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As vertical transmission of HIV-1 is an ongoing problem in East Africa, we analyzed HIV-1 strains of infected mothers, from Kisumu, Kenya. We sequenced the gag and gp120 regions from peripheral blood mononuclear cells (PBMC) of 15 HIV-infected mothers attending an antenatal clinic. PCR, cloning, bootscanning, using the program Simplot, and phylogenetic analyses were conducted to assign subtypes and identify recombinants. Our analyses showed two dual infections from patients who had infections with pure subtypes and recombinants subtype D. In addition, we also noted the presence of subsubtype A1 and A2, as well as unique recombinants in this area. These results imply that the HIV epidemic in western Kenya is a dynamic one and is continually evolving. Therefore, continued monitoring of the epidemic in this region is necessary if a vaccine for the area is to be developed.

The combination of unique single nucleotide polymorphisms in the CCR5 regulatory and in the CCR2 and CCR5 coding regions, defined nine CCR5 human haplogroups (HH): HHA-HHE, HHF*1, HHF*2, HHG*1, and HHG*2. Here we examined the distribution of CCR5 HH and their association with HIV infection and disease progression in 36 HIV-seronegative and 76 HIV-seropositive whites from North America and Spain [28 rapid progressors (RP) and 48 slow progressors (SP)]. Although analyses revealed that HHE frequencies were similar between HIV-seronegative and HIV-seropositive groups (25.0% vs. 32.2%, p > 0.05), HHE frequency in RP was significantly higher than that in SP (48.2% vs. 22.9%, p = 0.002). Survival analysis also showed that HHE heterozygous and homozygous were associated with an accelerated CD4 cell count decline to less than 200 cells/microL (adjusted RH 2.44, p = 0.045; adjusted RH = 3.12, p = 0.037, respectively). These data provide further evidence that CCR5 human haplogroups influence HIV-1 disease progression in HIV-infected persons.

The high genetic diversity of HIV-1 continues to complicate effective vaccine development. To better understand the extent of genetic diversity, intersubtype recombinants and their relative contribution to the HIV epidemic in Kenya, we undertook a detailed molecular epidemiological investigation on HIV-1-infected women attending an antenatal clinic in Kisumu, Kenya. Analysis of gag-p24 region from 460 specimens indicated that 310 (67.4%) were A, 94 (20.4%) were D, 28 (6.1%) were C, 9 (2.0%) were A2, 8 (1.7%) were G, and 11 (2.4%) were unclassifiable. Analysis of the env -gp41 region revealed that 326 (70.9%) were A, 85 (18.5%) D, 26 (5.7%) C, 9 (2.0%) each of A2 and G, 4(0.9%) unclassifiable, and 1 (0.2%) CRF02_AG. Parallel analyses of the gag-p24 and env-gp41 regions indicated that 344 (74.8%) were concordant subtypes, while the remaining 116 (25.2%) were discordant subtypes. The most common discordant subtypes were D/A (40, 8.7%), A/D (27, 5.9%), C/A (11, 2.4%), and A/C (8, 1.7%). Further analysis of a 2.1-kb fragment spanning the gag-pol region from 38 selected specimens revealed that 19 were intersubtype recombinants and majority of them were unique recombinant forms. Distribution of concordant and discordant subtypes remained fairly stable over the 4-year period (1996-2000) studied. Comparison of amino acid sequences of gag-p24 and env-gp41 regions with the subtype A consensus sequence or Kenyan candidate vaccine antigen (HIVA) revealed minor variations in the immunodominant epitopes. These data provide further evidence of high genetic diversity, with subtype A as the predominant subtype and a high proportion of intersubtype recombinants in Kenya.

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.

