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Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of DeltavpuSHIV(PPC), a live virus vaccine derived from SHIV(PPC). Macaques were administered two inoculations of DeltavpuSHIV(PPC), three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.

Chemokines and cytokines play a critical role in HIV infection, serving both to modulate virus replication and to recruit target cells to the site of infection. Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for a wide variety of cells, is secreted by macrophages. Since macrophages are the target cells for lentiviral infection in the brain and PDGF is a known inducer of macrophage chemoattractant protein-1 (MCP)-1, a potent chemokine closely associated with HIV encephalitis, we investigated the association of PDGF-B chain (PDGF-B) with encephalitis in macaques caused by simian human immunodeficiency virus (SHIV), a chimera of HIV and SIV. Northern blot analysis confirmed elevated expression of PDGF-B chain mRNA in the brains from encephalitic macaques. Validation of these in vivo studies was confirmed in rhesus macrophage cultures infected with SHIV(KU2) in which we demonstrated heightened expression of PDGF-B chain mRNA. Nuclear run-off analysis established transcriptional up-regulation of PDGF-B chain in virus-inoculated macrophage cultures. Reciprocally, addition of exogenous PDGF enhanced virus replication and MCP-1 expression in these cells. Inhibition of virus replication by tyrosine kinase inhibitor, STI-571, and by PDGF-B antisense oligonucleotides confirmed the specificity of the PDGF effect. Relevance of these findings was confirmed by analysis of archival brain tissue from SHIV encephalitic and non-encephalitic macaques for PDGF-B chain expression. PDGF-B chain protein expression was observed in the virus-infected cells in microglial nodules in the brains of SHIV-encephalitic macaques.

Neurological disease associated with lentiviral infection occurs mainly as a consequence of primary replication of the virus or a combination of the virus infection and replication of opportunistic pathogens in the central nervous system. Recent studies have shown that whereas the disease can be caused by CCR5 tropic viruses alone, its induction by CXCR4 (X4) tropic viruses occurred usually in association with infections caused by opportunistic pathogens and in the presence of a Th2 cytokine, interleukin (IL)-4. 1,2 Further, X4-mediated neurological disease developed preferentially in rhesus compared to pig-tailed macaques. Because macrophages are the target cells for lentiviral infection in the brain and because macrophage chemoattractant protein (MCP)-1 is one of the major chemokines that is closely associated with acquired immune deficiency syndrome (AIDS) dementia, we tested for correlations between MCP-1 production and virus tropism in macrophages from the two species of macaques. The studies showed that the higher susceptibility of rhesus macaques to X4 virus-mediated encephalitis correlated with heightened production of virus and MCP-1 in cultured macrophages from this species and that these effects were further enhanced with treatment with IL-4. However, the latter effect was restricted to macrophages infected with X4 viruses. IL-4 may therefore be a basic requirement for X4 viruses to cause central nervous system disease.

To evaluate the vaccine potential of SHIVs attenuated by deletion of viral accessory genes, seven rhesus macaques were sequentially immunized with Delta vpu Delta nefSHIV-4 (vaccine-I) followed by Delta vpuSHIV(PPC) (vaccine-II). Despite the absence of virological evidence of productive infection with the vaccine strains, based on analysis of infectivity among peripheral blood mononuclear cells (PBMC) of the vaccinated animals, all seven animals developed binding as well as neutralizing antibodies against both vaccine-I and -II. The animals also developed vaccine virus-specific CTLs that recognized homologous as well as heterologous pathogenic SHIVs and SIV, and also soluble inhibitory factors that blocked the in vitro replication of the vaccine strains and different challenge viruses. Virus-specific cellular and humoral responses were sustained throughout a 58-week prechallenge period. To model aspects of natural transmission, the animals received a mucosal (rectal) challenge, with a mixture of three challenge viruses, SHIV(KU), SHIV(89.6)P, and SIV(mac)R71/17E. Two mock-vaccinated control animals inoculated with the same mixture of challenge viruses developed large numbers of infectious PBMC, high plasma viremia, and precipitous loss of CD4(+) T cells. The control animals did not develop any immune responses and succumbed to AIDS between 6 and 7 weeks postchallenge. All seven vaccinated animals became infected with challenge viruses as indicated by the presence of infectious cells in the PBMC and/or viral RNA in plasma. However, peak plasma viremia in vaccinates was two to nearly five logs lower than in the control animals and later plasma viral RNA became undetectable in all vaccinates. Vaccinated animals maintained normal CD4(+) T cell levels throughout the study. Challenge with pathogenic viruses caused massive anamnestic responses as determined by quantitation of virus-specific CD4(+) and CD8(+) T cells by intracellular IFN-gamma staining, and these cells persisted for at least 74 weeks. The study is still in progress and at this time DNA of SIV has become undetectable in lymph nodes of six of the seven vaccinates, SHIV(89.6)P in five of the seven, and SHIV(KU) in three of the seven animals.

