Highly lytic and persistent lentiviruses naturally present in sheep with progressive pneumonia are genetically distinct.

Ovine and caprine lentiviruses share the capacity to induce slowly progressive and inflammatory diseases of the central nervous system (leukoencephalitis or visna), lungs (progressive pneumonia or maedi), and joints (arthritis) in their natural hosts. Studies on their replication indicated that ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) recently isolated in the United States establish persistent infection in ovine and caprine fibroblasts, whereas older prototype ovine lentiviruses such as Icelandic visna virus or American progressive pneumonia virus irreversibly lyse fibroblast cultures. Since all of the recent isolates were found to be persistent, Narayan et al. (J. Gen. Virol. 59:345-356, 1982) concluded that the highly lytic viruses were only tissue-culture-adapted strains. In the present report, we isolated new ovine lentiviruses from French sheep with naturally occurring progressive pneumonia which are either highly lytic (five isolates), as are the Icelandic strains of visna virus, or persistent (one isolate), as are CAEV or American persistent ovine lentiviruses. Protein and nucleic acid content analyses of these new highly lytic (type I) and persistent (type II) isolates indicated that type I and type II ovine lentiviruses were genetically distinct, type I and type II viruses being closely related to the Icelandic strains of visna virus and to CAEV, respectively. We conclude that (i) highly lytic ovine lentiviruses, such as the Icelandic prototype strains of visna virus and persistent lentiviruses more related to CAEV, are naturally present in the ovine species, and (ii) irreversible cell lysis induced by highly lytic viruses does not result from a tissue culture adaptation of field isolates that were originally persistent but is instead the consequence of a genetic content distinct from that of persistent viruses.     

Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.     

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.     

Antigenic variation in visna virus.

Two antigenic variants of visna virus were isolated sequentially from a single sheep inoculated with a plaque-purified strain of virus designated 1514. The genetically stable variants, LV1-1 and LV1-4, are of two classes: LV1-1 is partially neutralized by antibody to the inoculum strain 1514, while LV1-4 is not neutralized by antibody to 1514. The genetic mechanism responsible for generating the antigenic variants was investigated by comparing the chymotryptic and tryptic maps of the envelope glycoprotein gp135 and core polypeptides (p30, p16, p14), and by comparing the pattern of large oligonucleotides produced by digestion of the RNAs by T1 ribonuclease. We show that only the peptide maps of gp135 differ among strains, that the number of peptide fragments altered is small and that gp135 is the polypeptide that elicits neutralizing antibody. The maps of the RNAs are identical. We conclude that mutation in the glycoprotein gene rather than recombination is more probably responsible for antigenic variation, and speculate on the special aspects of visna virus replication relevant to this phenomenon.     

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.     

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.     

Serological definition of the lentivirus group of retroviruses.

The major polypeptides of visna viruses and other lentiviruses have been isolated and shown to be closely related if not identical in radioimmunoassays. By this criterion the lentiviruses form a distinct group of retroviruses unrelated to spuma viruses, mammalian and avian retroviruses that cause tumors, and unclassified retroviruses of cattle and horses. Two sera obtained from goats immunized with Mason-Pfizer monkey virus or squirrel monkey virus reacted with visna p30. Additional data suggest that this reaction represents infection of goats with a lentivirus or a new retrovirus closely related to the lentiviruses.     

Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A,an evolutionarily and structurally conserved protein,to degrade ovine and caprine APOBEC3 proteins

Mammals have co‐evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti‐viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core‐binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi–Visna virus [MVV]). However, the co‐evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif‐mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co‐factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co‐factor in degradation of ovine and caprine APOBEC3.     

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.     

Lack of neutralizing antibodies to caprine arthritis-encephalitis lentivirus in persistently infected goats can be overcome by immunization with inactivated Mycobacterium tuberculosis

The pathogenesis of the persistent progressive diseases of sheep (visna-maedi) and goats (arthritis-encephalitis) is dependent on continuous replication of the causative lentiviruses. One subgroup of these viruses, Icelandic visna virus, accomplishes this form of replication by undergoing antigenic mutation. Mutant viruses arising late in the infection escape neutralization by antibodies directed to the parental virus. In contrast, we show here that viruses obtained from persistently infected sheep and goats with natural disease in this country do not induce virus-neutralizing antibodies, although antibodies to virus core proteins were produced. The lack of neutralizing antibodies was not overcome by hyperimmunization of animals with concentrated preparations of live or inactivated virus. Thus, failure to produce these specific antibodies was not due to lack of sufficient antigen or interference with the immune response because of the ability of these viruses to infect macrophages. The hyporesponsive state, however, was overcome by immunization of animals with virus and large amounts of inactivated Mycobacterium tuberculosis. Induction of agglutinating and neutralizing antibodies by this method was probably due to a unique form of antigen processing by macrophages activated by M. tuberculosis. Neutralizing antibodies were produced for the first time against the caprine arthritis-encephalitis virus by this method. These antibodies have similar biological properties to those induced by Icelandic visna virus. They belong to the immunoglobulin G1 subclass, they are effective against a narrow range of caprine arthritis-encephalitis viruses, and they identify (for the first time) antigenic variants among these caprine agents.     

