HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996.

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.

Experimental infection of chimpanzees with hepatitis C virus of genotype 5a: genetic analysis of the virus and generation of a standardized challenge pool.

Six major genotypes (genotypes 1-6) of hepatitis C virus (HCV) have been identified. These genetic variants are being transmitted to chimpanzees, the only recognized animal model for the study of HCV. Genotype 5a (strain SA13), a variant found primarily in South Africa, has been transmitted to chimpanzees for the first time. Experimental infection of 2 chimpanzees was characterized by early appearance of viremia and peak virus titers of 10(5)-10(6) genome equivalents/mL. The HCV infection was resolved by week 15 after inoculation in 1 chimpanzee and persisted in the other. Both chimpanzees became anti-HCV-positive by week 14 after inoculation. Both chimpanzees developed viral hepatitis. The infectivity titer of a genotype 5a challenge pool prepared from the first passage of HCV in a chimpanzee was approximately 10(4) infectious doses/mL. Finally, sequence analysis of strain SA13 confirmed that genotype 5a is genetically distinct from other genotypes of HCV.

Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand.

Human immunodeficiency virus type 1 isolates of envelope genotype E are contributing substantially to the global pandemic. These strains appear to be mosaics, with the gag gene from clade A and the envelope from clade E; the parental clade E strain has not been found. Here we report the first full genomic sequence of one such mosaic virus, isolate CM240 from Thailand. Multiple breakpoints between the two parental genotypes have been found in a CM240 virus. The entire gag-pol region and most, if not all, of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41; thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be principally derived not from clade E, as previously thought, but from clade A. Another small segment not belonging to any recognized clade and presumably also contributed by the parental E strain has been found in the long terminal repeat. It may be significant that the implied virion structure resembles a pseudotype virus with the matrix and core from one clade and the outer envelope from another. In the long terminal repeat, differences were observed between CM240 and other clades in the number of NF-kappa B binding sites, the sequence of the TATA box, and the putative secondary structure of the transactivation response region stem-loop. The mosaic structure of a CM240 virion is suggestive of phenotypic differences which might have contributed to the emergence of this variant.

PIP: A new variant of human immunodeficiency virus (HIV)-1 with a mosaic genomic structure was identified in Thailand in 1992. This variant, termed genotype E, was characterized by an envelope gene sequence equidistant from genotypes A through D. The gag gene, encoding the matrix and core virus proteins, grouped with genotype A rather than forming a new clade. More than 500,000 Thais are estimated to be infected with the envelope clade E virus and its type 1 isolates, previously assumed to be rare outliers, are contributing substantially to the global acquired immunodeficiency syndrome pandemic. Reported here is the first complete genomic analysis of one such mosaic virus, isolate CM240 from Thailand. The entire gag-pol region and most of the accessory genes vif, vpr, tat, rev, and vpu appear to derive from clade A. The genotype switches to E shortly after the signal peptide of the envelope and back to clade A near the middle of gp41. Thus, the portion of the envelope that lies on the cytoplasmic side of the membrane appears to be derived from clade A. Another small segment presumably contributed by the parental E strain has been found in the long terminal repeat. The multiple crossover points detected in CM240 may reflect a common mechanism of frequent strand switching by reverse transcriptase. Full genomic analyses of other mosaic HIV-1 genomes are recommended to determine whether the breakpoints found in CM240 are recurrent and to identify the functional implications of the virion alterations.

Sequences and phylogenetic analysis of the nef gene from Thai subjects harboring subtype E HIV-1.

