Antigens of human T-lymphotropic virus type III/lymphadenopathy-associated virus

Antigens encoded by the gag and env genes of the human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) include a p55 gag polyprotein that yields p24 as the major virus core protein, and an env gene polyprotein, gp 160, that produces gp 120, the most immunogenic protein in humans, at the amino terminus. Although its use is limited to research laboratories due to the cost and specialized procedures involved, the analysis of sera by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the test providing the optimal balance of specificity and sensitivity. Because the gp 120 represents the external virus protein, it would be the most appropriate antigen for vaccine development. Also viruses serologically related to HTLV-III/LAV were detected recently in two species of Old World monkeys. Because about half the healthy African green monkeys appear to have been exposed to simian T-lymphotropic virus type III (STLV-III), a related agent of the species, a characterization of the STLV-III gp 120 and immune response of the host may provide additional information for vaccine development.     

Failure to detect evidence of human T-lymphotropic virus (HTLV) type I and type II in blood donors with isolated gag antibodies to HTLV-I/II.

Of the 267,650 blood donations from members of the US armed forces, 72 (0.027%) were serologically confirmed to be positive for human T-lymphotropic virus type I/II (HTLVpos) and 379 (0.14%) were Western blot (WB)-indeterminate with banding pattern restricted to the proteins encoded by the gag gene only (HTLVind). To determine whether these apparently healthy HTLVind blood donors are infected with HTLV-I or HTLV-II, coded specimens from randomly selected military blood donors (n = 73) were tested for antibodies to HTLV by WB and radioimmunoprecipitation assay (RIPA) using HTLV-I (MT-2) antigens, by enzyme immunoassay using synthetic peptides representing the immunodominant epitopes of HTLV, and for sequences of proviral HTLV DNA by the polymerase chain reaction (PCR). Of the 73 HTLVind donors, none showed presence of env reactivity by HTLV WB and RIPA. Minimal reactivity was observed with synthetic immunodominant motifs derived from the env protein of HTLV-I (Env-1(191-214) and Env-5(242-257)) or HTLV-II (Env-2(187-209) and Env-20(85-102)) and gag protein (Gag-1a(102-117) and Gag-10(364-385)). A peptide corresponding to the endogenous retroviral sequence with structural homologies to the gag protein of HTLVs (RTVLgag) reacted with antibodies not only in HTLVpos (88%) and HTLVind (42% to 66%) specimens, but also reacted with normal control subjects (60%). Furthermore, none of the 73 HTLVind specimens demonstrated presence of the HTLV genome when amplified with primers for the pol and tax/rex region. Six to 23 months from the initial test, 27 subjects still gave indeterminate WB patterns, and 13 of these repeat specimens were still negative for the presence of HTLV genome. We conclude that individuals at low risk for HTLV infection who have HTLVind WB reactivity are rarely, if ever, infected with HTLV-I or HTLV-II.     

Characterization of a new simian T-lymphocyte virus type 1 (STLV-1) in a wild living chimpanzee (Pan troglodytes verus) from Ivory Coast: evidence of a new STLV-1 group?

A new strain of simian T-lymphotropic virus type 1 in blood samples from a chimpanzee that lived in the tropical rainforest of Ivory Coast is described. The sequence obtained from the long terminal repeat region of the genome is significantly divergent from all known human and nonhuman primate T-lymphotropic virus type 1 strains (963% homology to the closest related strains from Central African subtype B) and clusters with none of the established clades. The tax sequences reveal two sequence differences that seem to be unique as they are not found in any of the HTLV-1 or STLV-1 tax sequences from the public databases.     

