Search PubMed⌕ Search

Biomedical subjects

O Narayan

Publications and source records attributed to O Narayan.

At least 73 records · Page 4Linked to original sources
PubMed1997

Animal model of mucosally transmitted human immunodeficiency virus type 1 disease: intravaginal and oral deposition of simian/human immunodeficiency virus in macaques results in systemic infection, elimination of CD4+ T cells, and AIDS.

Chimeric simian/human immunodeficiency virus (SHIV) consists of the env, vpu, tat, and rev genes of human immunodeficiency virus type 1 (HIV-1) on a background of simian immunodeficiency virus (SIV). We derived a SHIV that caused CD4+ cell loss and AIDS in pig-tailed macaques (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996) and used a cell-free stock of this virus (SHIV(KU-1)) to inoculate macaques by the intravaginal route. Macaques developed high virus burdens and severe loss of CD4+ cells within 1 month, even when inoculated with only a single animal infectious dose of the virus by the intravaginal route. The infection was characterized by a burst of virus replication that peaked during the first week following intravenous inoculation and a week later in the intravaginally inoculated animals. Intravaginally inoculated animals died within 6 months, with CD4+ counts of <30/microl in peripheral blood, anemia, weight loss, and opportunistic infections (malaria, toxoplasmosis, cryptosporidiosis, and Pneumocystis carinii pneumonia). To evaluate the kinetics of virus spread, we inoculated macaques intravaginally and euthanized them after 2, 4, 7, and 15 days postinoculation. In situ hybridization and immunocytochemistry revealed cells expressing viral RNA and protein in the vagina, uterus, and pelvic and mesenteric lymph nodes in the macaque euthanized on day 2. By day 4, virus-infected cells had disseminated to the spleen and thymus, and by day 15, global elimination of CD4+ T cells was in full progress. Kinetics of viral replication and CD4+ loss were similar in an animal inoculated with pathogenic SHIV orally. This provides a sexual-transmission model of human AIDS that can be used to study the pathogenesis of mucosal infection and to evaluate the efficacy of vaccines and drugs directed against HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Pathogenesis of ovine lentiviral encephalitis: derivation of a neurovirulent strain by in vivo passage.

The lentiviruses of sheep replicate almost exclusively in macrophages and cause chronic interstitial pneumonia, arthritis, and mastitis, but only rarely encephalitis. This study was undertaken to determine whether a non-neurovirulent field strain of ovine lentivirus isolated from joint fluid that replicated productively in lung and joint macrophages could be adapted to enter and replicate in the brain and cause encephalitis. The field isolate was passed seven times sequentially by intracerebral inoculation of sheep. The neuroadapted strain of virus caused severe encephalitis typical of visna in four of four sheep inoculated intracerebrally. The virus replicated to high titers in the brains of these animals and in cultured microglia. The inflammatory response in the brain was characterized by intense infiltrates of macrophages and CD8+ and CD4+ T cells. Many of the perivascular macrophages demonstrated TNF-alpha expression and there was upregulation of MHC Class II antigen expression on both inflammatory cells and endothelium. Inoculation of this neuroadapted virus into the bone marrow of three animals resulted in persistent infection and cell-associated viremia, but not encephalitis. Virus was not detected in brains from these animals, indicating that the virus was not neuroinvasive. These data suggest that neuroinvasiveness and neurovirulence are separate pathogenic determinants, both of which are required for the development of encephalitis during natural infection.

Animals↗
PubMed1997

Antibody to human MHC class I inhibits SIVsmmPBj1.9-induced proliferation of pigtailed macaque lymphocytes.