The molecular diversity and phylogenetic relationship of 22 HIV-1 group O strains were examined on the basis of the protease gene and the N-terminal region of gp41env. Analysis of the newly characterized protease sequences with 12 reference sequences revealed no specific clustering patterns, despite the distinct geographic locations of the specimens. In contrast, analysis of the newly sequenced gp41 sequences with 34 published sequences revealed two distinct clusters, each represented by one full-length sequence (MVP5180 and ANT-70). Further, four of the specimens classified as group O in the protease region clustered with group M in the gp41 region (three subtype A and one subtype G, respectively), suggesting dual and/or recombinant infections with HIV-1 groups M and O. The presence of two distinct clusters in the gp41 region indicates at least two possible subtypes within group O viruses, and this may provide useful information regarding molecular epidemiological studies of group O infections.

We analyzed HIV-1 genetic variability, phylogenetic relationships, and association with transmission modes among 58 HIV-1-infected patients from Buenos Aires City, Argentina. The 58 strains were classified as envgp41 HIV-1 group M subtype B (n = 34) and subgroup F1 of subtype F (n = 24). Potential recombinants combining parts of viral regions from different subtypes, Bprot/Fenv and Fprot/Benv, were found in two patients, and a dual infection with HIV-1 prot subtypes B and F was identified in one individual. Epidemiologic analysis of behavioral risks revealed that the frequency of infection with subtype F viruses was significantly higher (p < 0.0001) among heterosexual patients (71%) compared with homosexual patients (11%). The spread of non-B subtypes into heterosexual populations may be more common than previously thought. Our findings provide important information for monitoring the transmission of HIV-1 strains among different risk groups in Argentina as well as for vaccine development.

A longitudinal study, spanning 25 years and great demographic and cultural change, found a persistently high prevalence of human T-lymphotropic virus type II (HTLV-II) in the Xikrin Kayapo Indians of Brazil. More than 10% of the children continue to develop immune reactions to the virus in infancy, a sharp increase in seroprevalence occurs between ages 15 and 30 years, and prevalence in older women still approaches 100%. This suggests that the major modes of transmission (breast milk and sexual activity) have not changed. The demonstration of stable maintenance of HTLV-II in one ethnic group makes migration theories of its dispersal more plausible. However, the infection may not be a negligible burden on population survival: at least 1 of 62 persons followed until age 40 years died of possible tropical spastic paraparesis (TSP).

Long terminal repeat (LTR)-based restriction fragment length polymorphism (RFLP) analysis of human T cell lymphotropic virus type II (HTLV-II) from 17 seropositive Kayapo Indians from Brazil showed that all 17 samples contained a unique HTLV-IIa subtype (A-II). Additional RFLP screening demonstrated the presence of this subtype in two of three Brazilian blood donors and a Mexican prostitute and her child. In contrast, 129 samples from blood donors and intravenous drug users (IDUs) from the United States, two Pueblo Indian samples, five samples from Norwegian IDUs, and two samples from blood donors from Denmark were all found to be a different HTLV-IIa subtype (A-III). Phylogenetic analysis of two Kayapo and one Mexican LTR sequences showed that they cluster with a subtype A-II sequence from a Brazilian blood donor and with sequences from two prostitutes from Ghana and Cameroon. These results demonstrate that infection with the A-II subtype is endemic among the Kayapo Amerindians, has disseminated to non-Indian populations in Brazil, and is also present in Mexico. Furthermore, the A-II subtype does not appear to represent an origin for the HTLV-IIa infection in urban areas of the United States and Europe. This study provides evidence that HTLV-IIa may be a Paleo-Indian subtype as previously suggested for HTLV-IIb.