Human T-cell leukemia virus type I tax/rex DNA and RNA in cutaneous T- cell lymphoma

Peripheral blood mononuclear cells (PBMCs) and T-cell lines from patients with Sezary syndrome (SS) and skin lesions from patients with mycosis fungoides (MF) were examined by polymerase chain reaction (PCR) for DNA sequences homologous to the human retroviruses human T- lymphotropic virus (HTLV)-I and -II. Results obtained using primers and probes from the tax/rex region of HTLV-I indicate that 72% (18/25) of SS patients PBMCs, 80% (20/25) of T-cell lines established from SS- PBMC, and 30% (3/10) of skin lesions from MF patients were positive for HTLV-I tax/rex region DNA. Sequence analysis of the 127-bp fragment amplified by the tax/rex primers from 4 of these individuals was found to be identical to that in prototypic HTLV-I. Negative results were obtained using primers and probes from the HTLV-I gag region and the HTLV-II gag and tax regions. No PCR products were obtained using all primers and probes using DNA from 9 healthy blood donors and 10 cord bloods. Expression of HTLV-I tax/rex mRNA was found in 4 of 8 Sezary patients, as determined by RNA-PCR, indicating that this viral region is being transcribed in vivo. Exposure to Tax/Rex protein in SS- patients is supported by the fact that serum antibodies against p27rex and p40tax was observed in 43% and 29% of these SS patients, respectively. Although the causal relationship between the HTLV-I tax/rex region and cutaneous T-cell lymphoma (CTCL) remains unclear, these findings support the presence of a truncated HTLV-I retrovirus in CTCL patients.     

Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters

The human T-lymphotropic viruses (HTLVs) types 1 and 2 originated independently and are related to distinct lineages of simian T-lymphotropic viruses (STLV-1 and STLV-2, respectively). These facts, along with the finding that HTLV-1 diversity appears to have resulted from multiple cross-species transmissions of STLV-1, suggest that contact between humans and infected nonhuman primates (NHPs) may result in HTLV emergence. We investigated the diversity of HTLV among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. We show that this population is infected with a wide variety of HTLVs, including two previously unknown retroviruses: HTLV-4 is a member of a phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the phylogenetic diversity of STLV-3, a group not previously seen in humans. We also document human infection with multiple STLV-1-like viruses. These results demonstrate greater HTLV diversity than previously recognized and suggest that NHP exposure contributes to HTLV emergence. Our discovery of unique and divergent HTLVs has implications for HTLV diagnosis, blood screening, and potential disease development in infected persons. The findings also indicate that cross-species transmission is not the rate-limiting step in pandemic retrovirus emergence and suggest that it may be possible to predict and prevent disease emergence by surveillance of populations exposed to animal reservoirs and interventions to decrease risk factors, such as primate hunting.     

Highly divergent molecular variants of human T-lymphotropic virus type I from isolated populations in Papua New Guinea and the Solomon Islands.

To determine the molecular genetic relationship between Melanesian strains of human T-lymphotropic virus type I (HTLV-I) and cosmopolitan prototype HTLV-I, we amplified by PCR, then cloned, and sequenced a 522-base-pair region of the HTLV-I env gene in DNA extracted from uncultured (fresh) and cultured peripheral blood mononuclear cells obtained from six seropositive Melanesian Papua New Guineans and Solomon Islanders, including a Solomon Islander with HTLV-I myeloneuropathy. Unlike isolates of HTLV-I from Japan, the West Indies, the Americas, and Africa, which share greater than or equal to 97% sequence homology, the Melanesian strains of HTLV-I were only 91.8%-92.5% identical with a prototype Japanese HTLV-IATK-1. The nucleotide sequence of proviral DNA from the Solomon Islander with HTLV-I myeloneuropathy also diverged markedly from that of HTLV-I isolated from Japanese patients with HTLV-I-associated myelopathy and from Jamaican patients with tropical spastic paraparesis, suggesting that these variant viruses are capable of causing disease. The HTLV-I variants from Papua New Guineans, in turn, differed by nearly 4% from the Melanesian variants from Solomon Islanders, indicating the existence of another HTLV-I quasi-species. By contrast, HTLV-I strains from two residents of Bellona Island, a Polynesian Outlier within the Solomon Islands, were closely related to cosmopolitan prototype HTLV-I (greater than or equal to 97% sequence identity), suggesting recent introduction, possibly during this century. These findings are consistent with a proto-Melanesian HTLV-I strain of archaic presence, which evolved independently of contemporary cosmopolitan strains, and pose new questions about the origin and global dissemination of HTLV-I.     

