First molecular characterization of visna/maedi viruses from naturally infected sheep in Turkey.

Recent worldwide serological and genetic studies of small ruminant lentiviruses (SRLV) have led to the description of new genotypes and the development of new diagnostic tests. This study investigated the detection and molecular characterization of visna/maedi virus (VMV) infection in serum and blood samples from pure and mixed sheep breeds acquired from different regions in Turkey using ELISA and PCR techniques. The prevalence of VMV was 67.8 % by ELISA and/or LTR-PCR with both assays showing a medium level of agreement (kappa: 0.26; ± 0.038 CI). Positivity of VMV in sheep increased according to the age of the animal, although PCR positivity was higher than ELISA in young individuals. Phylogenetic analysis of 33 LTR sequences identified two distinct clades that were closely related to American and Greek LTR sequences. Phylogenetic analysis of 10 partial gag gene sequences identified A2, A3, A5, A9, A11 subtypes of genotype A SRLVs. In vitro culture of all isolates in fetal sheep lung cells (FSLC) showed a slow/low phenotype causing less or no lytic infection compared with infection with the WLC-1 American strain characterized by a rapid/highly lytic phenotype. Phylogenetic analysis revealed that Turkish VMV sequences preceded the establishment of American or Greek strains that were associated with the migration of sheep from the Middle East to Western Europe several centuries ago. This is the first study that describes Turkish VMV sequences with the molecular characterization of LTR and gag genes, and it strongly suggests that SRLV-genotype A originated in Turkey.

Perivascular inflammatory cells in ovine Visna/maedi encephalitis and their possible role in virus infection and lesion progression.

We examined the distribution in the perivascular spaces of Visna/maedi antigen, T cells (CD3+, CD4+ and CD8+), B cells and macrophages by immunohistochemistry in 22 natural cases of Visna/maedi encephalitis. Sheep showed lymphocytic or histiocytic lesions. In mild lymphocytic lesions, the viral antigen was detected in perivascular cuffs where CD8+ T cells predominated, but in severe lymphocytic lesions, sparse antigen was identified, and CD8+/CD4+ T cells appeared in a similar proportion in multilayer perivascular sleeves. In histiocytic lesions, vessels were surrounded by macrophages with abundant viral antigen, with CD8+/CD4+ T cells and B cells in the periphery. These results could reflect different stages of virus neuroinvasion and clarify the neuropathogenesis of Visna/maedi encephalitis.

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.

Visna/Maedi virus genetic characterization and serological diagnosis of infection in sheep from a neurological outbreak.

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.

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.

Vaccinia and other viruses with available vaccines show marked homology with the HIV-1 envelope glycoprotein: the prospect of using existing vaccines to stem the AIDS pandemic.

Cross-reactive immunity occurs when infection with or vaccination against one virus protects against another related family member. A search for homologues of the HIV-1 envelope glycoprotein revealed that it is composed of thousands of intercalating and overlapping viral matches of pentapeptide or longer gapped consensi, belonging to over 70% of the currently sequenced virome, infecting all kingdoms from bacteria to man. It was also highly homologous to proteins from the Visna/Maedi and other ovine viruses, while other proteins (nef/tat/gag/pol) were homologous to proteins from the equine infectious anaemia virus and HTLV-2/HTLV-3 viruses. This phenomenon suggests that horizontal gene transfer from coinfecting RNA and DNA viruses to retroviruses is extensive, providing a route for the subsequent insertion of non-retroviral genes into human and other genomes via retroviral integration. This homology includes all viruses for which vaccines already exist. Cross-reactive immunity may be operative in AIDS, as Vaccinia vaccination decreases viral replication in HIV-1 infected patients' cells, for the CCR5 tropic form. Measles, Dengue virus, or GB virus C infections also decrease the HIV-1 viral load. A resumption of Vaccinia/smallpox vaccination might be expected to have a significant effect on the AIDS pandemic, and a careful study of the potential uses of other existing viral and bacterial vaccines merits close attention. This phenomenon may also be relevant to other recalcitrant viruses, bacteria, and parasites for which no vaccine exists and the armory of existing vaccines may have a role to play in diseases other than those for which they were designed.

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.

Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus.

RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes.

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

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.

Role of alveolar macrophages in respiratory transmission of visna/maedi virus.

A major route of transmission of Visna/maedi virus (VMV), an ovine lentivirus, is thought to be via the respiratory tract, by inhalation of either cell-free or cell-associated virus. In previous studies, we have shown that infection via the lower respiratory tract is much more efficient than via upper respiratory tissues (T. N. McNeilly, P. Tennant, L. Lujan, M. Perez, and G. D. Harkiss, J. Gen. Virol. 88:670-679, 2007). Alveolar macrophages (AMs) are prime candidates for the initial uptake of virus in the lower lung, given their in vivo tropism for VMV, abundant numbers, location within the airways, and role in VMV-induced inflammation. Furthermore, AMs are the most likely cell type involved in the transmission of cell-associated virus. In this study, we use an experimental in vivo infection model that allowed the infection of specific segments of the ovine lung. We demonstrate that resident AMs are capable of VMV uptake in vivo and that this infection is associated with a specific up-regulation of AM granulocyte-macrophage colony-stimulating factor mRNA expression (P < 0.05) and an increase in bronchoalveolar lymphocyte numbers (P < 0.05), but not a generalized inflammatory response 7 days postinfection. We also demonstrate that both autologous and heterologous VMV-infected AMs are capable of transmitting virus after lower, but not upper, respiratory tract instillation and that this transfer of virus appears not to involve the direct migration of virus-infected AMs from the airspace. These results suggest that virus is transferred from AMs into the body via an intermediate route. The results also suggest that the inhalation of infected AMs represents an additional mechanism of virus transmission.