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian–human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV PPC and Δ vpuΔnef SHIV PPC , to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. Δ vpuΔnef SHIV PPC maintained a limited phase of productive replication in the four animals, with no loss of CD4 + T cells, whereas SHIV PPC became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4 + T cell loss. The nef, LTR, and env of the SHIV PPC viruses underwent numerous mutations, compared to Δ vpuΔnef SHIV PPC . This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.

Previous reports from our lab had shown that sera obtained from SIV mac -infected animals neutralized SIV mac infectivity in CD4 + T cells but failed to protect monkey primary macrophages from infection with the virus. However, the antibodies could inhibit completion of the viral life cycle in the macrophages at the postentry stage(s). In this report we examined the mechanisms of the late effect of the antibodies. Using monoclonal antibodies (MAbs), we demonstrated that only antibodies to the SIV envelope protein (KK17 and KK42) but not antibody to the viral core protein (FA2) had the same inhibitory effect as that of the anti-SIV sera. To identify the stage of the viral replication cycle that was inhibited by anti-SIV antibodies in macrophages, we used various PCR techniques to study viral entry/reverse transcription (by amplifying the viral gag gene), viral genome nuclear transport (by amplifying 2-LTR circular forms), viral integration (by Alu-PCR assay), and viral protein expression (by RIPA). We found that in macrophage cultures inoculated with SIV mac 251 that were preincubated with antienvelope MAbs, viral DNA was detected at 8 h postinoculation but the 2-LTR circular forms and integrated viral DNAs were undetectable, and viral proteins were not expressed in these infected macrophages. These results strongly suggested that anti-SIV antibodies inhibited SIV mac replication in macrophages by blocking nuclear transport of viral genomes since viral DNA could not be detected in the nuclei of treated cultures. Furthermore, we showed that although viral replication in macrophages was interrupted by the antibodies, when cocultured with permissive T cells, the viral genomes presented in the cytoplasm of the macrophages could readily transfer to T cells during cell–cell contact. Importantly, this transfer could not be prevented by the antibodies. These results might explain the failure of passive antibody immunization against SIV mac 251—a critical obstacle in AIDS vaccine development.

Four rhesus macaques were sequentially immunized with live vaccines Δ vpu Δ nef SHIV-4 (vaccine-I) and Δ vpu SHIV PPC (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV 89.6 P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4 + T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4 + T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV 89.6 P.

Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 pre-integration complex, activate transcription of cellular and viral promoters, and arrest the cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We now rename this cellular protein as Vpr-binding protein, or VprBP. In this report, we describe the cloning of the VprBP cDNA that encodes 1507 aa residues and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenous VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprBP suggests that the Vpr binding domain is located within the C-terminal half of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Subcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmic fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells also blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP mutants incapable of interacting with Vpr fail to block Vpr-GFP nuclear transport. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replication.

