Evidence for early local viral replication and local production of antiviral immunity upon mucosal simian-human immunodeficiency virus SHIV(89.6) infection in Macaca nemestrina.

Transmission of human immunodeficiency virus type 1 (HIV-1) is largely a result of heterosexual exposure, leading many investigators to evaluate mucosal vaccines for protection against intravaginal (i.vag.) transmission in macaque models of AIDS. Relatively little is known, however, about the dynamics of viral replication and the ensuing immune response following mucosal infection. We have utilized a simian-human immunodeficiency virus (SHIV) to study the differences in viremia, CD4 T-cell percentages, and mucosal and systemic anti-SHIV humoral and cellular immune responses during primary infection of animals infected either intravenously (i.v.) or i.vag. Positive viral cocultures, peripheral blood mononuclear cell viral load peaks, and CD4 cell declines were delayed by 1 week in the i.vag. inoculated animals compared to the animals infected i.v., demonstrating delayed viral spreading to the periphery. In contrast, mucosal anti-SHIV antibody levels were greater in magnitude and arose more rapidly and mucosal CD8(+) T-cell responses were enhanced in the i.vag. group animals, whereas both the magnitudes and times of onset of systemic immune responses for the animals in the two groups did not differ. These observations demonstrate that compartmentalization of viral replication and induction of local antiviral immunity occur in the genital tract early after i.vag. but not i.v. inoculation. Induction of mucosal immunity to target this local, contained replication should be a goal in HIV vaccine development.

Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina).

Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).

Detection of Kaposi's sarcoma-associated herpesvirus in oral and genital secretions of Zimbabwean women.

Kaposi's sarcoma-associated herpesvirus (KSHV) in oral and genital secretions of women may be involved in horizontal and vertical transmission in endemic regions. Nested polymerase chain reaction assays were used to detect KSHV DNA sequences in one-third of oral, vaginal, and cervical specimens and in 42% of peripheral blood mononuclear cell (PBMC) specimens collected from 41 women infected with human immunodeficiency virus type 1 who had Kaposi's sarcoma (KS). KSHV DNA was not detected in specimens from 100 women without KS, 9 of whom were seropositive for KSHV. A positive association was observed between KSHV DNA detection in oral and genital mucosa, neither of which was associated with KSHV DNA detection in PBMC. These data suggest that KSHV replicates in preferred anatomic sites at levels independent of PBMC viremia. Detection of genital-tract KSHV only among relatively immunosuppressed women may provide an explanation for infrequent perinatal transmission of KSHV.

Two distinct lineages of macaque gamma herpesviruses related to the Kaposi's sarcoma associated herpesvirus.

BACKGROUND: KSHV, Kaposi's sarcoma-associated herpesvirus, is a necessary cofactor for the development of Kaposi's sarcoma (KS). We have previously reported KSHV-related DNA sequences in retroperitoneal fibromatosis (RF) tissue from two species of macaque. The putative herpesvirus was called RFHV for RF-associated herpesvirus. These data suggested that KSHV is a human representative of a larger family of primate herpesviruses. OBJECTIVE: To identify and characterize other members of a putative family of KSHV-related herpesviruses in macaques in order to obtain information on the evolutionary history of KSHV infection in humans. STUDY DESIGN: Lymphoid tissue cells and blood leukocytes from rhesus-, cynomolgus- and pigtailed-macaques were tested for the presence of unknown herpesviruses using degenerate primer-driven PCR amplification. The sequences obtained were compared against known herpesvirus sequences. RESULTS: We have identified new herpesvirus DNA sequences in each of the three macaque species. Sequence comparisons indicate that these new viruses are most related to each other and form a separate phylogenetic lineage within the gamma herpesviruses. Screening of PBMC from Indonesian-origin quarantine animals suggests that these viruses (MGV, macaque gamma virus) are species-specific, and highly prevalent in the wild. They are readily cultured in vivo, and share a common tissue tropism with the previously identified RFHV. CONCLUSIONS: MGV and RFHV represent two independent introductions of an ancestral gamma herpesvirus into macaque precursors.

Isolation and partial characterization of a lentivirus from talapoin monkeys (Myopithecus talapoin).