PIP: It has been demonstrated that nef-defective SIV can cause attenuated disease in rhesus monkeys and that animals immunized with a similar construct can be protected when challenged with wild-type, pathogenic SIV. This finding has sparked interest in the nef gene of HIV-1. It remains unclear, however, whether nef serves an analogous role in human HIV-1 infection; Deacon et al. have reported an association of deletions in the nef gene with an attenuated disease course in humans, although other work does not support a strict correlation between the structure or function of nef and rates of disease progression. The nef gene product has been shown to influence the infectivity and pathogenicity of HIV-1. nef may also have a role in the immune response to HIV-1. The current database of nef sequences is derived almost exclusively from subtype B viruses. The authors therefore sought to develop a database of nef sequences from subjects infected with HIV-1 subtype E. Whole-blood samples were collected from 103 male commercial sex workers in Chiang Mai, Thailand. 17 tested positive for infection with HIV-1, 16 of whom were infected with subtype E by gp120 characterization. Intersubject variation in subtype E nef genes at the nucleotide level ranged from 2.1-7.8%. Thai subtype E nef sequences are more tightly clustered than subtype B nef sequences, analogous to what is seen in env. The authors note that their subtype E nef sequences do not share the genotypic polymorphism in the area of residues 10-30 noted with subtype B nef. These data confirm the need to develop a set of subtype E-specific reagents.^ieng

Dual infection with human immunodeficiency virus type 1 of distinct envelope subtypes in humans.

Multiple genetic subtypes of human immunodeficiency virus type 1 (HIV-1) have been identified among internationally collected isolates. The HIV-1 epidemic in Thailand is largely due to B and E subtypes of virus. Dual infection with distinct HIV-1 subtypes would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. Polymerase chain reaction typing and serologic typing were used to screen a panel of specimens from HIV-1-infected subjects in Thailand. Two persons simultaneously harbored HIV-1 of env subtypes B and E, and this was confirmed by colony hybridization with subtype-specific probes and nucleotide sequence analysis of a 630-bp fragment of gp120 from multiple molecular clones. In addition, both subtypes were identified in cocultured peripheral blood mononuclear cells from 1 individual. These data provide the first evidence of dual HIV-1 infection in humans and reinforce the need for polyvalent vaccines.

Two antigenically distinct subtypes of human immunodeficiency virus type 1: viral genotype predicts neutralization serotype.

At least five distinct genetic subtypes (genotypes) of human immunodeficiency virus type 1 (HIV-1) have been identified by DNA sequencing. Current vaccine candidates are based on virus strains from North America and Europe that represent only one subtype. The extent to which distinct genotypes of HIV-1 correspond to antigenically distinguishable serotypes is largely unknown and may be critically important to vaccine design. Cross-neuralization studies were done with viruses and plasma from two different genotypes. Based on neutralization susceptibility, 10 primary HIV-1 isolates from Thailand and the United States were classified into one of two antigenic subtypes that correlated with viral genotype. The existence of serotypes of HIV-1 suggests that a broadly effective vaccine may have to include strains from multiple subtypes. Neutralization of these primary HIV-1 isolates differed substantially from results with laboratory strains. Future neutralization studies using primary isolates and multiple genotypes may be important for assessment of HIV-1 antigenic diversity.

Genetic variants of HIV-1 in Thailand.

Serosurveys conducted prior to 1988 indicated a very low level of HIV-1 infection in Thailand, even among high-risk groups. The Ministry of Health has reported a dramatic increase in HIV-1 infection during the last three years. The geographic and demographic distribution of the epidemic is broad, involving multiple provinces and risk groups. Foci of higher incidence and prevalence have been noted in the urban center of Bangkok and in the northern provinces of Chiang Mai and Chiang Rai. Here we report the results of genetic characterization of 16 HIV-1 isolates from Thailand using a combination of polymerase chain reaction (PCR) typing and DNA sequencing. The complete sequence of gp160 (env) of five isolates, partial env sequence of six additional isolates, and the gag gene of two isolates were determined. Two highly distinct HIV-1 variants were found. One variant resembled those prevalent in North America and Europe; five of the isolates were of this type. The remaining eleven isolates were very similar to one another and represented a variant unlike any previously described. Phylogenetic tree analysis of complete env and gag genes placed the two variants on widely separated branches. Protein sequence comparisons indicate both general and specific features that distinguish the Northern Thailand variant both from the Bangkok variant and from virtually all previously sequenced HIV-1 isolates. A simple PCR test for distinguishing the two variants has been developed for use in epidemiologic surveys.