Previously we have shown that the simian immunodeficiency virus SIVsmmPBj1.9, a molecular clone of SIVsmmPBj14, induces proliferation of human peripheral blood mononuclear cells (PBMC). We have extended this observation to show that SIVsmmPBj1.9 induces proliferation of PBMC from pigtailed macaques. This proliferative response was markedly inhibited by mAbs against human class I MHC, class II MHC and CD4 antigens, and partially inhibited by mAbs against integrin beta 2 subunit (CD18) and LFA-1 (CD11a). However, these antibodies differed in their ability to inhibit in vitro viral infectivity of PBMC. While anti-CD4, MHC class II, and LFA-1 strongly inhibited viral infectivity, antibodies to MHC class I demonstrated little effect on viral infectivity. A control antibody (PLM2) against porcine CD18 inhibited neither virus-induced proliferation nor viral infectivity. Based on these results, we suggest that SIVsmmPBj1.9-induced proliferation requires the participation of class I MHC, class II MHC and CD4 molecules. In addition, the observation that anti-class I MHC Ab inhibited proliferation of macaque PBMC induced by mitogen (PHA) and bacterial superantigens, such as Staphylococcus enterotoxin A and toxin shock syndrome toxin-1, suggests that SIVsmmPBj1.9 also contains a viral superantigen similar to that previously demonstrated in SIVsmmPBj14.

Animals↗
PubMed1996

Genetic characterization of two phenotypically distinct North American ovine lentiviruses and their possible origin from caprine arthritis-encephalitis virus.

Ovine and caprine lentiviruses are closely related genetically and antigenically although the diseases that these viruses cause in their respective host animals can vary greatly. In sheep, syndromes consist primarily of interstitial pneumonia with rare occurrences of arthritis and encephalitis, whereas in goats, the disease expresses mainly as arthritis in adult animals with rare cases of encephalitis in newborns. Experimentally, viruses from either sheep or goats can infect animals of the reciprocal species and many field strains of ovine lentivirus have biological properties similar to those of caprine viruses. However, a molecular correlation for the phenotypic differences between ovine and caprine lentivirus strains is unknown. To investigate this, we examined genetic characteristics of two phenotypically distinct North American ovine lentiviruses. Nucleotide sequence analysis of the envelope regions from virus strains 85/34 and 84/28 showed that despite significant biological differences, these viruses are closely related to each other and are genotypically more homologous to caprine arthritis-encephalitis virus (CAEV) than to visna virus of sheep. Furthermore, analysis of the nucleotide substitutions in their env regions indicated that when differences between the two ovine viruses and CAEV were found, the changes often resulted in nucleotides homologous with visna virus. These results suggest that the two field strains of ovine lentivirus may have originated from a cross-species infection of sheep by a CAEV-like virus and, evolution of their genomes toward that of ovine lentivirus may be reflective of adaptation of these viruses to the new ovine host.

Animals↗
PubMed1996

Characterization of a leucine-zipper-like domain in Vpr protein of human immunodeficiency virus type 1.

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.

Amino Acid Sequence↗
PubMed1996

Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.

Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

Animals↗
PubMed1996

Neutralization of SIVmac239/17E in lymphocyte cultures involves virus strain-specific linear and conformational epitopes encoded by different regions of the env gene including the "V3" domain.

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 the env gene 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 gp 120. Site-directed mutagenesis of the env gene 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.

Animals↗
PubMed1996

Antigenic variation of SIV: mutations in V4 alter the neutralization profile.