The simian human immunodeficiency virus (SHIV) macaque model of AIDS has provided a very useful system for evaluation of envelope-based candidate vaccines against HIV-1. Eight rhesus macaques were immunized with monomeric recombinant gp120 of HIV-1(LAI) (rgp120) and used to evaluate whether this vaccine conferred protection against challenge with pathogenic SHIVs (SHIV(KU-2) and SHIV(89.6)P). The vaccinated macaques developed high titers of antibodies against rgp120 that reacted efficiently with the envelope proteins of homologous SHIV (SHIV(KU-2)) and poorly with the SHIV(89.6)P envelope, a heterologous strain of SHIV. This vaccine also induced neutralizing antibodies but only against SHIV(KU-2). Vaccine-induced antibodies were of high avidity and predominantly against linear epitopes on the protein. Vaccinated macaques developed gp120-specific T-helper cells but no consistent cytotoxic T lymphocytes. However, cellular immune responses were short-lived in all eight vaccinates. At week 22 postimmunization, four vaccinates were challenged with SHIV(KU-2) and the other four with SHIV(89.6)P. Four unvaccinated control macaques were also infected: two with SHIV(KU-2) and two with SHIV(89.6)P. Vaccinated macaques generally showed anamnestic antibody and T-helper cell responses. However, T-helper responses were again short-lived. Upon challenge, the level of productive virus replication was indistinguishable between vaccine and control groups, suggesting that rgp120 did not confer protection against virus replication when animals were challenged with homologous or heterologous SHIV viruses.

HIV-1 causes cognitive and motor deficits and HIV encephalitis (HIVE) in a significant proportion of AIDS patients. Neurological impairment and HIVE are thought to result from release of cytokines and other harmful substances from infected, activated microglia. In this study, the quantitative relationship between microglial activation and neurological impairment was examined in the simian immunodeficiency model of HIVE. Macaque monkeys were infected with a passaged, neurovirulent strain of simian immunodeficiency virus, SIV mac 239(R71/17E). In concurrent studies, functional impairment was assessed by motor and auditory brainstem evoked potentials and by measurements of cognitive and motor behavioral deficits. Brain tissue was examined by immunohistochemistry using two markers of microglia activation, MHC-II and matrix metalloproteinase-9 (MMP-9). The inoculated animals formed two groups: rapid progressors, which survived 6–14 weeks postinoculation, and slow progressors, which survived 87–109 weeks. In the rapid progressors, two patterns of MHC-II expression were present: (1) a widely disseminated pattern of MHC-II expressing microglia and microglial nodules in cortical gray matter and subcortical white matter, and (2) a more focal pattern in which MHC-II expressing microglia were concentrated into white matter. Animals exhibiting both patterns of microglial activation showed mild to severe changes in cognitive and motor behavior and evoked potentials. All rapid progressors showed expression of MMP-9 in microglia located in subcortical white matter. In the slow progressors MHC-II and MMP-9 staining was similar to uninoculated control macaques, and there was little or no evidence of HIVE. These animals showed behavioral deficits at the end of the disease course, but little changes in evoked potentials. Thus, increases in MHC-II and MMP-9 expression are associated with development of cognitive and motor deficits, alterations in evoked potentials, and rapid disease progression.