Until recently, HTLV-I was considered to be an Old World virus and HTLV-II was thought to be endemic in the Americas. However, the presence of HTLV-II among Pygmies and other populations of Africa has raised doubts as to whether HTLV-II is primarily a New World virus. The large serosurveys conducted in the urban and rural areas of southern Ghana have identified a 1-2% prevalence for HTLV-I/II. To define the HTLV type, we have used a Western blot assay (HTLV-2.3 blot) that allows simultaneous confirmation and differentiation between HTLVs. Samples (n = 139) were chosen on the basis of previous reactivity with either an enzyme immune assay or r21e-spiked WB results. The WB 2.3 analysis of these specimens identified 55 (40%) to be HTLV positive, 70 (50%) to be HTLV indeterminant, and 14 (10%) to be HTLV negative for HTLV. HTLV seroindeterminant patterns ranged from both gag and env (14 were r21+, p24+, and/or p19+ [all were RIPA negative]) to gag only (21 were p24+/p19+, 16 were p19+, and 7 were p24+), and env only (8 were r21+ and 4 were rgp46+) reactivities. Of the 55 HTLV-positive specimens, 41 were typed as HTLV-I, 9 were HTLV-II, and 5 could not be typed (HTLV-I/II). Of the nine HTLV-II-positive specimens, three were from patients with Burkitt's lymphoma and six were from healthy individuals (two pregnant women) with no obvious risk factors for HTLV-II.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary amino acid sequences derived from the gag, pol, and env gene products of human T-cell lymphotropic virus type I (HTLV-I) and type II (HTLV-II) and the env protein of simian T-cell lymphotropic virus type I (STLV-I) were aligned and computer algorithms were used to predict B- and T-cell epitopes. Structural B- and T-cell motifs that showed amino acid sequence conservation of antigenic determinants in HTLV-I, HTLV-II, and STLV-I, as well as different antigenic determinants of HTLV-I and HTLV-II, were identified. Several of these B and T epitopes have been shown experimentally to be immunodominant and two of the B epitopes have been used for type-specific serology. These predictive epitopes provide a guide to develop improved diagnostic assays and for the development of potential subunit vaccines for HILV-I and HTLV-II.

As the HIV-1 pandemic becomes increasingly complex, the genetic characterization of HIV strains bears important implications for vaccine research. To better understand the molecular evolution of HIV-1 viral diversity, we performed a comparative molecular analysis of HIV strains collected from high-risk persons in Kinshasa, Democratic Republic of Congo (DRC). Analysis of the gag-p24, env-C2V3 and -gp41 regions from 83 specimens collected in 1999-2000 revealed that 44 (53%) had concordant subtypes in the three regions (14 subsubtype A1, 10 subtype G, 8 subtype D, 5 subtype C, 2 each subsubtype F1 and CRF01_AE, and one each of subtypes H and J, and subsubtype A2, while the remaining 39 (47%) had mosaic genomes comprising multiple subtype combinations. Similar multisubtype patterns were also observed in 24 specimens collected in 1985. Sequence analysis of the gag-pol region (2.1 kb) from 21 discordant specimens in the gag-p24, env-C2V3 and -gp41 regions in 1985 and 1999-2000 further confirmed the complex recombinant patterns. Despite the remarkable similarity in overall subtype distribution, the intra- and intersubtype distances of major subtypes A1 and G increased significantly from 1985 to 1999-2000 (p=0.018 and p=0.0016, respectively). Given the complexity of HIV-1 viruses circulating in DRC, efforts should focus on the development of vaccines that result in cross-clade immunity.

The effect of CCR2 polymorphism on HIV-1 mother-to-child transmission and disease progression has not been explored in depth within Africa. As the CCR2-64I variant of this putative HIV coreceptor has been associated with slower progression to AIDS in adults, the current study was undertaken to examine the relationship between CCR2 polymorphism and HIV-1 perinatal transmission and child survival in western Kenya. CCR2 genotype was determined for 445 HIV-seropositive mothers and their infants. The CCR2-64I allele frequency of both mothers and children did not differ by HIV-1 transmission status, regardless of maternal viral load, viral subtype, immune status, or placental malaria status. For infants who acquired HIV perinatally (n = 78), there was no association between CCR2 genotype and viral load upon infection or survival rate over the 2-year follow-up. Our results do not indicate an effect of CCR2-64I on perinatal HIV transmission and survival in Kenyan children.