Antigenic variation is a characteristic feature of lentiviral infection. The SIV/macaque model of AIDS provides an ideal system in which to investigate the molecular basis of antigenic variation. The purpose of this study was to genetically map the nucleotide changes in env that alter the neutralization phenotype of SIV. Serum taken from an SIVmac239-infected macaque (2D) at 30 weeks postinoculation was found to neutralize the input virus (SIVmac239) and an isolate, P9, obtained at 10 weeks p.i., but did not neutralize two other isolates, P13 and P23, obtained at 20 and 52 weeks, respectively. Sequence analysis of these virus variants revealed clustered amino acid changes in V1 and single base pair changes in V2-V4 of P13 and P23. Infectious recombinant viruses in which the V1 and V1-V3 sequences of SIVmac239 were replaced with those of P13 or P23 retained the neutralization profile of SIVmac239; both were neutralized by macaque 2D serum. Recombinants containing the entire surface glycoprotein (gp120) (V1-V5) and the 5' portion of gp41 of P13 and P23 and those containing gp120 sequences from V4 through the 5' portion of the transmembrane glycoprotein (gp41) were not neutralized by 2D serum. Using a panel of monoclonal antibodies in radioimmunoprecipitation assays, P23 and recombinants containing V4 and V5 of P23 were shown to be antigenically distinct from P13 and SIVmac239. The majority of the amino acid changes in the antigenically distinct viruses were clustered in V4 (amino acids 413-418) and these changes created new potential N-linked glycosylation sites. This study demonstrates that a small number of specific amino acid changes (amino acids 412 to 418 in the env gene) in the V4 region of the SIV envelope glycoprotein can alter antibody recognition and neutralization and that these phenotypic changes may be associated with altered glycosylation of the envelope.

Amino Acid Sequence↗
PubMed1996

Prolonged infection in rhesus macaques with simian immunodeficiency virus (SIVmac239) results in animal-specific and rarely tissue-specific selection of nef variants.

We analyzed the sequence of nef genes from different tissues of three rhesus macaques that had been infected with molecularly cloned SIVmac239 for 88 to 92 weeks. Comparison of the predicted amino acid sequences revealed that each macaque had selected out specific amino acid substitutions and that most of this variation (70%) was confined to four regions, amino acids 39 to 75, 90 to 105, 153 to 167, and 191 to 217, comprising 36% of the protein. The nef genes in these animals underwent extensive genetic variation with average nucleotide and amino acid substitution rates varying from 0.86 to 2.84% and 2.47 to 6.27%, respectively, although tissue-specific selection of nef variants occurred in only 1 of 14 tissues examined in this study. Comparison of the rate of nucleotide and amino acid substitutions in the nef genes to those previously reported in the env in the central nervous system (CNS) and lymph node (LN) revealed that the predicted amino acid substitution rates for Nef were much higher than for the gp120 region of env in the CNS and LN tissues for one macaque. In the two other macaques, the predicted amino acid substitution rates were similar between these two proteins in LN tissues, but the amino acid substitution rates in Nef were significantly higher than in the gp120 from the CNS. Comparison of the nucleotide substitutions in the region of overlap between the env and the nef revealed that approximately 83% of the nucleotide substitutions in this area resulted in a Nef amino acid sequence change, 26% of the nucleotide substitutions resulted in a gp41 amino acid change, and 9.5% of nucleotide substitutions resulted in amino acid sequence changes in both proteins, suggesting a preference for the selection of amino acid substitutions in the Nef in these animals. Our results indicate that in animals infected with SIVmac239 for prolonged periods, variation in the nef occurs at rates similar to or exceeding that observed for the env gene.

Amino Acid Sequence↗
PubMed1996

Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep.

Seventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.

Amino Acid Sequence↗
PubMed1996

Initial characterization of viral sequences from a SHIV-inoculated pig-tailed macaque that developed AIDS.

In this study, we report on the derivation of a pathogenic SIV-HIV chimeric virus (SHIV) and the initial characterization of the viral sequences from the first (macaque PPc) of a series of pig-tailed macaques that developed CD4+ T cell loss and AIDS. Viral genes were amplified by PCR from the brain, lymphoid, and kidney tissues and their sequences compared to the original SHIV used to initiate passages in macaques. Our results show that the vpu gene, which was nonfunctional in the original SHIV, now coded for functional protein in macaque PPc. The tat and rev genes had no consensus changes but the nef gene had 4-5 consensus changes, depending on the tissue examined. The gp 120 gene had the highest number of nucleotide and amino acid substitution rates that varied from 0.64% to 1.44% and 1.17% to 3.71%, respectively, again depending on the tissue examined. These results suggest that a constellation of changes accumulated at the genomic level during the derivation of a SHIV that was pathogenic for pig-tailed macaques.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Lymphocyte-tropic simian immunodeficiency virus causes persistent infection in the brains of rhesus monkeys.

