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.

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.

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.

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo.

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.

Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen.

The small ruminant lentiviruses, namely caprine arthritis encephalitis virus (CAEV) and Maedi Visna virus (MVV) are grown currently in secondary synovial membrane cells. Primary and secondary cell cultures are sometimes difficult to obtain and support a low number of passages and, therefore, permissive cell lines are needed. A transformed cell line was obtained by transfection of ovine synovial membrane secondary cell culture with a plasmid containing the SV40 large T antigen gene. The transformed cell culture described in this paper showed a higher growth rate and a more homogenous population of fibroblast-like cells when compared to the original ovine synovial membrane secondary cell cultures. Karyotype analysis has indicated the induction of many random chromosome changes, leading to a decrease in chromosome number. The SV40 DNA was detected in the nucleus and in the cytoplasm of transformed cells. The putative expression of large T antigen was presumed by the detection of the corresponding mRNA by PCR. Finally, the transformed ovine synovial membrane cells were shown to be permissive to small ruminant lentiviruses, and these are suggested as a cell line for in vitro isolation and propagation of these viruses.

Identification and visualization of the dimerization initiation site of the prototype lentivirus, maedi visna virus: a potential GACG tetraloop displays structural homology with the alpha- and gamma-retroviruses.

Dimerization of retroviral genomic RNA is essential for efficient viral replication and is mediated by structural interactions between identical RNA motifs in the viral leader region. We have visualized, by electron microscopy, RNA dimers formed from the leader region of the prototype lentivirus, maedi visna virus. Characterization by in vitro assays of the domains responsible for this interaction has identified a 20 nucleotide sequence that functions as the core dimerization initiation site. This region is predicted to form a GACG tetraloop and therefore differs significantly from the kissing loop palindromes utilized to initiate dimerization in primate lentiviruses. The motif is strongly conserved across the ovine and caprine lentiviruses, implying a critical functional role. Furthermore, the proposed GACG tetraloop exhibits marked structural homology with similar structural motifs present in the leader regions of the alpha- and gamma-retroviruses, and the maedi visna virus dimer linkage region is capable of forming heterodimeric species with the Moloney murine leukemia virus Psi domain. This may be indicative of commonality of origin of the two viruses or convergent evolution.

Genomic characterization of a slow/low maedi visna virus.

The complete genomic sequence of a sheep lentivirus isolate that presents a slow/low phenotype in vitro has been determined. The virus, designated P1OLV, was isolated from lung cells of a naturally infected sheep in Portugal. Three overlapping DNA fragments amplified by PCR, and encompassing the entire viral genome were cloned and sequenced. This isolate has an overall similarity of approximately 80% with the K1514 Maedi Visna virus (MVV) and approximately 70% with the caprine arthritis encephalitis virus (CAEV) Co strain. Phylogenetic analysis based on SU and RT nucleotide sequences grouped P1OLV with previously reported ovine MVV. To determine the virus replication rate, sheep choroid plexus (SCP) and lung cells, macrophages (MØ), and goat synovial membrane (GSM) cells were inoculated with either P1OLV or with the lytic North American strain WLC-1. Viral RNA in culture supernatants was measured by one-tube real time quantitative RT-PCR. Significant differences were observed between the replication rates of the two viruses, with WLC-1 growing rapidly and to high levels in all the cells tested, while P1OLV replicated more slowly and to lower levels inducing persistent infections in lung and SCP cells. The U3 region of the LTR of P1OLV lacks the sequence repeats that are present in the LTRs of WLC-1 and MVV prototype K1514 and that contain additional binding sites for the AML(vis) transcriptional factor. To evaluate the contribution of LTR in the virus replication rate in vitro, we measured the basal activity of the promoter from P1OLV and WLC-1 in a luciferase-driven gene expression assay and lower levels of expression were achieved for P1OLV. The genetic and biological properties of P1OLV will be useful for the study of virus transcriptional factors and genes that may be responsible for the slow/low phenotype.

Construction and characterization of a recombinant ovine lentivirus carrying the optimized green fluorescent protein gene at the dUTPase locus.