Rhesus macaques express three theta-defensins (RTDs 1-3), cyclic octadecapeptides with antiviral and lectin-like properties. Corresponding theta-defensin genes exist and are expressed in humans, but a signal sequence mutation prevents the formation of mature theta-defensin peptides. Retrocyclin-1 is a theta-defensin peptide whose precursor is encoded by human theta-defensin pseudogenes. It can protect human peripheral blood lymphocytes from infection by R5 and X4 strains of HIV-1, and provides a molecular template for designing novel antiviral agents. In this study, we used JC53-BL reporter cells to assess the activity of retrocyclin-1 (RC-100) and several analogues against primary HIV-1 isolates, including R5 and R5X4 strains of subtypes A-D, CRF-01_AE, and recombinants. Each analogue differed from retrocyclin-1 by a single amino acid substitution: Gly --Tyr in RC-106, RC-115, and RC-116, and Arg --Lys in RC-101. Although the modification in RC-101 was chemically conservative, this peptide was significantly more potent than retrocyclin-1 across the panel of primary isolates. We performed surface plasmon resonance binding studies, using recombinant gp120 and CD4 produced in insect cells. Although RC-100 and RC-101 bound gp120 LAV/IIIB with a K(d) of 30-35 nM, they bound gp120 from CRF-01_AE strains (CM 235 and 93TH975.15) with K(d) values of 200-750 nM. Overall, our findings suggest that clade-related differences in gp120 glycosylation impact the ability of retrocyclin-1 to bind this viral glycoprotein, and modulate the peptides' ability to prevent HIV-1 infection. The performance of RC-101 suggests that additional "engineering" could further enhance the antiviral properties of theta-defensins.

Resistance to HIV-1 infection despite repeated exposures has been associated with one or more HIV-specific responses, enhanced nonspecific immune modifications, and/or host genetic polymorphisms in certain individuals (highly exposed, persistently seronegative, HEPS). In the present investigation, we focused on the CCR5 gene polymorphisms and the association of such mutations to resistance to HIV-1 infection among 12 HEPS women in Chiang Rai, northern Thailand, and compared our findings with data from 10 HIV-1-infected and 9 HIV-1-uninfected unexposed women from the same geographic area. Although we have previously shown that none of the Thai women carried the Delta32 mutation, further analysis of the CCR5 coding gene region revealed that none of the women had other mutations that affect coreceptor activity (C101X or FS299) or chemokine responses (C20S, A29S, L55Q, C178R). Analysis of the CCR5 promoter region revealed that the CCR5 haplogroup C (HHC; 60%) was the predominant haplogroup among these women. Comparative analysis of the frequencies of different haplogroups among the three groups did not reveal any statistically significant differences (p0.05). However, we did find that two individuals from the HEPS group were homozygous for HHF*2 (the CCR2b- 64I bearing haplogroup) compared to none from the HIV-1-infected and -uninfected groups. There was no detectable difference in specific CCR5 haplogroups and their ability to mediate env fusion or to mediate HIV-1 infection in vitro. These data suggest that homozygosity of the HHF*2 haplogroup may be one of the factors that mediate resistance to HIV-1 infection in this cohort of HEPS women.

HIV-1 coreceptors CCR5 and CXCR4 play an important role in viral entry and pathogenesis. To better understand the role of viral tropism in HIV-1 transmission, we examined the coreceptor utilization of viral isolates obtained from men enrolled in a study of heterosexual transmission in northern Thailand. Viral isolates were obtained from HIV-1-positive males who had either HIV-1-infected spouses (RM; n = 5) or HIV-1-uninfected spouses (HM; n = 10). Viral isolates from 1 of the 5 RM males and 2 of the 10 HM males were CCR5 tropic, whereas isolates from 3 RM males and 6 of the HM male isolates were CXCR4 tropic. Of the nine X4-tropic isolates, seven also used at least one of the following coreceptors: CCR8, CCR1, CCR2b, or CX3CR1, and none employed CCR5 as an additional coreceptor. More importantly, three isolates, RM-15, HM-13, and HM-16 (one from a transmitter and two from nontransmitter), did not infect GHOST4.cl.34 cells expressing any of the known coreceptors. Further analysis using MAGI-plaque assays, which allow visualization of infected cells, revealed that RM-15 had low numbers of infected cells in MAGI-R5 and MAGI-X4 cultures, whereas HM-13 and HM-16 had high levels of plaques in MAGI-X4 cultures. Replication kinetics using activated lymphocytes revealed that these three isolates replicated in CCR5(+/+) as well as CCR5(-/-) peripheral blood mononuclear cells, suggesting that these isolates did not have an absolute requirement of CCR5 for viral entry. All three isolates were sensitive to the X4-antagonistic compounds T-22 and AMD3100. Analysis of the C2V3 region did not reveal any significant structural differences between any of the Thai subtype E isolates. Thus, there was no association between the pattern of coreceptor usage and transmissibility among these subtype E HIV-1 isolates.

