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Summary. To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV KU-2-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage. [ABSTRACT FROM AUTHOR]

Summary. A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-? (SHIV-IFN-?) was constructed and the effect of co-expression of IFN-? on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24?h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-a and IFN-? was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-? (Th1-type cytokine) was more rapidly induced by SHIV-IFN-? vaccination, and IFN-?-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-? with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity. [ABSTRACT FROM AUTHOR]

CD4-bearing T cells are the primary targets for human immunodeficiency virus type 1(HIV-1)/simian immunodeficiency virus (SIV) infection. However, it is unclear whether the susceptibility of CD4-bearing T cells including CD4 single positive and CD4/8 double positive T cells to HIV/SIV infection is the same or not. In this study, we compared the susceptibility to SIV infection between CD4(+) and CD4(+)8(+) T cells, using Herpesvirus saimiri (HVS)-transformed CD4(+) and CD4(+)8(+) T cells established from peripheral blood mononuclear cells (PBMC) of rhesus macaques. Although there was little difference between the two CD4-bearing T cell population in the expression level of CD4 molecules and chemokine receptors such as CXCR4 and CCR5, SIV replicated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells. Moreover, we found that reverse transcription initiated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells and that the cell lysates from CD4(+) T cells impaired the RT activity more strongly than that from CD4(+)8(+) T cells. These findings suggest that intracellular environment in CD4(+) 8(+) T cells is better for reverse transcription and that the infection of those CD4(+)8(+) T cells might play critical and different roles in HIV-1/SIV infection and dissemination.

To clarify the involvement of primitive non-specific immune responses in the protective effects of a live, attenuated virus, each two rhesus macaques were intravenously immunized with an attenuated chimeric simian and human immunodeficiency virus (SHIV) in which the nef gene was deleted (SHIV-NI) or a SHIV having human IFN-gamma inserted into the deleted nef region (SHIV IFN-gamma). These immunized monkeys were intravenously challenged with a heterologous pathogenic SHIV (SHIV-C2/1) at four weeks post immunization (wpi). After vaccination, one of each SHIV-NI- or SHIV IFN-gamma-immunized monkeys showed a low level of SIV Gag-specific lymphocyte proliferative response but did not have neutralizing antibodies to both the parental and challenge viruses. After the challenge, the plasma viral RNA loads of the challenge virus were suppressed in all the immunized monkeys and the severe CD4+ T cell loss observed in the unimmunized monkeys was not found. Thus, both SHIV IFN-gamma and SHIV-NI infections could prevent from disease progression by a pathogenic virus early after immunization, suggesting that primitive non-specific immune response elicited by attenuated virus infection, in addition to highly acquired virus-specific immunity, contributes to the protective effect against a pathogenic virus.

To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV(KU-2)-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage.

A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-gamma (SHIV-IFN-gamma) was constructed and the effect of co-expression of IFN-gamma on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24 h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-gamma (Th1-type cytokine) was more rapidly induced by SHIV-IFN-gamma vaccination, and IFN-gamma-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-gamma with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes, because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs containing Env from two strains of HIV-1, CMR304 and CMR306, which belong to subtype F and A, respectively, were newly obtained. These SHIVs essentially showed a coreceptor usage and a neutralization pattern that were similar to those of the parental HIV-1s. In macaque PBMC, SHIVcmr304 replicated with kinetics similar to that of prototypic SHIV-NM-3rN with HIV-1 NL432 Env, but SHIVcmr306 replicated poorly. Inoculation of four rhesus macaques with SHIVcmr304 resulted in an increase of plasma viral load in all the macaques, though viral RNA copies were 100-fold lower than that in the infection with NM-3rN. This SHIV containing Env from HIV-1 subtype F will be a valuable source for the analysis of HIV-1 subtype F and the evaluation of vaccine candidates as a genetically divergent challenge virus.

Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6 P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6 P challenge virus using PCR amplification and restriction enzyme analysis of the env region of the viruses. Analysis of the 3'-end of the env region and 5'-half of the nef region using a heteroduplex mobility assay revealed that the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys had quite an abundant and similar heterogeneous quasispecies population. In contrast, the viruses isolated from the vaccinated monkeys had different and fewer quasispecies indicating a selective immune pressure in the vaccinated monkeys. The in vitro replication of the viruses isolated from the vaccinated monkeys in human and macaque peripheral blood mononucular cells (PBMCs) as well as in established cell lines such as M8166 and HSC-F cells, were slow and delayed when compared to the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys. Further comparison revealed that in HSC-F cells the viruses from vaccinated monkeys again showed delayed and weak CD4(+) cell down-modulation as well as having little or no effect on cell growth or cell viability on HSC-F cells and monkey PBMC. Thus we noticed that these re-isolated 89.6 P viruses from the vaccinated monkeys had changed or had been selected for low pathogenic viruses in the monkeys. This suggests that though the vaccination did not completely prevent the replication of the challenge virus in the monkeys it did contain the challenge virus by suppressing the pathogenic variants. This further enhances the prospects of this nef-deleted SHIV as the bases for effective anti-HIV vaccine candidates.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.