Recently, we developed a highly pathogenic variant of simian–human immunodeficiency virus, SHIV-4 (containing the tat, rev, vpu, and env of the HXB2 strain of HIV-1 in a genetic background of SIV mac 239), through a series of four bone marrow-bone marrow passages—first in rhesus monkeys and then in pig-tailed macaques [Joag et al. (1996) J. Virol. 70, 3189–3197]. Inoculation of pig-tailed macaques with this pathogenic virus (SHIV KU-1 ) causes subtotal elimination of CD4 + T cells and fatal opportunistic infections, usually within 6 months. Genetic characterization of SHIV KU-1 showed that it has a functional vpu gene (the first codon is ATG vs ACG for the vpu of SHIV-4) and several amino acid substitutions in Env and nef [Stephens et al. (1997) Virology 231, 313–321]. Two pig-tailed macaques, PPc and PQc, were the first to develop a severe loss of CD4 + T cells and the acquired immune deficiency syndrome and were euthanized at 26 and 105 weeks, respectively. In this report, we analyzed the changes that occurred in the vpu, nef, and env (gp120) genes of the virus used to inoculate macaques PPc and PQc and established the chronology of changes that occurred in these viral genes as these two animals lost their CD4 + T cells and progressed to develop acquired immune deficiency syndrome. Compared with SHIV-4, the virus used to inoculate macaques PPc and PQc had 0, 3, and 0 consensus amino acid changes in the Vpu, gp120, and Nef, respectively. An analysis of the viral sequences amplified from peripheral blood mononuclear cells samples taken at various times after inoculation of PPc revealed that the vpu had not reverted to an open reading frame (closed vpu, ACG) at 4 weeks after inoculation, but by 16 weeks vpu had reverted to an open reading frame (open vpu, ATG). Macaque PQc, which had a longer course of disease, had a closed vpu at 4 and 16 weeks, but by 28 weeks, both closed and open vpu were detected. From 39 to 105 weeks, only an open vpu was detected. In both macaques, the reversion to an open vpu correlated well with the second phase (major) of CD4 + T cell loss. An analysis of the nef and env sequences isolated from the same times after inoculation revealed an association between the reversion of vpu to an open reading frame and the accumulation of increased numbers of consensus changes in these two viral proteins. These data suggest that the concomitant reversion of vpu to an open reading frame along with increased substitutions in Nef and gp120 were important genetic changes in the viral genome that were responsible for the increased and highly efficient rate of replication of the virus in CD4 + T cells and macrophages, which in turn led to elimination of the CD4 + T cells and profound loss of immunocompetence in the infected animals.

Image texture analysis is used in a wide variety of applications in medical research. Neurovirulent simian immunodeficiency virus (SIV) infection in monkeys is considered a good model for HIV-1 infection in humans and causes neuropathological changes in white matter which can include diffuse myelin pallor, subtle white matter astrocytosis, perivascular macrophage infiltrates, and microglial nodules with multinucleated giant cells. The ability of image texture analysis to quantify these changes was evaluated. Sections of thionin-stained brain tissue from eight male rhesus macaques ranging in age from 42-59 months were used. Four animals served as controls and four animals were infected with neurovirulent SIVmac239/17E-R71 by bone marrow inoculation. Images of cerebral white matter were captured and analyzed by calculating 13 textural features based on statistical analysis of spatial co-occurrence matrices. Statistical analysis of the results included multiple comparisons using the Newman-Keuls multiple range test. The effect of variation in background illumination used at image acquisition was also evaluated. Ten of the 13 textural features used in this study successfully discriminated between tissue from control and SIV-infected animals and were consistent with independent neuropathological assessment. Three textural features were highly sensitive to variation in background illumination and found not useful in this application.

SIVmac251 and its closely related derivatives SIVmac239 and SIVmac239/17E vary greatly in their susceptibility to neutralization with homologous and heterologous antisera. Whereas SIVmac251 induces homologous neutralizing antibodies, the antibodies induced by SIVmac239 rarely neutralize infectivity of this virus in lymphocyte cultures. In contrast, SIVmac239/17E is remarkably susceptible to neutralization with homologous and heterologous antisera induced by other strains of SIVmac. In this study, we studied the molecular basis for the neutralization of SIVmac239/17E. Using chimeric viruses in which different regions of theenvgene of both SIVmac239 and SIVmac239/17E were inserted into a background of either of the parental genomes, we showed that the newly acquired neutralization properties of SIVmac239/17E were attributable to amino acid substitutions between the V2 and V4 regions of gp120. Site-directed mutagenesis of theenvgene in this region showed that the arginine substitutions at positions 334 and/or 340 within the “V3” domain were fundamental to virus neutralization but other substitutions in the V2–V4 region added to the ease of its neutralization since it became neutralizable with much higher dilutions of serum. The molecular determinants for neutralization of this virus are distinct from those reported as responsible for neutralization of SIVmac251 and both are distinct from SIVmac239.

