High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.     

Isolation and Characterization of a Neuropathogenic Simian Immunodeficiency Virus Derived from a Sooty Mangabey

Transfusion of blood from a simian immunodeficiency virus (SIV)- and simian T-cell lymphotropic virus-infected sooty mangabey (designated FGb) to rhesus and pig-tailed macaques resulted in the development of neurologic disease in addition to AIDS. To investigate the role of SIV in neurologic disease, virus was isolated from a lymph node of a pig-tailed macaque (designated PGm) and the cerebrospinal fluid of a rhesus macaque (designated ROn2) and passaged to additional macaques. SIV-related neuropathogenic effects were observed in 100% of the pig-tailed macaques inoculated with either virus. Lesions in these animals included extensive formation of SIV RNA-positive giant cells in the brain parenchyma and meninges. Based upon morphology, the majority of infected cells in both lymphoid and brain tissue appeared to be of macrophage lineage. The virus isolates replicated very well in pig-tailed and rhesus macaque peripheral blood mononuclear cells (PBMC) with rapid kinetics. Differential replicative abilities were observed in both PBMC and macrophage populations, with viruses growing to higher titers in pig-tailed macaque cells than in rhesus macaque cells. An infectious molecular clone of virus derived from the isolate from macaque PGm (PGm5.3) was generated and was shown to have in vitro replication characteristics similar to those of the uncloned virus stock. While molecular analyses of this virus revealed its similarity to SIV isolates from sooty mangabeys, significant amino acid differences in Env and Nef were observed. This virus should provide an excellent system for investigating the mechanism of lentivirus-induced neurologic disease.     

Development of Neurological Disease Is Associated with Increased Immune Activation in Simian Immunodeficiency Virus-Infected Macaques

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.     

Role of microglial cells in selective replication of simian immunodeficiency virus genotypes in the brain

An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (Macaca nemestrina) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (n = 3), moderate (n = 5), or mild (n = 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (P = 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (P = 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.     

Extensive envelope heterogeneity of simian immunodeficiency virus in tissues from infected macaques.

The extent of virus genetic variation within tissues and peripheral blood mononuclear cells (PBMC) from two simian immunodeficiency virus (SIV)-infected macaques was analyzed. The products of PCR amplification of two regions, region 1 (SIV V1 region) and region 2 (region corresponding to the human immunodeficiency virus V3 cysteine loop and part of the C3 region immediately downstream), of the SIV envelope were examined for single-stranded conformation polymorphism followed by sequence analysis of selected clones. The V1 region of the SIV envelope of viruses present within lymphoid tissues displayed extensive heterogeneity, while viral populations within the PBMC and brain appeared to be less variable. Region 2 heterogeneity in both animals was generally confined to three residues in a tissue-specific manner. In addition, virus from the brains of both animals appeared to be distinct compared with viruses present in other tissues and PBMC of the same animal, both in the pattern of PCR-single-stranded conformation polymorphism SCP and in the sequence of region 2. These studies revealed that the tissues of SIV-infected macaques were a reservoir for viral variants distinct from those seen in PBMC.     

A Quantitative Measurement of Antiviral Activity of Anti-Human Immunodeficiency Virus Type 1 Drugs against Simian Immunodeficiency Virus Infection: Dose-Response Curve Slope Strongly Influences Class-Specific Inhibitory Potential

Simian immunodeficiency virus (SIV) infection in macaques is so far the best animal model for human immunodeficiency virus type 1 (HIV-1) studies, but suppressing viral replication in infected animals remains challenging. Using a novel single-round infectivity assay, we quantitated the antiviral activities of antiretroviral drugs against SIV. Our results emphasize the importance of the dose-response curve slope in determining the inhibitory potential of antiretroviral drugs and provide useful information for regimen selection in treating SIV-infected animals in models of therapy and virus eradication.     

Molecular characterization and comparison of simian immunodeficiency virus isolates from macaques, mangabeys, and African green monkeys

Simian immunodeficiency virus (SIV)/Mne has been inoculated into three species of macaques and into baboons. Virus was isolated from all the macaques who subsequently died at 15 to 120 weeks (mean 80 weeks) with various manifestations of immune deficiency. Individual animals varied in their viral antibody profile as a function of time after infection. Independent SIV isolates obtained from African green monkeys and magabeys were compared to SIV/Mne for their ability to replicate in lymphocytes and macrophages and with respect to the immunological relatedness of their viral proteins. Antibodies present in human immunodeficiency virus-2 (HIV-2)-infected individuals were readily detected by the virus produced by a single-cell clone of SIV/Mne.     

Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.     

Distinct Evolutionary Pressures Underlie Diversity in Simian Immunodeficiency Virus and Human Immunodeficiency Virus Lineages

Simian immunodeficiency virus (SIV) infection of rhesus macaques causes immune depletion and disease closely resembling human AIDS and is well recognized as the most relevant animal model for the human disease. Experimental investigations of viral pathogenesis and vaccine protection primarily involve a limited set of related viruses originating in sooty mangabeys (SIVsmm). The diversity of human immunodeficiency virus type 1 (HIV-1) has evolved in humans in about a century; in contrast, SIV isolates used in the macaque model evolved in sooty mangabeys over millennia. To investigate the possible consequences of such different evolutionary histories for selection pressures and observed diversity in SIVsmm and HIV-1, we isolated, sequenced, and analyzed 20 independent isolates of SIVsmm, including representatives of 7 distinct clades of viruses isolated from natural infection. We found SIVsmm diversity to be lower overall than HIV-1 M group diversity. Reduced positive selection (i.e., less diversifying evolution) was evident in extended regions of SIVsmm proteins, most notably in Gag p27 and Env gp120. In addition, the relative diversities of proteins in the two lineages were distinct: SIVsmm Env and Gag were much less diverse than their HIV-1 counterparts. This may be explained by lower SIV-directed immune activity in mangabeys relative to HIV-1-directed immunity in humans. These findings add an additional layer of complexity to the interpretation and, potentially, to the predictive utility of the SIV/macaque model, and they highlight the unique features of human and simian lentiviral evolution that inform studies of pathogenesis and strategies for AIDS vaccine design.     

Human and simian immunodeficiency virus capsid proteins are major viral determinants of early,postentry replication blocks in simian cells

The cells of most Old World monkey species exhibit early, postentry restrictions on infection by human immunodeficiency virus type 1 (HIV-1) but not by simian immunodeficiency virus of macaques (SIV(mac)). Conversely, SIV(mac), but not HIV-1, infection is blocked in most New World monkey cells. By using chimeric HIV-1/SIV(mac) viruses capable of a single round of infection, we demonstrated that a major viral determinant of this restriction is the capsid (CA) protein. The efficiency of early events following HIV-1 and SIV(mac) entry is apparently determined by the interaction of the incoming viral CA and species-specific host factors.     

A rapid progressor-specific variant clone of simian immunodeficiency virus replicates efficiently in vivo only in the absence of immune responses

A subset of simian immunodeficiency virus (SIV)-infected macaques progresses rapidly to disease with transient SIV-specific immune responses and high viral loads. Unique SIV variants with convergent Env mutations evolve in these rapid progressor (RP) macaques. To address the pathogenic significance of RP-specific variants, we generated infectious molecular clones from the terminal-phase plasma of an RP macaque. Inoculation of macaques with a representative clone, SIVsmH635FC, resulted in a persistent viremia, comparable to that produced by pathogenic SIVsmE543-3, and a chronic disease with progressive loss of CD4(+) T cells. However, SIVsmH635FC did not reproduce the rapid-disease phenomenon. Molecular analyses of viruses from these macaques revealed rapid reversion to the wild-type SIVsmE543-3 sequence at two RP-specific sites and slower reversion at another three sites. SIVsmH635FC infection was not sufficient to cause rapid progression even following coinoculation with SIVsmE543-3, despite acute depletion of memory CD4(+) T cells. SIVsmH635FC competed efficiently during primary infection in the coinoculated macaques, but SIVsmE543-3 predominated after the development of SIV-specific immune responses. These data suggest that the replication fitness of the RP variant was similar to that of SIVsmE543-3 in a na?ve host; however, SIVsmH635FC was at a disadvantage following the development of SIV-specific immune responses. Consistent with these findings, neutralization assays revealed that SIVsmH635FC was highly sensitive to neutralization but that the parental SIVsmE543-3 strain was highly resistant. This study suggests that the evolution of RP-specific variants is the result of replication in a severely immunocompromised host, rather than the direct cause of rapid progression.     