During the progression of AIDS, there is a shift in abundance of immune cells from Th1-producing cells to Th2-producing cells. To determine whether this change might have an effect on HIV-1 replication in vivo, we constructed simian/human immunodeficiency chimeric viruses having the human IL-5 gene (a Th2-type cytokine) and examined the effect of the inserted gene on viral replication, IL-5 production and viral stability in vitro. The DNA of human IL-5 was inserted into vpr-deleted and nef-deleted infectious SHIVs. The obtained replication-competent viruses were used to infect human T-cell lines and monkey peripheral blood mononuclear cells. As a result, at the time of peak NI-IL5 virus production, IL-5 was produced with a significantly higher titer than 3sj-IL5. The functionality of the produced IL-5 was confirmed by IL-5-dependent cells. The replication of both SHIVs having IL-5 appeared to be faster than that of the parental viruses without the IL-5 gene. These results show that co-expression of IL-5 stimulates SHIV replication in vitro. Thus, it is expected that expression of IL-5 will also have an effect on viral replication and pathogenicity in vivo.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) act quite specifically on human immunodeficiency virus type 1 (HIV-1). In general, they are not effective on human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus (SIV). Only SIV strains from African green monkeys are sensitive to several NNRTIs. Here we isolated NNRTI- and 3TC-resistant SIVagm variants. Viruses resistant to delavirdine contained V112I and M231I substitutions, while those resistant to 3TC contained a M 185I substitution. These amino acids are highly conserved in HIV-1, HIV-2, SIVmac and SIVagm, and the M184I (M185I in SIVagm) substitution was observed in 3TC-resistant HIV-1 and SIVmac. The roles of the observed mutations in NNRTI-resistance of SIVagm and HIV-1 were further confirmed by site-directed mutagenesis. The present results have provided a new insight into the common mechanism of sensitivity of HIV- 1 and SIVagm to NNRTIs.

We analyzed 57 HIV-1 isolates from Cameroon and the Republic of Congo, with respect to the env C2V3 and/or the pol integrase regions. The results indicated that the topology of the pol tree correlated well with that of the env tree for four clusters of subtype D, F G and H, suggesting that these trees reflect the true evolution of the overall genome structures of these subtypes. However, of 22 Cameroonian isolates that were classified as subtype A based on env, 20 of them diverged in their pol sequence into two lineages that were completely different from the prototypical subtype A, tentatively designated as subtypes A1 and A2. The subtype A1 isolates (6 out of 22) were related in their env C2V3 regions with prototypical subtype A strain, but in their pol regions, they formed an independent cluster that diverged from known HIV-1 subtypes so far reported (except for subtypes I and J). The subtype A2 isolates (14 out of 22), which represent the major epidemic type of HIV-1 in Cameroon, clustered distinctly in both the env and pol trees with the recently described A/G mosaic strains from Nigeria and Djibouti. These two lineages were not spreading in the neighboring Republic of Congo.

To investigate the role of apoptosis in the pathogenesis of HIV infection we used macaques infected with simian immunodeficiency virus (SIV) as a primate model and examined the characteristics of the apoptosis of lymphocytes in SIV mac-infected macaques. In vitro apoptosis was more strongly induced in peripheral blood mononuclear cells (PBMC) from SIV mac239-infected macaques than those from uninfected controls. We found that the frequency of Fas antigen-positive cells was higher in PBMC from SIV mac-infected macaques than from uninfected controls, and in vitro apoptosis of PBMC was suppressed by an inhibitor of the interleukin-1 beta converting enzyme (ICE) family proteases. In biopsied lymph nodes, the number of apoptotic nuclei in T cell-dependent areas was higher in SIV mac-infected macaques than in uninfected controls. A higher number of apoptotic nuclei in lymph nodes of SIV mac-infected macaques was observed in the stage of persistent general lymphadenopathy than in those with AIDS-related complex, while there was no significant difference in the extent of apoptosis of cultured PBMC among the SIV mac-infected macaques. These results suggest that in vitro apoptosis is mediated by the Fas/Fas ligand and ICE system and that apoptosis in lymph nodes may be more closely related to the stage of SIV mac infection than is that of cultured PBMC.