Molecularly cloned SIVmac239 is the prototypical SIVmac lymphocyte-tropic virus that replicates productively in lymphocytes but poorly in macrophages. In macaques, the virus causes activation and productive infection of T lymphocytes which invade the central nervous system (CNS) early after infection in the animal. However, infected animals develop immunosuppression and AIDS but rarely overt neurological disease. In this study, we examined multiple regions of the brain and spinal cord for the presence of SIV env sequences and histological lesions in five macaques that had been infected with SIVmac239 for 1.7 to 2.25 years. Histopathological examination of the brain revealed no lesions consistent with encephalitis; however, viral DNA was found in all five brains. In one animal the virus caused infection in a widely disseminated pattern from the frontal cortex to the distal end of the spinal cord, whereas in the other four animals infection in the CNS occurred in a nonspecific, focal pattern. Sequence analyses were performed on gp120 sequences isolated from selected regions of the CNS and compared to gp120 sequences isolated from corresponding lymph nodes, a tissue known to support productive replication of SIVmac239. Examination of the viral sequences from the CNS tissue from two animals (macaques 10F and 14F) revealed a low mutation rate when compared to the sequences isolated from the lymph node tissues. The percentage change in the amino acid sequence was approximately 1% for CNS clones versus > or = 3% for clones isolated from the lymph node. The majority of the CNS viral sequences of macaques 10F and 14F had none of the genetic markers shown in a previous study to be associated with macrophage-tropic variants and indeed retained a nucleotide sequence of similar to the original lymphocyte-tropic virus used for inoculation despite almost 2 years of persistent infection in the animals. Construction of chimeric viruses with V1-V5 regions of selected macaque 10F and macaque 14F CNS-gp120 clones confirmed the predicted lymphocyte-tropic nature of these env genes. In contrast, the gp120 sequences isolated from the CNS tissue of one of the other three animals (macaque 13F) had a mutation rate comparable to that observed for the lymph node clones. The CNS clones from this animal had amino acid substitutions that were previously shown to be associated with macrophage tropism. Compared to the chimeric viruses constructed with V1-V5 sequences from macaques 10F and 14F, viruses constructed with the V1-V5 sequences of several macaque 13F brain clones did not yield infectious virus.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and trans-activation of viral long terminal repeat.

Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4+ T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-kappa B, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS.

Base Sequence↗
PubMed1995

Induction of protection against Borna disease by inoculation with high-dose-attenuated Borna disease virus.

Borna disease is a chronic neurological disease caused by an enveloped negative-strand RNA virus (BDV). Experimental disease can be reproduced in rats with brain homogenates derived from infected animals or with virus derived from infected cells in culture. The virus replicates in cultured cells without evidence of cytopathic effect or production of significant levels of cell-free virus. Borna disease is caused by an immunopathological response to viral infection of neural cells. To further investigate the pathogenesis of Borna disease, rats were inoculated with different doses of BDV attenuated by culture in MDCK cells. Low doses of attenuated BDV (10(2)-10(4) TCID50) resulted in typical clinical disease and severe encephalitis; however, the lag period between inoculation and disease was considerably longer than that with virulent BDV. In contrast, animals inoculated with a high dose of attenuated BDV (10(5)-10(6) TCID50) did not develop clinical disease, although a mild encephalitic response was present that did not progress beyond the mild encephalitis. Animals inoculated with a high dose of BDV developed high titers of anti-BDV antibody and were protected against virulent challenge. Protection was correlated with the rapid induction of an immune response in the animals and the lack of any biologically detectable virus in the CNS.

Animals↗