We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early part of the HIV-1 epidemic among commercial sex workers (CSWs) in Kinshasa, Democratic Republic of Congo (formerly Zaire). Serologic analysis revealed that 27 (28.7%) of the 94 specimens were seropositive by both peptide and whole-virus lysate EIAs and that 24 were positive by molecular screening assays, using generic primers that can detect all known groups of HIV-1. Phylogenetic analyses of the gag(p24), C2V3, and gp41 regions of these 24 specimens showed that all were group M; none of them had any evidence of group O, N, or SIVcpz-like sequences. On the basis of env sequence analysis, the 24 group M specimens were classified as subtypes G (37.5%), A (21%), F1 (12.5%), CRF01_AE (8%), D (4%), and H (4%); 3 (12.5%) were unclassifiable (U). Similar analysis of the gag(p24) region revealed that the majority of infections were subtype A; however, one-third of the specimens were subtype G. Parallel analysis of gag(p24) and env regions revealed discordant subtypes in many specimens that may reflect possible dual and/or recombinant viruses. These data suggest a predominance of subtype G (both pure G and recombinant CRF02_AG) during the early part of the epidemic in Kinshasa. Infections with group N or SIVcpz-like viruses were not present among these CSWs in Kinshasa.

It is known that certain individuals remain persistently seronegative despite repeated exposure to HIV-1. Studies have shown that some exposed uninfected (EU) individuals who are homozygous for a 32-bp deletion in the CCR5 gene are resistant to infection with non-syncytium-inducing (R5) viruses. In the present investigation, we provide evidence that a highly exposed-uninfected individual with the CCR5 32-bp deletion (EUdelta32-1) also has partial resistance to syncytium-inducing (R5X4) HIV-1 viruses, when compared with unexposed-uninfected individuals with (UUdelta32-1 and UUdelta32-2) and without (UU-1 and UU-2) the 32-bp deletion. The partial resistance of EU cells was due neither to altered coreceptor expression, nor to specific mutation or deletion in the coding region of chemokine coreceptors CXCR4 and CCR3. While SDF-1, the ligand for CXCR4, blocked entry of R5X4 viruses to a similar extent in EUdelta32 and UUdelta32, there was a differential production of soluble factors by EUdelta32. Both CD4+ and CD8+ cells from EUdelta32-1 produced soluble factors that efficiently suppressed infection by HIV-1 R5X4 viruses when compared with supernatant from UUdelta32. These data provide evidence that additional soluble factors are involved in resistance to infection with R5X4 viruses.