In order to examine whether the viral population is affected by intramucosal transmission, we analyzed the viral genotypes first detected in peripheral blood mononuclear cells (PBMCs) after intravaginal inoculation, before virus antibodies were detectable, and compared them with those in the inoculum. Three female cynomolgus macaques were inoculated intravaginally and a fourth was inoculated intravenously with polyclonal simian immunodeficiency viruses (SIVmac32H). The provirus genomes which first appeared in PBMC were sequenced in the V1 to V2 region of the SIV envelope gene. A comparison of the sequences obtained from each monkey revealed a homogeneous or heterogeneous viral population depending on the infection route. In the intravenously inoculated monkey, the viral population was heterogenous and was similar to that in the virus inoculum. On the other hand, in the intravaginally inoculated monkeys, single genotypes (in two monkeys) and one genotype with a slight variation (in one monkey) were found, but they were different from each other, having no characteristic sequences in the V1 to V2 region in common. None of the genotypes found in the PBMC were major genotypes in the virus inoculum. These results suggest that some selective mechanism, which differs among individuals, restricts the viral population during mucosal transmission.

To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.

We constructed an infectious molecular clone of simian immunodeficiency virus from an African mandrill (SIVMND). Upon transfection, this clone directed the production of progeny virus particles infectious to and cytopathic for CD4+ human leukemia cells. Thirteen frameshift proviral mutants with an alteration in the eight open reading frames of SIVMND were generated by recombinant DNA techniques, and were analyzed biologically and biochemically. While mutations in the structural genes gag, pol, and env abolished viral growth and induction of cytopathology, mutants of the vif, vpr, and nef genes were fully biologically active. Of the tat and rev mutants, only one rev mutant grew in CD4+ cells with delayed kinetics. In reporter-based transient expression systems, transactivation potentials of the tat and rev mutants were evaluated. A mutant lacking 2nd coding exon of tat gene exhibited tat activity similar to that of the wild type clone. The infectious rev mutant was partially defective for rev gene activity.

The gag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed in Escherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIV gag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.

We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, in gag, pol, and env genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96,95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly, tat- and rev-like short open reading frames contained inframe stop codons in the FIV Petaluma but not in the FIV TM2.

Mutants of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), which have been shown to be infectious in established cell lines, were tested for ability to replicate and induce syncytium formation in human peripheral blood mononuclear cells (PBMC). The vpu mutant of HIV-1 showed depressed kinetics of replication in an established T cell line, as reported previously, but in PBMC, its replication was similar to that of the wild type virus. The vpx gene of HIV-2 was required for efficient virus propagation in PBMC, but not in an established T cell line, as previously reported. However, the growth rates of the vpx mutant in PBMC preparations from two individuals were different. The results of experiments on infection of PBMC with the vif and vpr mutants of HIV-1 and HIV-2 were essentially consistent with previous results of infection of established T cell lines. No negative effect of the nef gene products of HIV-1 and HIV-2 was observed. The abilities of the wild type virus and the mutants of HIV-1 to induce syncytium formation in both PBMC and established cell lines were similar. In contrast, neither the wild type nor any of the mutants of HIV-2 induced syncytium formation in PBMC. These results suggest that the functions of some genes can be detected only in mixed populations or primary cells such as PBMC. Studies on the roles of these genes in PBMC may provide a better understanding of their functions in vivo.

Feline leukemia virus (FeLV) is a horizontally transmitted agent of the domestic cat which is known to be associated with wide spectrum of diseases of the hematopoietic system. In the present study, proviral DNAs of FeLV proviruses were examined in the tumor cells of natural killer cell lineage which is very rare in cats. In the chromosomal DNA of the tumor cells, 5 distinct bands corresponding to exogenous FeLV provirus genomes were detected by digestion with EcoRI which does not cut most FeLV isolates. Five clones of pLC1, pLC2, pLC3, pLC4, and pLC5 obtained from the 5 respective bands were analysed by restriction endonuclease mapping and Southern blot hybridization using gene-specific probes of FeLV. The results have clearly demonstrated that pLC4 and pLC5 contained large deletions in the pol and part of gag regions, while the full-length proviruses could be observed in pLC1 and pLC2. Furthermore, pLC3 contained part of a variant FeLV genome having an EcoRI site in its gag region. The molecular clones of defective and variant FeLV in this study may be useful for the further examination of tumorigenesis of large granular lymphoma in the cat.

Growth of two measles virus strains, the TYCSA and CAM, was compared in three continuous cell lines derived from the nervous tissues, human neuroblastoma IMR-32, human glioma 118MGC, and rat glioma C-6. The two human neural cells were shown to support the growth of both measles virus strains as efficiently as in the non-neural Vero cells. Different types of cytopathic effect (CPE) between the two virus strains were noticed in IMR-32 cells; the CAM strain induced strand-forming type CPE and the TYCSA strain giant-cell type CPE. As a difference of growth pattern between IMR-32 and 118MGC cells, virus antigen was demonstrated in both the nucleus and cytoplasm of 118MGC cells whereas virus antigen was present only in the cytoplasm of IMR-32 cells. In contrast to the productive infection in human neural cells, growth of both virus strains was restricted in rat glioma C-6 cells without showing CPE although the prolonged presence of virus antigens was demonstrated by the immunofluorescent technique.