We have identified a novel lentivirus prevalent in talapoin monkeys (Myopithecus talapoin), extending previous observations of human immunodeficiency virus-1 cross-reactive antibodies in the serum of these monkeys. We obtained a virus isolate from one of three seropositive monkeys initially available to us. The virus was tentatively named simian immunodeficiency virus from talapoin monkeys (SIVtal). Despite the difficulty of isolating this virus, it was readily passed between monkeys in captivity through unknown routes of transmission. The virus could be propagated for short terms in peripheral blood mononuclear cells of talapoin monkeys but not in human peripheral blood mononuclear cells or human T cell lines. The propagated virus was used to infect a naive talapoin monkey, four rhesus macaques (M. mulatta), and two cynomolgus macaques (M. fascicularis). All animals seroconverted and virus could be reisolated during a short period after experimental infection. A survey of SIVtal-infected captive talapoin monkeys revealed a relative decrease in CD4(+) cell numbers in chronically (>2 years) infected animals. No other signs of immunodeficiency were observed in any of the infected animals. PCR amplification followed by DNA sequencing of two fragments of the polymerase gene revealed that SIVtal is different from the presently known lentiviruses and perhaps most related to the SIV from Sykes monkeys.

Activation in vivo of retroperitoneal fibromatosis-associated herpesvirus, a simian homologue of human herpesvirus-8.

Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposi's sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans.

In vitro HIV-1 infection in Macaca nemestrina PBMCs is blocked at a step beyond reverse transcription.

Various stages in the lifecycle of HIV-1 were investigated in Macaca nemestrina and humans in vitro. Early events were analyzed by end point dilution DNA PCR with HIV-1 and SHIV infected PBMCs, while p24 and p27 ELISA assays were used to analyze core antigen production from infected cells. The results demonstrate that a step in the virus life cycle, beyond reverse transcription is blocked for HIV-1 infection in macaque cells.

Serial in vivo passage of HIV-1 infection in Macaca nemestrina.

In an earlier study we found that pigtailed macaques (Macaca nemestrina) that were experimentally infected with human immunodeficiency virus type 1 (HIV-1) initially became viremic and seroconverted, but HIV-1 replication diminished markedly over time. In an attempt to develop a longer term pathogenic model, blood from HIV-1-infected macaques was serially transfused into three groups of naive macaques. Transfer was successful through two transfusions as shown by repeated virus isolations and confirmed by the development of cell-free plasma viremia and by seroconversion. Three to five weeks after transfusion, plasma levels of HIV-1 RNA from several macaques in the first two groups exceeded those of the initially inoculated macaques. However, animals in the third group had diminished RNA levels, were virus culture negative, and did not seroconvert. Sequence analyses of env-region clones from infected animals revealed only minimal changes over the course of the passages. These results confirm HIV-1 replication in M. nemestrina during the acute phase of infection. However, adaptation of HIV-1 to a macaque-pathogenic variant did not occur during serial passage, possibly because the animals were able to restrict HIV-1 replication below a level required for a pathogenic variant to emerge. Whether such containment is a function of the host's immune response or a virus cell incompatibility remains to be determined.

Infection of Macaca nemestrina neonates with HIV-1 via different routes of inoculation.

OBJECTIVES: Receptive anal intercourse but not orogenital sex has been identified as a major risk factor for transmission of HIV-1. Recent studies using simian immunodeficiency virus (SIV) in rhesus macaques have demonstrated relatively efficient infection following oral administration, indicating that modes of transmission may vary between HIV-1 and SIV. Here, we investigate whether HIV-1 infection of macaques via the oral route is more efficient than via the rectal route. DESIGN: Eleven Macaca nemestrina neonates were exposed to HIV-1 via different routes (four oral, two intravenous, and five rectal). One animal was orally inoculated with a sham inoculum and two control animals were not exposed. METHODS: All animals were followed for virological signs of infection, and for pathogenesis associated with HIV-1 infection by general physical examinations, complete blood cell counts and lymphocyte subset analysis, and full necropsies. RESULTS: Three out of five rectally exposed macaques and both of the intravenously inoculated animals became infected with HIV-1, whereas none of the orally exposed animals showed evidence of HIV-1 infection. Clinical observations following exposure included failure to thrive in the orally inoculated animals and low CD4/CD8 ratios in the rectally exposed macaques. CONCLUSIONS: The finding that, contrary to what has been reported for SIV, transmission of HIV-1 via the oral route is not more efficient than via the rectal route, indicates important biological differences between HIV-1 and SIV, with direct implications for the spread of HIV and associated AIDS, and for development of anti-HIV-1 vaccines.

Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species.