Human immunodeficiency virus type 1 (HIV-1) replicates productively in vitro in CD4(+)-T cells and/or macrophages. In the host, however, HIV-1 replication may be restricted by the quiescence of susceptible cells. Vpr is a 15-kDa late viral gene product, which is assembled in the virion and suspected to enhance HIV-1 replication in the infected host. We demonstrated previously that Vpr interacted specifically with the cellular transcription factor Sp1, and activated transcription from the HIV-1 long-terminal-repeat. Both Vpr-Sp1 interaction and trans-activation by Vpr required a central Leu/Ile-rich domain (LR domain, aa 60-81) in Vpr. This domain of Vpr was also found critical for Vpr interaction with another cellular protein of 180 kDa. We now provide biochemical evidence that the Vpr LR-domain has a leucine-zipper-like structure. The leucine-zipper structure has been found in a variety of cellular transcription factors, which use the leucine-zipper domain to form a specific dimer before they can bind to DNA through an upstream basic domain. The LR domain of HIV-1 Vpr, when fused to the basic domain of the cellular transcription factor CREB, was capable of supporting specific DNA binding by the CREB basic domain. Point mutational analysis of the Leu/Ile residues in the LR domain suggested that multiple Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues critical for Vpr interaction with the cellular 180-kDa protein. These results suggested that the leucine-zipper-like domain may be an important functional determinant for HIV-1 Vpr.

Since the pathogenesis of SIV mac disease complex is thought to be explained by the tropism of the infecting virus for either CD4 + T-lymphocytes or macrophages or both types of cells, we compared the infection in primary macaque macrophages with molecularly cloned, lymphocyte-tropic SIV mac 239 and a cloned, macrophage-tropic chimeric virus (SIV mac 239/17E) whose env gene was derived from brain of a macaque (17E) dying from SIV-induced encephalopathy. SIV mac 239/17E caused a productive, syncytial cytopathic infection accompanied by accumulation of virus particles within cytoplasmic esicles of the macrophages. Pulse-chase and immune precipitation studies showed that both the viral glycoprotein precursor (gp160) and the gag precursor (p57) were cleaved into gp120 and p27, respectively, and both were released into the culture medium of infected cells, although most of the p27 remained cell associated. SIV mac 239 also infected macrophages, but in comparison to SIV mac 239/17E, minimal virus replication occurred. Immunocytostaining revealed that while occasional syncytia were observed in cultures, the majority of the infected cells were not associated with syncytium formation. Ultrastructural studies did not reveal the accumulation of virions within infected macrophages. Pulse-chase studies showed that both gp160 and p57 were produced but were cleaved inefficiently and only minimal amounts of gp120 and p27 were released into the culture medium, even after prolonged incubation times. The processing of proteins of the two viruses was indistinguishable in lymphocytes. Since these two viruses are identical except for changes within the env gene, these results indicate that efficient assembly and release of SIV from blood-derived macrophages is mediated by changes in the envelope glycoprotein.

The human immunodeficiency virus, type 1 (HIV-1) genome encodes a 15-kDa accessory gene product, Vpr, that is essential for virus replication in primary monocytes/macrophages. Being present in the virion, Vpr is believed to function in the early phases of HIV-1 replication, including nuclear migration of the pre-integration complex and/or transcription of the provirus genome. By gel filtration analysis of highly purified Vpr protein and its mutants, we demonstrate that HIV-1 Vpr exists as an oligomer. The N-terminal domain of Vpr (amino acids (aa) 1-42) is sufficient for oligomerization; however, deletion of aa 36-76 from Vpr disrupts oligomerization, suggesting that aa 36-42 are critical for Vpr oligomerization. As a result of Vpr oligomerization, basic aa residues within Vpr aa 1-73 are highly resistant to trypsin digestion, while those within Vpr aa 74-96 are easily accessible. Mutations within the leucine-/isoleucine-rich domain (aa 60-81), which was previously identified to be involved in Vpr interaction with a host cellular protein, rendered Arg62 more susceptible to trypsin digestion. Thus, the Vpr oligomeric structure must be extended into this domain. These results suggest a novel feature of HIV-1 Vpr that may be important for its functions.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIV mac 239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIV mac 239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of vital variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol. , 66, 3550-3556, 1992). The neurotropic virus SIV mac 239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIV mac 239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIV mac 239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIV mac 239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIV mac 239. Construction of an infectious recombinant virus containing the tat, rev , and env genes from 17E and the remainder of the genome from the parental virus SIV mac 239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIV mac 239.