AdUTPase gene ( du) deleted ovine lentivirus (OvLV(Deltadu)) mutant, derived from Visna/maedi virus (VMV) molecular clone KV1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein ( egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV(Deltadu-egfp). OvLV(Deltadu) reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p < 0.05). By contrast, OvLV(Deltadu-egfp) RT activity and virus titer were lower than for KV1772 and OvLV(Deltadu) (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV(Deltadu) and OvLV(Deltadu-egfp) (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV(Deltadu-egfp) at a low level. OvLV(Deltadu-egfp) retained egfp after 10 passages in cell culture.OvLV(Deltadu-egfp) was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV(Deltadu-egfp)-inoculated lambs, but by contrast to the in vitro experiments OvLV(Deltadu-egfp) lost the insert. Priming with OvLV(Deltadu-egfp) did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV(Deltadu-egfp)-primed lambs. OvLV serum antibody titers increased steadily in OvLV(Deltadu-egfp)-inoculated lambs, but in a lamb from which OvLV(Deltadu-egfp) was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV(Deltadu-egfp) is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV(Deltadu-egfp) as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.

Maedi-visna virus detection in ovine third eyelids.

Maedi-visna is a systemic disease of sheep caused by a lentivirus, maedi-visna virus (MVV), which mainly affects the lungs and central nervous system but may also affect the mammary glands, joints and other tissues. The aim of the present study was to determine whether the third eyelid was affected in cases of systemic infection. Third eyelid and lung samples from sheep naturally infected with maedi were used. Total DNA was extracted from paraffin-wax-embedded tissues, and a nested polymerase chain reaction (PCR) was performed to amplify MVV proviral DNA. The samples were also tested by in-situ PCR and immunohistochemical methods specific for the detection of MVV proviral DNA and p25, respectively. All sheep showed moderate to severe chronic lymphoproliferative inflammation in the third eyelids. Products of the expected size were obtained by PCR from both lung and third eyelid tissue. In the nictitating membrane, MVV proviral DNA was detected in situ within macrophages, and glandular, ductal and surface epithelia. Immunohistochemistry demonstrated that the infection was productive. Taken together, these results indicate that the third eyelid may represent a target for natural MVV infection and may play a role in disease transmission.

Implication of caspases during maedi-visna virus-induced apoptosis.

Maedi-visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep. In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.

Inhibition of nitric oxide enhances ovine lentivirus replication in monocyte-derived macrophages.

Ovine lentivirus (OvLV) also known as maedi-visna virus, infects and replicates primarily in macrophages. This investigation examined the role of nitric oxide in the replication of OvLV in cultured macrophages. Peripheral blood mononuclear cells were collected from OvLV-free sheep and cultured in Teflon coated flasks at a high concentration of lamb serum. The cells were subsequently infected with OvLV strain 85/34. OvLV replication was assessed under different experimental treatments by comparison of reverse transcriptase (RT) activity in culture supernatant. Cultures that were treated with exogenous nitric oxide via S-nitroso-acetylpenicillamine did not have altered levels of RT activity compared to cultures treated with the inactive control compound, acetylpenicillamine. However, blockage of nitric oxide production by treatment with aminoguanidine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), led to a significant rise in RT activity. This rise in RT activity was partially reversed in aminoguanidine treated cultures by L-arginine, the normal substrate for iNOS. Finally, the number of viral antigen producing cells was also quantified after aminoguanidine treatment and found to be significantly higher than untreated cultures. Collectively, these results indicate that nitric oxide is a negative regulator of OvLV replication in macrophages.

Differential receptor usage of small ruminant lentiviruses in ovine and caprine cells: host range but not cytopathic phenotype is determined by receptor usage.

The ovine maedi-visna (MVV) and caprine arthritis-encephalitis (CAEV) small ruminant lentiviruses (SRLV) exhibit differential species tropism and cytopathic effects in vitro. Icelandic MVV-K1514 is a lytic SRLV which can infect cells from many species in addition to ruminants, whereas a lytic North American MVV strain (85/34) as well as nonlytic MVV strain S93 and CAEV can infect only ruminant cells. In the present study, we determined if differential receptor usage in sheep and goat cells is the basis of differential species tropism or cytopathic phenotype of SRLV. Infection interference assays in sheep and goat synovial membrane cells using pseudotyped CAEV vectors showed that North American MVV strains 85/34 and S93 and CAEV use a common receptor (SRLV receptor A), whereas MVV-K1514 uses a different receptor (SRLV receptor B). In addition, human 293T cells expressing CAEV but not MVV-K1514 envelope glycoproteins fused with a goat cell line persistently infected with MVV-K1514, indicating that MVV-K1514 does not use SRLV receptor A for cell-to-cell fusion. Therefore, our results indicate that the differential species tropism of SRLV is determined by receptor usage. However, receptor usage is unrelated to cytopathic phenotype.

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.

Adenine and deazaadenine nucleoside and deoxynucleoside analogues: inhibition of viral replication of sheep MVV (in vitro model for HIV) and bovine BHV-1.