Replication and cytopathic effects of ovine lentivirus strains in alveolar macrophages correlate with in vivo pathogenicity.

Ruminant lentiviruses share genomic sequences and biologic properties with human immunodeficiency viruses. Four ovine lentivirus strains were assessed for cytopathic effects and virus replication. Lentivirus isolate H/24 produced high virus titers and lysis of synovial cells but replicated slowly and caused no fusion of alveolar macrophages. Lentivirus isolates 84/28 and 85/14 produced low virus titers, less syncytia, and limited or no cell lysis in synovial cells and macrophages. In contrast, ovine lentivirus isolate 85/34 produced early peak virus titers and caused rapid fusion and lysis of both macrophages and synovial cells. Ovine lentivirus isolates which were cytopathic for macrophages induced lymphoproliferative disease when inoculated into lambs.     

Isolation and partial characterization of ovine lentivirus in Czech Republic

The first isolation and partial characterization of ovine lentivirus in Czech Republic is described. The virus was isolated in a tissue culture system derived from plexus choroideus from sheep. The new isolate was compared with prototypic K1514 Maedi-Visna strain; these two viruses shared antigenic determinants as determined by serological testing. Both viral strains reacted in a PCR reaction with primers situated in thegag andpol gene. Based on similarities of growth characteristics, antigenic determinants and primer binding sites it can be concluded that the isolate OPM is an ovine lentivirus and is at least partly related to the prototypic Maedi-Visna strain K1514.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.     

Establishment of a novel ovine kidney cell line for isolation and propagation of viruses infecting domestic cloven-hoofed animal species

A sheep kidney-derived cell line, FLK-N3, was successfully established after serial (>100) passages. Persistent infection of this cell line with viruses and mycoplasma was not detected. The cells grew well and showed susceptibility to a wide variety of viruses derived from ovine, bovine, and porcine species, including orf virus, maedi visna virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine viral diarrhea viruses 1 and 2, bovine coronavirus, bovine respiratory syncytial virus, bovine enterovirus, suid herpesvirus 1, and porcine enterovirus. These results suggest that the FLK-N3 cell line could be useful for isolation and propagation of viruses that affect cloven-hoofed animals.     

Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein

The complete surface glycoprotein (SU) nucleotide sequences of three French isolates of caprine arthritis-encephalitis virus (CAEV) were determined and compared with those of previously described isolates: three American isolates and one French isolate. Phylogenetic analyses revealed the existence of four distinct and roughly equidistant evolutionary CAEV subtypes. Four conserved and five variable domains were identified in the SU. The fine specificities of antibodies produced against these domains during natural infection were examined using a pepscan analysis. Nine immunogenic segments were delineated throughout the conserved and variable domains of SU, two of them corresponding to conserved immunodominant epitopes. Antigenic determinants which may be involved in the immunopathogenic process induced by CAEV were identified. These results also provide sensitive and specific antigen peptides for the serological detection and differentiation of CAEV and visna/maedi virus infections.     

Caprine arthritis-encephalitis virus envelope surface glycoprotein regions interacting with the transmembrane glycoprotein: structural and functional parallels with human immunodeficiency virus type 1 gp120

A sequence similarity between surface envelope glycoprotein (SU) gp135 of the lentiviruses maedi-visna virus and caprine arthritis-encephalitis virus (CAEV) and human immunodeficiency virus type 1 (HIV-1) gp120 has been described. The regions of sequence similarity are in the second and fifth conserved regions of gp120, and the similarity is highest in sequences coinciding with beta-strands 4 to 8 and 25, which are located in the most virion-proximal region of the gp120 inner domain. A subset of this structure, formed by gp120 beta-strands 4, 5, and 25, is conserved in most or all lentiviruses. Because of the orientation of gp120 on the virion, this highly conserved virion-proximal region of the gp120 core may interact with the transmembrane glycoprotein (TM) together with the amino and carboxy termini of full-length gp120. Therefore, interactions between SU and TM of lentiviruses may be structurally related. Here we tested whether the amino acid residues in the putative virion-proximal region of CAEV gp135 comprising putative beta-strands 4, 5, and 25, as well as its amino and carboxy termini, are important for stable interactions with TM. An amino acid change at gp135 position 119 or 521, located in the turn between putative beta-strands 4 and 5 and near beta-strand 25, respectively, specifically disrupted the epitope recognized by monoclonal antibody 29A. Thus, similar to the corresponding gp120 regions, these gp135 residues are located in close proximity to each other in the folded protein, supporting the hypothesis of a structural similarity between the gp120 virion-proximal inner domain and gp135. Amino acid changes in the amino- and carboxy-terminal and putative virion-proximal regions of gp135 increased gp135 shedding from the cell surface, indicating that these gp135 regions are involved in interactions with TM. Our results indicate structural and functional parallels between CAEV gp135 and HIV-1 gp120 that may be more broadly applicable to the SU of other lentiviruses.