Simian retroperitoneal fibromatosis (RF) is a vascular fibroproliferative neoplasm which has many morphological and histological similarities to human Kaposi's sarcoma (KS). Like epidemic KS in AIDS patients, RF is highly associated with an immunodeficiency syndrome (simian acquired immunodeficiency syndrome [SAIDS]) caused by a retrovirus infection. Recently, a new gammaherpesvirus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), has been identified in KS tumors, suggesting that KS has a viral etiology. Our previous experimental transmission studies and epidemiological data suggest that RF also has an infectious etiology. In order to determine whether a similar virus is also associated with RF, we have assayed for the presence of an unknown herpesvirus using degenerate PCR primers targeting the highly conserved DNA polymerase genes of the herpesvirus family. Here we provide DNA sequence evidence for two new herpesviruses closely related to KSHV from RF tissues of two macaque species, Macaca nemestrina and Macaca mulatta. Our data suggest that KSHV and the putative macaque herpesviruses define a new group within the subfamily Gammaherpesvirinae whose members are implicated in the pathogenesis of KS and KS-like neoplasms in different primate species.

Antigenicity and immunogenicity of recombinant envelope glycoproteins of SIVmac32H with different in vivo passage histories.

Shortly after infection of two rhesus monkeys (Macaca mulatta) either with a SIVmac32H challenge stock or with the same virus that had been passaged in another rhesus monkey for 11 months, SIV-envelope genes were cloned from their peripheral blood mononuclear cells and subsequently expressed by recombinant vaccinia viruses. The molecular weights and antigenicities of the thus produced envelope glycoproteins were largely identical to those of the native SIV. The envelope glycoprotein derived from the in vivo passaged virus proved to be poorly recognized by virus neutralizing monoclonal antibodies directed against one of the seven antigenic sites for which monoclonal antibodies were available. Immunization studies in rats showed that this protein was also less efficient in inducing antibodies against this antigenic site, and that it induced significantly lower levels of virus neutralizing antibodies than the other SIV-envelope glycoprotein. The immunogenicity of the SIV-envelope glycoprotein incorporated into immune stimulating complexes (iscoms) was compared to that of the same protein presented with Quil A or MDP-tsl.

Neutralization of feline immunodeficiency virus by polyclonal feline antibody: simultaneous involvement of hypervariable regions 4 and 5 of the surface glycoprotein.

Sites involved in antibody-mediated neutralization of feline immunodeficiency virus were mapped by reciprocal exchange of envelope fragments or amino acids between molecular clones of feline immunodeficiency virus with different susceptibilities to neutralization by a polyclonal cat serum. Combinations of mutations within HV-4 or within HV-4 and HV-5 changed the susceptibility of the viruses to neutralizing antibody.

Impact of natural sequence variation in the V2 region of the envelope protein of human immunodeficiency virus type 1 on syncytium induction: a mutational analysis.

Several studies have demonstrated a functional role for the V1-V2 region of the human immunodeficiency virus type 1 (HIV-1) envelope surface glycoprotein gp120 in the membrane fusion processes underlying viral entry and syncytium induction. In a study with chimeric primary envelope genes, we have previously demonstrated that the exchange of V2 regions was sufficient to transfer syncytium-inducing capacity to a non-syncytium-inducing envelope protein. The exchanged V2 regions, comprising a number of variable amino acids, conferred changes to both the predicted secondary structure and to the net positive charge of the V2 loops. In a syncytium-forming assay based on transient envelope protein expression in CD4+ SupT1 cells, we have extended this observation by mutating the variable positions of the V2 region to determine the relative contribution of individual amino acids to syncytium formation. It can be shown that simultaneous mutation of multiple amino acids is needed to interfere with the V2 region-determined syncytium-inducing phenotype. Single amino acid changes either influencing charge of predicted secondary structure of the V2 loop proved to be insufficient to abolish V2 region-controlled syncytium formation. This robust V2 organization may allow the virus to accumulate mutations, while retaining its biological phenotype.

Two different mutations in the envelope protein of feline immunodeficiency virus allow the virus to escape from neutralization by feline serum antibodies.

Viral progeny of two molecular clones of feline immunodeficiency virus (FIV), 19k1 and 19k32, were tested in a virus neutralization assay. In this assay the infection of thymocytes with FIV19k1 was neutralized by serum S1422, derived from an SPF cat 22 weeks after infection with FIV19k1. We previously reported that a point mutation at position 560 in hypervariable region-5 (HV-5) of 19k1 confers resistance to virus neutralization (Siebelink et al., 1993, J. Virol. 67:2202-2208). Viral progeny of the other molecular clone, FIV19k32, which differs in the envelope protein in only six amino acids from 19k1, was not neutralized. In order to map sites involved in virus neutralization we constructed chimeric clones by reciprocal exchange of 19k1 and 19k32 envelope gene fragments. Reciprocal exchange of a 1662 bp fragment, encoding almost the whole surface protein, which differs in five amino acids between these two clones, resulted in exchange of the phenotype. Amino acids of the envelope protein of 19k1 and 19k32, in which these clones differ, were substituted by point mutation. We demonstrated that one of these mutations, a substitution of leucine to serine at position 483 in HV-4, also conferred resistance of 19k1 to neutralization by serum S1422.