479 BOTH HUMAN IMMUNODEFICIENCY VIRUS (HIV) and simian immunodeficiency virus (SIV) use their external envelope glycoprotein in conjunction with the CD4 cell surface protein of lymphocytes and monocyte-macrophages, and one of the chemokine coreceptors (CCR1-5, CXCR4, Bob and Bonzo) to achieve viral fusion and cell entry.1±3 These chemokine receptors belong to the seven transmembrane (TM)-domain G protein-coupled family of cell surface receptors.1 Experimental studies on interspecies transmission of SIV between patas and tantalus monkeys from Central Africa suggest that although these African nonhuman primate species are susceptible to SIV infection from other species, they do not develop any SIV-related disease.4 In contrast, the Asian nonhuman primate species, free from SIV infection because of their geographic segregation, develop similar AIDS-like symptoms when experimentally infected with SIV. The underlying host genetic reasons for the differences in disease manifestation between African and and Asian monkeys remain unclear. Genetic differences in the CCR5 gene (in particular, a 32-bp deletion) have been thought to play a crucial role in resistance to HIV disease in some patients.5 Similarly, an amino acid mutation, V64I, encoded in the CCR2 gene, has also been found to confer some protection in HIV-1-infected individuals. Despite the availability of data on single amino acid mutations and deletions in the chemokine receptor genes, understanding of the natural resistance to HIV disease in some humans and to SIV disease in African primates remains incomplete. To determine molecular changes in the CCR5 gene of African and Asian nonhuman primates that might suggest the reasons for resistance to SIV disease in African monkeys, and disease susceptibility to SIV in Asian macaques, we sequenced the genes from several representative monkey species of African and Asian origin. The CCR5 genes were amplified by polymerase chain reaction (PCR) from DNA extracted from the peripheral blood mononuclear cells (PBMCs) of 11 different monkeys or apes (belonging to 7 different species) from Africa and Asia: (1) chimpanzee (Pan troglodytes, Gabon, Africa)7; (2) gorilla (Gorilla gorilla, Gabon, Africa); (3) vervet subspecies of African green monkeys (AGMs) (Cercopithecus aethiops pygerythrus, South Africa); (4) baboons (Papio aneubis, Africa); (5) two Asian pigtail macaques (Macaca nemestrina, Indonesia); (6) one stumptail macaque (Macaca arctoides, Indonesia); and (7) one rhesus macaque (Macaca mulatta, India). The PCR, sequencing, and phylogeny protocols used for the analyses of the CCR5 genes are discussed in Fig. 1A and Fig. 2. We identified 12 nucleotide changes in the CCR5 gene, relative to the human sequence, that were specific for each of the 7 nonhuman primate species analyzed (data not shown). By contrast, it has been previously shown that there is relative homogeneity of this gene in Homo sapiens.11 All 12 cysteine residues (critical in protein folding) present at positions 20, 58, 101, 178, 213, 224, 269, 290, 291, 321, 323, and 324 and all 7 TM domains in the CCR5 protein showed a tight evolutionary conservation in both human and nonhuman primates (Fig. 1A). We also observed molecular changes specific to humans and chimpanzees only; humans and AGMs only; rhesus macaques and AGMs only; and pigtail stumptail macaques and AGMs (Table 1). The CCR5 gene of Asian macaques was the most divergent, differing by 2.4±2.7% from humans and apes at the nucleotide level, and by 1.9±4.2% at the peptide level (Fig. 1A), suggesting the antiquity of macaques as a species. This was also reflected in the phylogenetic tree (Fig. 2), where all three macaque species, chosen from two different parts of Asia, formed a distinct cluster. Similarly, the vervet monkeys and baboons also formed distinct clusters

The relevance of a TNF-alpha promoter polymorphism, a G-to-A polymorphic sequence at position-308, was examined to test whether variant alleles of TNF-alpha affect susceptibility to infection with HIV-1 and progression to AIDS. Analysis of specimens from cohorts of HIV-1 positive homosexual men demonstrated that 3 of the 32 (9.4%) HIV-1-infected long-term nonprogressors (LTNPs) were homozygous for the uncommon TNF-2 allele compared with 3 of the 196 (1.5%) HIV-1-seronegative blood donors and uninfected homosexual men (p < 0.05). There was no difference in heterozygosity among HIV-1-seropositive or -seronegative groups, although some of the seropositive men heterozygous for the TNF2 genotype were also heterozygous for CCR5delta32. However, no significant association was found between TNF genotypes and time of survival, CD4 slopes, or viral loads when seroincident (n = 109) and seroprevalent cases (n = 442) from the Chicago MACS were analyzed. Functional analysis of lymphocytes from the seronegative group revealed no difference in endogenous or mitogen-induced TNF-alpha production, as well as susceptibility to in vitro HIV-1 infection between different TNF-genotype donors. These data suggest that TNF genotypes do not play a direct role in HIV-1 disease progression; however, they could potentially be part of a multigenic linkage that may be involved in delaying progression to AIDS.

