interstitial pneumonia. The severity of lung lesions was determined by clinical sign, lung weight, and lesion sore in the lungs measured by three pathologic parameters. Sheep with lung lesions showed hyperelevated levels of granulocyte-macrophage colony-stimulating factor upregulated gamma-interferon, interleukin 2 receptor, and interleukins 1 beta, 4, and 10 mRNAs. Class II mRNAs were found not to be elevated in the lungs of sheep with lung lesions. Tumor necrosis factor alpha and transforming growth factor beta 1 mRNA levels were similar in all sheep lungs studied. We discuss the major roles played by granulocyte-macrophage colony-stimulating factor and type 2 cytokines in the pathogenesis of this disease and the possible stimulation of the production of these cytokines by viral surface glycoproteins.

Expression of the SU glycoprotein of maedi visna virus in baculovirus.

The envelope glycoprotein, gp 135, of the ovine lentivirus maedi visna virus is the main target for a specific antibody response in vivo, however, little is known about the specific regions of gp 135 which elicit this response. Research on the function of gp 135 has been hampered by the lack of reagents to study such structure/function relationships. We have used a baculovirus expression system to express gp 135 lacking the viral signal sequence. This recombinant protein is glycosylated and recognised by immune sera from clinically affected animals.

The phenotype and phagocytic activity of macrophages during maedi-visna virus infection.

Macrophages from maedi-visna virus (MVV) infected sheep have been shown to have an activated phenotype from sites of lesions in vivo. Here we have looked at the direct effect of virus infection on macrophage phenotype and activity in vitro by flow cytometry. There was no significant difference in the expression of several surface markers (CD4, CD8, MHC Class I, MHC Class II, lymphocyte function associated antigen(LFA)-1 and LFA-3) on monocyte-derived macrophages (MDM) by 5 days post MVV infection. In contrast the phagocytic activity of MVV-infected MDM for the yeast Candida utilis and erythrocytes was decreased by 5 days p.i. although the surface binding of erythrocytes was not affected. Interestingly, an activated phenotype was seen on alveolar macrophages (AM) from sheep with maedi (surface expression of MHC Class I, Class II and LFA-1 was increased), but there was no difference in the binding and phagocytosis of erythrocytes by these cells. However the binding and phagocytosis of the bacterium, Pasteurella hemolytica was increased with AM from MVV-infected sheep without lesions. Similarly there was no significant difference in the phagocytic and erythrocyte rosetting activity between fresh monocytes from MVV-infected and uninfected control sheep. Therefore the phenotype of macrophages taken from sites of lesions caused by MVV does not correspond to a direct effect by the virus on these cells or to particular activities of the macrophages.

Circulating cytotoxic T lymphocyte precursors in maedi-visna virus-infected sheep.

Maedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.

Early events in the experimental interstitial lung disease induced in sheep by the Visna-maedi virus.

Visna-maedi virus is a lentivirus closely related to the human immunodeficiency virus type I (HIV-I). During spontaneous infection of sheep by Visna-maedi virus an interstitial lung disease is observed. It is characterized by an alveolitis, peribronchovascular lymphoid nodules, alveolar wall thickening and myomatosis. In order to decipher the pathology of this lentiviral infection we have induced this disease in colostrum-deprived newborn lambs.

Early events in immune evasion by the lentivirus maedi-visna occurring within infected lymphoid tissue.

Infections caused by lentiviruses, including human immunodeficiency virus, are characterized by slowly progressive disease in the presence of a virus-specific immune response. The earliest events in the virus-host interaction are likely to be important in determining disease establishment and progression, and the kinetics of these early events following lentiviral infection are described here. Lymphatic cannulation in the sheep has been used to monitor both the virus and the immune response in efferent lymph after infection of the node with maedi-visna virus (MVV). Viral replication and dissemination could be detected and consisted of a wave of MVV-infected cells leaving the node around 9 to 18 days postinfection. No cell-free virus was recovered despite the fact that soluble MVV p25 was detected in lymph plasma. The maximum frequency of MVV-infected cells was only 11 in 10(6) but over the first 20 days of infection amounted to greater than 10(4) virus-infected cells leaving the node. There was a profound increase in the output of activated lymphoblast from the lymph nodes of infected sheep, characterized by an increased percentage of CD8+ lymphoblasts. All of the CD8+ lymphoblasts at the peak of the response expressed both major histocompatibility complex class II DR and DQ molecules but not interleukin-2 receptor (CD25). The in vitro proliferative response of efferent lymph cells existing the node after challenge with MVV to both recombinant human interleukin-2 and the mitogen concanavalin A was decreased between days 8 and 16 postinfection, and a specific proliferative response to MVV was not detected until after day 15. Despite the high level of CD8+ lymphoblasts in efferent lymph, direct MVV-specific cytotoxic activity was demonstrated in only one of the five MVV-challenged sheep. MVV-specific antibody responses, including neutralization and MVV p25 immune complexes in efferent lymph, were detectable during the major period of virus dissemination. The relationship of these findings to the evasion of the host's acute immune response by MVV is discussed.

Phenotypic analysis of cells in bronchoalveolar lavage fluid and peripheral blood of maedi visna-infected sheep.

A phenotypic analysis of bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) cells in maedi visna virus (MVV)-infected sheep has been performed. The differential cell count in BALF from MVV-infected animals was characterized by a significant increase (P < 0.05) in lymphocytes and neutrophils. Lymphocyte phenotyping in BALF from MVV-infected sheep showed a significant decrease (P < 0.05) of CD4+ cells, a significant increase (P < 0.05) of CD8+ cells and a significant inversion (P < 0.001) of the CD4+/CD8+ ratio. CD5+ lymphocytes were also significantly decreased (P < 0.05). Gamma delta T cells and B cells did not differ significantly when compared with the controls. No correlation was observed between BALF and PB lymphocyte phenotypes. BALF macrophages from MVV-infected animals showed increased MHC class II expression and BALF lymphocytes from the same animals demonstrated up-regulation of LFA-1 and LFA-3 expression. These findings and their relationship with lentiviral pathogenesis are discussed.