Human Immunodeficiency Virus Neurotropism: an Analysis of Viral Replication and Cytopathicity for Divergent Strains in Monocytes and Microglia

Productive replication of human immunodeficiency virus type 1 (HIV-1) in brain macrophages and microglia is a critical component of viral neuropathogenesis. However, how virus-macrophage interactions lead to neurological disease remains incompletely understood. Possibly, a differential ability of virus to replicate in brain tissue macrophages versus macrophages in other tissues underlies HIV-1 neurovirulence. To these ends, we established systems for the isolation and propagation of pure populations of human microglia and then analyzed the viral life cycles of divergent HIV-1 strains in these cells and in cultured monocytes by using identical viral inocula and indicator systems. The HIV-1 isolates included those isolated from blood, lung tissue, cerebrospinal fluids (CSF), and brain tissues of infected subjects: HIV-1_ADA and HIV-1_89.6 (from peripheral blood mononuclear cells), HIV-1_DJV and HIV-1_JR-FL (from brain tissue), HIV-1_SF162 (from CSF), and HIV-1_BAL (from lung tissue). The synthesis of viral nucleic acids and viral mRNA, cytopathicity, and release of progeny virions were assessed. A significant heterogeneity among macrophage-tropic isolates for infection of monocytes and microglia was demonstrated. Importantly, a complete analysis of the viral life cycle revealed no preferential differences in the abilities of the HIV-1 strains tested to replicate in microglia and/or monocytes. Macrophage tropism likely dictates the abilities of HIV-1 to invade, replicate, and incite disease within its microglial target cells.     

Viral Genetic Evolution in Macaques Infected with Molecularly Cloned Simian Immunodeficiency Virus Correlates with the Extent of Persistent Viremia

Genetic evolution of the simian immunodeficiency virus (SIV) envelope glycoprotein was evaluated in a group of six macaques (Macaca nemestrina) infected with the molecularly cloned, moderately pathogenic SIVsm62d. The extent of envelope evolution was subsequently evaluated within the context of the individual pattern of viremia and disease outcome. Two macaques in this cohort developed AIDS by 1.5 years postinoculation (progressors), whereas the remaining four macaques remained asymptomatic (nonprogressors). Compared with the nonprogressor macaques, the two progressor macaques exhibited higher persistent plasma viremia, higher homologous neutralizing antibody titers, and more extensive mutation and evolution in the V1 region of envelope. Although clearly distinct in each of these parameters from the progressors, the four nonprogressors exhibited more individual variability with respect to the extent of persistent viremia and genetic evolution of the V1 region of envelope. The extent of V1 envelope varied from no apparent V1 evolution in a macaque with good viral containment to extensive evolution in one macaque with persistent viremia. This study underscores the critical role of persistent replication in the genetic evolution of SIV.     

The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.     

Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.     

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.     

Zeta Chain of the T-Cell Receptor Interacts with nef of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2

A truncated version of the nef gene of simian immunodeficiency virus SIVmac239 capable of encoding amino acids 98 to 263 was used as bait to screen a cDNA library from activated lymphocytes in a yeast two-hybrid system. The zeta chain of the T-cell receptor (TCR_ζ) was found to interact specifically not only with truncated SIV nef in yeast cells but also with full-length glutathione S-transferase (GST)-SIVnef fusion protein in vitro. Coimmunoprecipitation of TCR_ζ with full-length SIV nef was demonstrated in transfected Jurkat cells and in Cos 18 cells which express the cytoplasmic domain of TCR_ζ fused to the external domain of CD8 via the CD8 transmembrane domain. Using a series of nef deletion mutants, we have mapped the binding site within the central core domain of nef (amino acids 98 to 235). Binding of TCR_ζ was specific for nef isolated from SIVmac239, SIVsmH4, and human immunodeficiency virus (HIV)-2ST and was not detected with nef from five different HIV-1 isolates. An active tyrosine kinase was coprecipitated with nef-TCR_ζ complexes from Jurkat cells but not from J.CAM1.6 cells which lack a functional Lck tyrosine kinase. These results demonstrate a specific association of SIV and HIV-2 nef, but not HIV-1 nef, with TCR_ζ.