Lack of BLV and PTLV DNA sequences in the majority of patients with large granular lymphocyte leukaemia

The primate T-cell lymphoma/leukaemia viruses (PTLV) and bovine leukaemia virus (BLV) comprise a unique genus of retroviruses, infection with which induces seroreactivity in the host against conserved epitopes in their p24 gag and gp21 env cognate proteins. Herein, we have confirmed this serocrossreactivity. Patients with large granular lymphocyte (LGL) leukaemia have frequent seroreactivity to the p24 and gp21 env proteins of human T-cell lymphoma/leukaemia virus I (HTLV-I), one of the species in the genus. However, only a small minority of patients are actually infected with prototypic HTLV-I or HTLV-II, another species within the group. In an attempt to determine whether LGL leukaemia might be associated with other members of the PTLV/BLV genus, we examined the peripheral blood mononuclear cell DNA of 22 HTLV p24 and/or gp21 seropositive LGL leukaemia patients via PCR using degenerate and specific primer pair/probe systems capable of detecting all known members of the PTLV/BLV genus. None of the samples was positive. These data indicate that although HTLV-II may be associated with some cases of LGL leukaemia most patients are not infected with a PTLV or BLV virus.     

Defective human T-lymphotrophic virus type I provirus in T-cell prolymphocytic leukaemia

T-cell prolymphocytic leukaemia (T-PLL) is a rare form of post-thymic T-cell neoplasm, the aetiology of which remains unknown. We examined human T-lymphotropic virus (HTLV) provirus in five HTLV-I/II seronegative patients with T-PLL. Southern blotting did not show monoclonal integration of the HTLV-I genome in any of the DNA samples. However, two of the five DNA samples contained an HTLV-I tax sequence. Other sets of oligonucleotide primers for HTLV-I gag, pol, env and LTR regions were all negative. HTLV-I tax gene expression and p40tax antibody were not detected in samples from cases with HTLV-I tax sequence. Our findings suggest that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis, in which HTLV-I genome insertion triggers T-PLL but the deletion of various regions of the integrated provirus subsequently prevents active replication and the expression of the virus.     

New insights in HTLV-I phylogeny by sequencing and analyzing the entire envelope gene

The HTLV-I envelope plays a major role in the process of target cell infection. It is implied in the recognition of the viral receptor(s), penetration of the viral genetic material, and induction of host immunity to the virus. It is thus important to study the genetic variability of the viral env gene as well as its variation in terms of evolution. In a new approach to these features, we sequenced the entire env gene of 65 HTLV-I isolates originating from Gabon, French Guiana, West Indies, and Iran, such isolates representing all major HTLVI phylums but the Australo-Melanesian one. The sequences obtained and all PTLV-I (HTLV-I and STLV-I) env sequences available in the literature were analyzed. Phylogenetic studies using different algorithms (minimum evolution, neighbor joining, maximum parsimony, and maximum likelihood) gave the same clear-cut results. Newly sequenced HTLV-I isolates described in this report allocated in three well-defined subtypes: Cosmopolitan, Central African, and a new distinct one that we termed "Maroni" subtype (present in the Maroni Basin, French Guiana, and West Indies). Clearly, the most divergent PTLV-I strains present in Asia- Australo-Melanesia as well as African and Asian STLV-I derived from the same node in the phylogenetic tree as isolates of the Central African subtype. In addition, we showed that within each PTLV-I subtype, groups of isolates may be characterized by nonrandom and systematically associated mutations.     

Evaluation of enzyme immunoassay using a recombinant envelope protein expressed in insect cells for serological confirmation of HTLV-I infection.

A recombinant human T-lymphotropic virus type I (HTLV-I) envelope protein expressed in insect cells using a recombinant baculovirus was employed as the antigen in an enzyme immunoassay (renvEIA). Peripheral blood samples were obtained from asymptomatic carriers or healthy individuals. Plasma was tested for HTLV-I antibody by renvEIA, particle agglutination, and Western immunoblot (WB), and lymphocyte DNA was tested for HTLV-I proviral DNA amplification by polymerase chain reaction (PCR). Of 61 people aged 9 months or older, 23 were positive (gag+, env+) and 19 others were in the "indeterminate" category (gag+, env-) when their WB results were interpreted according to the WHO-proposed criteria. Thirty-seven cases, including all of the WB+ cases and 14 of 19 WB indeterminate cases, were positive by renvEIA. In 34 of 37 renvEIA-positive cases, the presence of long terminal repeat (LTR) and tax/rex region of HTLV-I proviral DNA was detected by polymerase chain reaction (PCR) and following Southern blot hybridization. Thus, renvEIA would be a useful supplemental assay to confirm the presence of HTLV-I antibody in HTLV-I asymptomatic carriers.     