In vitro infection of T cells with human T lymphotropic virus types I and II (HTLV-I and HTLV-II) resulted in constitutive expression of ICAM-1. Higher levels of ICAM-1 mRNA were expressed in HTLV-transformed cell lines (MT-2, MoT, C8166) when compared with uninfected T cell lines (A301). We demonstrate that this activation is conferred through a site on the ICAM-1 promoter that is activated in trans by the Tax protein of HTLV-I and HTLV-II. Enhanced promoter activity was detected when the ICAM-1 construct (-1162/+1) was transfected into HTLV-I-infected (MT-2), HTLV-II-infected (MoT, AI 1050), or an HTLV-I Tax-only-expressing (C8166) cell line as compared to the uninfected T cell line (A3.01). Cotransfection of the uninfected T cell line A3.01 with the ICAM construct along with Tax-I and Tax-II expression plasmid also resulted in increased promoter activity. Furthermore, experiments with deletion constructs of the ICAM-1 promoter region indicated that a region between -88 and -53 bp relative to the transcription start site is sufficient for Tax-inducible CAT expression. This segment includes an 11-bp palindromic segment (TTTCCGGGAAA) that has homology with the IFN-gamma and IL-6 response element. An 11-bp segment containing this regulatory region proved to be sufficient to confer Tax-I and Tax-II inducibility on a heterologous promoter (TK-CAT). Taken together these findings indicate that constitutive expression of ICAM-1 by HTLV-infected cells is influenced by the viral trans-activator protein Tax. This increased expression of ICAM-1 in response to the Tax protein may play an important role in the lymphoproliferation associated with HTLV infection.

Accumulation of unintegrated human T lymphotropic virus (HTLV) DNA was analyzed in long-term T cell lines infected with HTLV type I (HTLV-I) or type II (HTLV-II). By using a polymerase chain reaction-based assay, amplified products of expected size were obtained in all of the HTLV-I-infected (n = 7) and HTLV-II-infected (n = 8) cell lines. The signal intensities of the hybridizing band varied greatly among the cell lines and did not correlate with HTLV p24gag antigen production. Further analysis of HTLV-I-infected clones demonstrated considerable variability in the unintegrated DNA accumulation, suggesting that either the epigenetic status of the host cell or some environmental factor determines the occurrence of unintegrated DNA. The presence of lower levels of unintegrated DNA in most of the HTLV-infected, long-term cell lines presumably results in persistent noncytopathic infection.

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and employed to detect p24 capsid antigen from human T-cell lymphotropic viruses type I and II (HTLV-I, HTLV-II), simian T-cell lymphotropic virus type I (STLV-I)-infected cell lines, and from mononuclear cell cocultures of HTLV-infected humans and STLV-I infected monkeys. A monoclonal antibody specific for HTLV p24 and p53 capsid antigens was coated onto 96-well microtiter plates to capture HTLV/STLV antigen. Captured antigen was then detected by the addition of a polyclonal, biotinylated human anti-HTLV-I antibody, and color developed with tetramethyl benzidine/H2O2 substrate. As little as 15 pg/ml of HTLV-I p24 antigen could be detected in this assay. Culture supernatants from HTLV-I-infected cell lines (HUT-102, MT-2, C5/MJ, HTLV-II-infected cell lines (Mo-T, Mo-B, PanG 12.1, NRA) and STLV-I-infected cell lines (Matsu, NEPC M39) were all positive in the assay. In addition, p24 was detected from peripheral blood mononuclear cell (PBMC) cocultures of 8 of 8 (100%) HTLV-I diseased patients, 14 of 20 (70%) HTLV-I and HTLV-II-infected, asymptomatic persons, and 8 of 8 (100%) STLV-I-infected, asymptomatic monkeys. Culture supernatants of cells infected with human immunodeficiency virus type (HIV-1), simian immunodeficiency virus (SIV), Chlamydia trachomatis, cytomegalovirus (CMV), herpes simplex I and II (HSV), feline leukemia virus (FELV), bovine leukemia virus (BLV), and bovine immunodeficiency virus (BIV) were all negative. Similarly, normal human peripheral blood mononuclear cells and uninfected, transformed human T cells, were also negative in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)