Eight rhesus macaques inoculated with molecularly cloned SIV mac 239 developed viremia and virus-binding antibod-ies, but only one (macaque 2D) developed neutralizing antibodies to the virus. Viremia persisted in macaque 2D even in the presence of neutralizing antibodies. Neutralizing antibodies in the plasma collected from macaque 2D late in infection neutralized virus isolated early in infection. In contrast, these antibodies failed to neutralize the plasma viruses isolated after the appearance of neutralizing antibodies. Only antigenic variants were isolated from blood, spleen, and lymph nodes. Viruses isolated from other macaques that did not develop neutralizing antibodies were neutralized by 2D serum and were of the parental (SIV mac 239) phenotype. The variant viruses maintained their strict tropism for lymphocytes, similar to the parental virus.

Visna virus is a lentivirus which causes chronic, progressive encephalitis in sheep. The virus is highly tropic for cells of the monocyte/macrophage lineage and completion of the viral replicative cycle occurs only in mature macrophages. Combined immunohistochemistry and in situ hybridization studies have demonstrated an association between the number of infected macrophages in the brain and the severity of the lesions. A neurovirulent visna virus strain, developed by multiple sequential intracerebral passages in sheep, had an enhanced ability to replicate in macrophages. Visna virus-infected animals produce several cytokines, one of which augments inflammation by inducing expression of MHC class II antigens on macrophages.

The immunopathogenesis of lentiviral lesions in sheep and goats requires continuous replication of the virus in tissues of the animal. This entails escape from various defense mechanisms of the host. Viral expression occurs mainly in tissue-specific macrophage populations and viral proteins produced by the cells induce and combine with antibodies to form immune complexes. These may be pathogenic locally. Infected macrophages also present lentiviral antigens to T lymphocytes and this results in a cascade of cellular responses including proliferation and accumulation of CD8 cells. Cytokines including interferon(s) are produced by lymphocytes and these enhance the antigen-presenting capacity of the macrophages. These lymphoproliferative cellular responses vary from those in human immunodeficiency virus- and simian immunodeficiency virus-infected hosts, mainly because CD4 cells of sheep and goats are not killed by the viruses. These cells, therefore, respond immunologically to viral antigens and this leads to active-chronic inflammation.

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.

Lentiviruses are exogenous, nononcogenic retroviruses that cause persistent infections in monocytes and macrophages. Among the clinical manifestations of such infections in sheep and goats is a slowly progressive arthritis, primarily involving the carpal joints of adult animals. Initially, clinical disease begins as synovitis; this progresses to involve surrounding connective tissues and osseous structures as inflammation increases and progresses. Inflammatory cells include macrophages, lymphocytes, and plasma cells. Macrophages and monocytes are the only cells that are infected with virus and in the inflamed joint may represent 50 per cent of the cells in the synovial fluid. However, only a small number of these cells are infected. Cytotoxic T lymphocytes predominate over helper T lymphocytes in the synovial fluid. The role of these cells in the pathogenesis of disease is not currently known. The pathogenesis of disease caused by these lentiviruses is related to the infection of the monocyte-macrophage cells in the animal. This event, along with virus-specific factors, render the host incapable of eliminating the virus. Despite persistence of the virus, viral replication is maintained at a tightly restricted level at all times in the infected animal. This is achieved by factors that regulate the maturation of monocytes. Basically, the infection is latent in monocytes and its precursors and becomes more productive as the cells mature. The maturing infected macrophage presents a portion of the viral proteins that it synthesizes on its cell surface in close association with class II major histocompatibility complex antigens. Lymphocytes react with this cell and produce interferon. This lymphokine induces further expression of Ia antigens. This feedback loop of Ia expression, together with persistence of the virus, maintains macrophages in a constant state of antigen presentation. This forms the trigger for the inflammatory condition that eventually leads to the degenerative lesions seen in the joint.