A determinant of feline immunodeficiency virus involved in Crandell feline kidney cell tropism.

Viral progeny of the molecular clone 19k1 of feline immunodeficiency virus (FIV) can infect feline T-cells but not Crandell feline kidney (CrFK) cells. In contrast, the biological isolate FIV-AM6c, which was CrFK adapted by co-cultivation of FIV-AM6 infected thymocytes with CrFK cells, can infect both thymocytes and CrFK cells. The envelope gene of FIV-AM6c was amplified by polymerase chain reaction using DNA from infected CrFK cells, and subsequently cloned and sequenced. To map viral determinants of CrFK cell tropism, chimeric viruses with a 19k1 background containing envelope gene fragments of FIV-AM6c were constructed. CrFK cells were transfected with DNA of these chimeric clones and co-cultivated with thymocytes. After 3 days the CrFK cells and the thymocytes were cultured separately. FIV antigen could be detected in most of the thymocyte cultures within 14 days and in one of the CrFK cultures after 52 days. The resulting virus from this CrFK culture can infect both CrFK cells and thymocytes. The results of this study indicate that the envelope region contains determinants of CrFK tropism. The delay in replication indicates that also determinants other than those identified here are involved in CrFK cell tropism. More chimeric clones are being studied at present to map these determinants.

Enhancement of infectivity of a non-syncytium inducing HIV-1 by sCD4 and by human antibodies that neutralize syncytium inducing HIV-1.

Enhancement of virus infectivity after sCD4 treatment has been documented for SIVagm and HIV-2. It has been suggested that a similar phenomenon may play a role in HIV-1 infection. In the present study we have analysed biological activities of virus neutralizing polyclonal and monoclonal human antibodies and of sCD4, towards HIV-1 chimeras with envelope proteins derived from one donor, which display different biological phenotypes. The antibodies, which recognize the V3 and/or the CD4 binding domains of the glycoproteins of these viruses and also sCD4 showed different levels of virus neutralizing activity toward the syncytium inducing HIV-1 strains. In contrast, they all dramatically enhanced the infectivity of an HIV-1 chimera with an envelope glycoprotein displaying the non-syncytium-inducing phenotype. Given the relatively conserved nature of non-syncytium-inducing HIV-1 surface glycoproteins early after infection, these data suggest a major role for antibody mediated enhancement of virus infectivity in the early pathogenesis of HIV-1 infection.

Insertion of N-linked glycosylation sites in the variable regions of the human immunodeficiency virus type 1 surface glycoprotein through AAT triplet reiteration.

Variable regions with sequence length variation in the human immunodeficiency virus type 1 envelope exhibit an unusual pattern of codon usage with AAT, ACT, and AGT together composing > 70% of all codons used. We postulate that this distribution is caused by insertion of AAT triplets followed by point mutations and selection. Accumulation of the encoded amino acids (asparagine, serine, and threonine) leads to the creation of new N-linked glycosylation sites, which helps the virus to escape from the immune pressure exerted by virus-neutralizing antibodies.

Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation.

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.

A single amino acid substitution in hypervariable region 5 of the envelope protein of feline immunodeficiency virus allows escape from virus neutralization.

We infected a specific-pathogen-free cat (cat 14) with molecularly cloned feline immunodeficiency virus clone 19k1 (FIV19k1 [K. H. J. Siebelink, I. Chu, G. F. Rimmelzwaan, K. Weijer, A. D. M. E. Osterhaus, and M. L. Bosch, J. Virol. 66:1091-1097, 1992]). Serum of this cat obtained 22 weeks postinfection (serum 1422) neutralized FIV19k1 but not FIV19k32, which is 99.3% identical to FIV19k1 in the envelope gene. Serum 1422 also neutralized virus isolated from cat 14 at weeks 2 and 32 postinfection. We then cultured FIV19k1 in the continuous presence of serum 1422, which resulted in a delay in virus replication of 6 weeks. The resulting virus population appeared to be resistant to virus neutralization by serum 1422. Nucleotide sequencing of the env open reading frame of this presumed escape mutant revealed the presence of one silent and two substitution mutations, both of the latter in hypervariable region 5. Through the construction of chimeric viruses and site-directed mutagenesis, we demonstrated that one of these mutations, the substitution of lysine to glutamine at amino acid position 560 in hypervariable region 5, was sufficient to allow the escape of FIV19k1 from neutralization by serum 1422.