Salmonella infection of afferent lymph dendritic cells.

The interactions of Salmonella enterica subspecies I serotype Abortusovis (S. Abortusovis) with ovine afferent lymph dendritic cells (ALDCs) were investigated for their ability to deliver Maedi visna virus (MVV) GAG p25 antigens to ALDCs purified from afferent lymph. Salmonellae were found to enter ALDC populations by a process of cell invasion, as confirmed by electron and confocal microscopy. This led to phenotypical changes in ALDC populations, as defined by CD1b and CD14 expression. No differences in the clearance kinetics of intracellular aroA-negative Salmonella from CD1b+ CD14lo and CD1b+ CD14(-) ALDC populations were noted over 72 h. ALDCs were also shown to present MVV GAG p25 expressed by aroA-negative S. Abortusovis to CD4+ T lymphocytes. Thus, the poor immune responses that Salmonella vaccines elicited in large animal models compared with mice are neither a result of an inability of Salmonella to infect large animal DCs nor an inability of these DCs to present delivered antigens. However, the low efficiency of infection of ALDC compared with macrophages or monocyte-derived DCs may account for the poor immune responses induced in large animal models.

Association of CD80 and CD86 expression levels with disease status of Visna/Maedi virus infected sheep.

In small ruminant lentivirus infections, cellular immune responses are diminished in clinically affected animals. The underlying mechanisms for this are unknown. In this study, we tested the hypothesis that alterations in expression of the co-stimulatory molecules B7-1 and B7-2 are involved in infections with Visna/Maedi virus (VMV), the prototype lentivirus of sheep. We studied B7 expression levels ex vivo in peripheral blood mononuclear cells (PBMCs), determining B7 RNA levels by real time reverse transcriptase polymerase chain reaction in asymptomatic as well as clinically affected VMV-seropositive sheep. The levels of both B7 molecules were increased in VMV-seropositive asymptomatic sheep. However, in VMV clinically affected sheep, the level of CD80 (but not CD86) was low compared with the level in uninfected sheep (p < 0.05). CD80 and CD86 RNA levels were associated with the ability of PBMCs to respond to VMV gag antigens (p14, p17, and p25) by proliferation, with most seropositive asymptomatic sheep showing positive proliferative responses but clinically affected sheep showing no response. The response to p25 in clinically affected animals was increased by the addition of interleukin-2 to the cultures. Decreased recall responses to unrelated antigens (assessed by production of interferon-gamma) were also found in clinically affected sheep. Thus, among seropositive sheep, decreased B7-1 (CD80) RNA levels and diminished antigen-specific cellular immune responses in PBMCs point to a VMV disease status, whereas increased CD80 and CD86 levels and augmented cellular responses are linked to asymptomatic infection.

Vaccination of sheep with Maedi-visna virus gag gene and protein, beneficial or harmful?

In spite of intense efforts no vaccine is yet available that protects against lentiviral infections. Sheep were immunised eight times over a period of 2.5 years with the maedi-visna (MVV) gag gene on two different vectors, 2 sheep with VR1012-gag-CTE and 2 sheep with pcDNA3.1-gag-CTE. All sheep responded to some of the mature MVV Gag proteins in Western blot (WB). Three of them responded to the virus in lymphocyte proliferation test. The sheep received a boost with recombinant Gag protein resulting in elevated antibody response. However, when they were challenged intratracheally with MVV they all became immediately infected as judged by a strong rise in antibody titer and virus isolation from blood. It is therefore clear that the vaccination gave no protection. It is even possible that it facilitated infectivity since virus was isolated earlier from all the vaccinated sheep than from any of the unvaccinated sheep infected in the same way with the same dose.

Expression of the gp150 maedi visna virus envelope precursor protein by mammalian expression vectors.

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.

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.

Detection of maedi-visna virus in the liver and heart of naturally infected sheep.

Maedi-visna virus (MVV) in sheep, which infects mainly cells of the monocyte/macrophage lineage, produces changes in the lung, mammary gland, brain and joints. In this study, however, the liver and heart of six naturally infected sheep were examined for the presence of the virus. MVV proviral DNA was demonstrated by polymerase chain reaction (PCR) analysis, and immunohistochemical examination revealed viral antigens in the cytoplasm of hepatocytes and cardiac myocytes. Although histopathological examination showed mild to moderate, chronic lymphocytic cholangiohepatitis and myocarditis and the presence of small lymphoid aggregates, the typical maedi lymphoproliferative lesions (lymphoid follicle-like structures of considerable size with germinal centres) were not seen in the liver and heart. These novel findings suggest that, although the macrophage is the main cell for productive viral replication, the liver and heart represent additional MVV targets.

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.

Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes.

Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection. This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens.

Immune response to individual maedi-visna virus gag antigens.

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individual gag antigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+ T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all three gag antigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinant gag antigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+ T lymphocytes. All three gag antigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.