This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.

A herd of 15 dairy goats with a previous history of neurological disease in kids was screened for caprine arthritis-encephalitis (CAE) virus infection. Virus was isolated from the PBL of all animals as well as milk from lactating does. All animals had antibody detected by ELISA and gel immuno-diffusion. Two animals from this herd were examined for responses to infection. One was an adult female with poor milk production and the other was a 3-month-old female kid which developed acute neurological disease. Both animals had a chronic mastitis compatible with a viral aetiology, and CAE virus was isolated from the mammary tissue of both. The mammary gland must thus be considered an important target organ for virus infection and dissemination.

Lentiviruses are unique retroviruses which cause diseases with long incubation periods and prolonged clinical courses. The prototype lentiviruses, visna/maedi of sheep and arthritis-encephalitis virus of goats (CAEV), infect cells of the monocyte-macrophage system and replicate at a restricted level in these cells. The virus life cycle is closely associated with maturational factors in the cells; monocytes support the early stages of the replication cycle which goes to completion only when the cells mature to macrophages. Virus replication in the monocyte-macrophage results in lesions characterized by mononuclear cell infiltration of the central nervous system (CNS), lungs, synovium and mammary gland and their draining lymph nodes. Co-cultivation of sheep or goat lymphocytes with macrophages infected with visna or CAE viruses results in production of a unique interferon (LV-IFN). LV-IFN is a non-glycosylated protein of 54,000 to 64,000 daltons and has biological properties which have several implications for pathogenesis. Firstly, it retards the rate of maturation of monocytes and thus indirectly slows the rate of virus replication. Second, it restricts the rate of virus replication in mature macrophages by preventing virus maturation. Third, it induces expression of class II (Ia) antigens of the major histocompatibility complex on cells of macrophage lineage. Thus, by curtailing virus replication and enhancing expression of MHC class II antigens, LV-IFN may contribute to the induction and augmentation of the host's lymphoproliferative response to the virus.

Naturally occurring antigenic variants of visna virus isolated from two persistently infected sheep have been compared using RNAase T 1 fingerprint analysis of the viral RNAs. All the viruses examined have oligonucleotide changes in the 3′-terminal region of the viral genome and many of the alterations are common among the variants of both sheep. There is progressive accumulation of point mutations in the 3′-terminal region of antigenically altered viruses isolated from persistently infected animals. In addition, antigenic variants isolated independently from another sheep contain the same altered oligonucleotides; the nucleotide sequences of these oligonucleotides are identical. These data strongly suggest that the alterations observed in the nucleotide sequence of the variants are related to the change in their antigenic character.

Temperature-sensitive mutants of visna virus have been isolated from N-methyl-N′-nitro-N-nitroso-guanidine mutagenized virus. These mutants have been partially characterized and have been separated into three classes based on the time after infection when the temperature-sensitive function was expressed.

The nucleic acid of an SV40-like virus isolated from a patient with progressive multifocal leukoencephalopathy has been compared with the DNA of a laboratory strain of SV40. DNA of this isolate (designated PML-2) is a covalently closed circular duplex with the same sedimentation rate as SV40 DNA. Denatured PML-2 DNA hybridizes with denatured SV40 DNA at about the same rate as homologous DNA. When digested with Hemophilus influenzae restriction endonuclease, PML-2 DNA yielded 11 fragments separable by electrophoresis, nine of which co-migrated with fragments of SV40 DNA, and two of which did not. We conclude that the PML-2 virus is a variant of SV40.