A series of N(6)-cycloalkyl-2',3'-dideoxyadenosine derivatives has been prepared by coupling of 2,6-dichloropurine to protected 2,3-dideoxyribose, followed by reaction with appropriate cycloalkylamines. Synthesized compounds, along with other purine nucleoside analogues previously synthesized in our laboratory, have been tested for their antiviral activity against Bovine herpesvirus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV), the latter being an in vitro and in vivo model of Human Immunodeficiency Virus (HIV). All compounds showed good antireplicative activity against MVV, with the N(6)-cycloheptyl-2',3'-dideoxyadenosine (5b) being the most active [effective concentration (EC(50)) causing 50% reduction of cytopatic effects (CPE)=27 nM]. All compounds showed also a from low to very low cell toxicity, resulting in a cytotoxic dose 50 (CD(50))/EC(50) ratio in some cases higher than 1000.

Visna virus-induced activation of MAPK is required for virus replication and correlates with virus-induced neuropathology.

It is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression of gag and env, but not rev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.

Functional murine leukemia virus vectors pseudotyped with the visna virus envelope show expanded visna virus cell tropism.

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.

Restricted species tropism of maedi-visna virus strain EV-1 is not due to limited receptor distribution.

The distribution of receptors for maedi-visna virus (MVV) was studied using co-cultivation assays for virus fusion and PCR-based assays to detect the formation of virus-specific reverse transcription products after virus entry. Receptors were present on cell lines from human, monkey, mouse, chicken, quail, hamster and ovine sources. Thus, the distribution of the receptor for MVV is more similar to that of the amphotropic type C retroviruses than to that of other lentiviruses. The receptor was sensitive to proteolysis by papain, but was resistant to trypsin. Chinese hamster ovary (CHO) and lung cells (V79 TOR) did not express functional receptors for MVV. The receptor was mapped to either chromosome 2 or 4 of the mouse using somatic cell hybrids. This allowed several candidates (e.g. MHC-II, CXCR4) that have been proposed for the MVV receptor to be excluded.

Infection of dendritic cells by the Maedi-Visna lentivirus.

The early stages of lentivirus infection of dendritic cells have been studied in an in vivo model. Maedi-visna virus (MVV) is a natural pathogen of sheep with a tropism for macrophages, but the infection of dendritic cells has not been proven, largely because of the difficulties of definitively distinguishing the two cell types. Afferent lymphatic dendritic cells from sheep have been phenotypically characterized and separated from macrophages. Dendritic cells purified from experimentally infected sheep have been demonstrated not only to carry infectious MVV but also to be hosts of the virus themselves. The results of the in vivo infection experiments are supported by infections of purified afferent lymph dendritic cells in vitro, in which late reverse transcriptase products are demonstrated by PCR. The significance of the infection of afferent lymph dendritic cells is discussed in relation to the initial spread of lentivirus infection and the requirement for CD4 T cells.

Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages.

Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.

Visna-maedi virus induces interleukin-8 in sheep alveolar macrophages through a tyrosine-kinase signaling pathway.

The mechanisms leading to the severe lung damage seen in some sheep naturally infected with the visna-maedi virus, and to pulmonary lesions in other lentiviral diseases, appear to involve the recruitment of large numbers of uninfected inflammatory cells. Only a few alveolar macrophages from experimentally infected lambs express virus, but high levels of interleukin (IL)-8 mRNA are present in the macrophage population. In vitro infection with visna-maedi virus at low multiplicity of alveolar macrophages from uninfected sheep also strongly induced the expression of IL-8 mRNA and the accumulation of IL-8 in the extracellular medium. An initial peak of IL-8 mRNA expression at 3 or 6 h after infection was followed by a fall, then a more persistent expression lasting at least 48 h after infection. The early peak was accompanied by expression of mRNA for IL-1beta, and a possible rise in tumor necrosis factor alpha (TNFalpha) mRNA, although this was frequently elevated in uninfected ovine alveolar macrophages. Interestingly, these events occurred identically in cells treated with non-infectious heat-treated virus, suggesting that interaction between viral components and cellular membrane receptors could suffice for both early and late IL-8 induction. The level of IL-8 mRNA induced by treatment with live or inactivated virus could be severely reduced by pretreatment of the macrophages with genistein but not with staurosporine, suggesting the involvement of a tyrosine-kinase signaling pathway. The early induction of IL-1beta and possibly of TNFalpha may explain the occurrence of a later persistent expression of IL-8 mRNA through an autocrine mechanism.