Deleted HTLV provirus in peripheral blood cells of a patient with T-cell prolymphocytic leukaemia

We describe the first case of T-cell prolymphocytic leukaemia (T-PLL) in which the peripheral blood cells contained a human T-lymphotropic virus (HTLV) related tax sequence. Serum screening tests for anti-HTLV-I/II antibodies were negative. Polymerase chain reaction disclosed the presence of an HTLV-I tax sequence in the peripheral blood. Other sets of oligonucleotide primers for HTLV-I gag , pol , env and the long terminal repeat regions and for the HTLV-II pol region were negative in the DNA of the cells. Although patients with T-PLL have been reported to be seronegative for HTLV-I, our findings point to the possibility that HTLV-I infection might be involved in the aetiology of at least some cases of T-PLL and that there may be alternative mechanisms involved in HTLV-associated leukaemogenesis.     

Nucleotide sequence analyses of partial envgp46 gene of human T-lymphotropic virus type I from inhabitants of Fujian Province in Southeast China

Partial sequences from the env(gp46) gene of two human T-lymphotropic virus type I (HTLV-I) isolates (LIN and WEN) obtained from inhabitants of Fujian Province in southeast China were analyzed. A phylogenetic tree was constructed from these sequence data and those of other known HTLV-I isolates from all over the world. Comparisons of the LIN and WEN nucleotide sequences with other HTLV-I isolates showed diversity ranging from 0.73 to 7.00% for LIN and from 0.87 to 7.00% for WEN. Sequences of isolates LIN, WEN, MT-2, TSP1, and CH were most closely related, and the phylogenetic tree showed that all belong to the widespread subtype A of the cosmopolitan group. These preliminary data indicate that HTLV-I isolates from Fujian Province, China are closely related to HTLV-I strains from Japan and the Caribbean.     

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).     

Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.     

Isolation and characterization of simian immunodeficiency viruses from two subspecies of African green monkeys

Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.     

Predominance of subtype A and G HIV type 1 in Nigeria, with geographical differences in their distribution

The purpose of this study was to generate data on the relative prevalences of the HIV-1 subtypes circulating in Nigeria. A total of 252 HIV-1-positive samples collected during an epidemiologic survey conducted in April 1996 were genetically characterized by HMA (heteroduplex mobility assay) and/or sequencing. Samples were collected in Lagos, Calabar, Kano, and Maiduguri. Overall, the predominant env subtypes were A (61.3%) and G (37.5%). Subtype A is more prevalent in the south (p < 0.001), about 70% in Lagos and Calabar, whereas a quarter of the samples was classified as subtype G in these states. In contrast, subtype G is predominant in the north ( < 0.001), representing 58% of the samples in Kano. In the northeastern region, Maiduguri, almost similar proportions of subtype A and G were seen, 49 and 47.4%, respectively. A total of 37 samples was also sequenced in the p24 region from the gag gene; 13 (35%) had discordant subtype designations between env and gag. The majority of the gag (12 of 17) and env (14 of 22) subtype A sequences clustered with the A/G-IBNG strain. Within subtype G, three different subclusters were seen among the envelope sequences. These different subclusters are observed among samples obtained from asymptomatic individuals and AIDS patients from the four Nigerian states studied. In conclusion, we observed a limited number of HIV-1 subtypes circulating in Nigeria, with subtypes A and G being the major env subtypes responsible for the HIV-1 epidemic. Nevertheless, the high rate of recombinant viruses (A/G) and the different A/G recombinant structures indicate a complex pattern of HIV-1 viruses circulating in this country.