Molecular cloning and analysis of full-length genome of HIV type 1 strains prevalent in countries of the former Soviet Union

The HIV-1 epidemic among injecting drug users (IDUs) in countries of the former Soviet Union (FSU) was caused mainly by two HIV-1 variants: subtype A and CRF03-AB. To date only three full-length HIV-I genomes from the FSU have been sequenced: one subtype A from Byelorussia and two CRF03-AB from Russia. We report the full-length genome cloning and analysis of two more HIV-1 strains from the FSU countries (98UA0116 of subtype A and 98BY10443 of CRF03-AB). Isolate 98UA0116 is the second cloned and sequenced full-length HIV-1 genome of subtype A lineage from the FSU, which may be a novel subsubtype within sub-type A. Isolate 98BY10443 is the third full-length HIV-1 genome of CRF03-AB in the world to be cloned and sequenced. Additionally, it is the first CRF03-AB strain discovered in Byelorussia. Cloned genomic sequences of the FSU HIV-1 isolates are being used for the development of a region-specific HIV-1 vaccine.     

New insights into the origin of the HIV type 1 subtype A epidemic in former Soviet Union's countries derived from sequence analyses of preepidemically transmitted viruses

The HIV-1 subtype A epidemic affecting injecting drug users (IDU) in former Soviet Union (FSU) countries started dramatically in Odessa, southern Ukraine, in 1995, and is caused by a variant of monophyletic origin, often designated IDU-A. We phylogenetically analyzed one near full-length genome and two partial sequences of three HIV-1 subtype A viruses collected in St. Petersburg, Russia, heterosexually transmitted in 1992-1994. The sequences branched basally to the IDU-A clade, together with eight viruses from Odessa collected in 1993, all presumably acquired heterosexually, and two viruses from the Democratic Republic of Congo. Of all other FSU sequences in databases, only those from three recently collected viruses, one from Ukraine and two from northwestern Russia, at least one of them acquired heterosexually, branched basally to the IDU-A cluster. The results indicate that the FSU IDU-A variant derives from a strain that initially propagated heterosexually in Ukraine and originated in central Africa.     

HIV-1 genetic diversity and genotypic drug susceptibility in the Republic of Georgia

The genetic diversity and genotypic drug susceptibility of HIV-1 strains circulating in the Republic of Georgia, formerly part of the Soviet Union, were investigated for first time. Forty-eight HIV-positive drug-naive Georgian individuals contributed PBMC DNA between 1998 and 2003. On the basis of phylogenetic analyses of partial pol sequences, the predominant HIV-1 genetic forms were subtype A (70%), followed by subtype B (26%); both genetic forms were carried by injecting drug users and heterosexuals. There was also one subtype C (2%) and one CRF18_cpx (2%). The Georgian subtype A strains clustered with subtype A from Russia, designated A(FSU). Twelve of the subtype A strains (25%) contained the secondary protease inhibitor mutation V77I and 9 also had two other silent mutations. This "V77I haplotype" marks one particular genetic lineage of the epidemic in the former Soviet Union. Two strains (4%) carried antiretroviral (ARV) drug resistance mutations. Nearly full-length genome sequences of five Georgian strains were also completed. Two, 98GEMZ011 (subtype A) and 98GEMZ003 (subtype B), closely resembled the parental strains that recombined to create CRF03_AB. The use of these parental strains in the analysis revealed an additional segment of subtype A in CRF03_AB. Thus, the HIV-1 epidemic in Georgia was composed of a mixture of subtype A(FSU) and subtype B.     

Simultaneous introduction of HIV type 1 subtype A and B viruses into injecting drug users in southern Ukraine at the beginning of the epidemic in the former Soviet Union

The vast majority of HIV-1 strains from the epidemic in the former Soviet Union (FSU) belong to subtype A (IDU-A) and CRF03_AB (IDU-A/B), for which IDU-A is one of parental strains; no epidemic by another parental virus, belonging to subtype B (IDU-B), has yet been identified. To characterize viruses present during the early stage of the epidemic in southern Ukraine, where the first outbreaks in the FSU were registered, we obtained partial env and pol sequences from IDUs from Odessa and Nikolaev and compared them with viruses from other outbreaks. All viruses from Odessa belonged to the IDU-A type, which is in accord with previous studies. At the same time, we found that the outbreak in Nikolaev was caused by IDU-B viruses, indicating that this outbreak is the result of an independent virus introduction. Phylogenetic analysis of viruses from the FSU supported the epidemiological data suggesting that the HIV-1 epidemic in the FSU started in southern Ukraine.     

The rate of epidemiological and virological changes during the transition from nascent to concentrated HIV epidemic stage in the former Soviet Union countries

The rate of processes accompanying the transition of the HIV-1 epidemic from nascent stage to concentrated one in the Former Soviet Union (FSU) during intravenous drug user (IDU)-associated HIV infection outbreaks in 1994-1999 has not been analyzed. To define the rates, we studied susceptible populations and circulating viruses before, during, and after the outbreaks. Our findings included the following: (1) the pattern of high HIV-1 genetic diversity characteristic of the nascent epidemic changed to a concentrated one within 1 year in St. Petersburg and in Moscow; (2) different FSU regions were at different stages of the HIV-1 epidemic in 1994-1996; (3) the change of serotypic patterns characteristic of different stages of the HIV/AIDS epidemic for the non-IDU risk group occurred within 1 year in Moscow, suggesting an extremely high rate of IDU-associated epidemic pattern distributions in regions and susceptible populations in the FSU.     

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.     

Different distribution of HIV type 1 genetic variants in European patients with distinct risk practices

The use of two genetic markers has permitted the analysis of the distribution of two different human immunodeficiency virus type 1 (HIV-1) variants in patients of the homosexual (HO) and intravenous drug user (IDU) groups in distinct European countries. In Germany, Holland, and Italy the variants circulating in each risk group of HO and IDU patients were genetically distinguishable according to the genetic markers used. In contrast, in France and Spain, the same variant has been recovered from patients with different risk practices. These data highlight the diversity of the HIV-1 epidemic in Europe and the different patterns of HIV-1 variant distribution in European countries.     

Apparent founder effect during the early years of the San Francisco HIV type 1 epidemic (1978-1979)

HIV-1 envelope sequence variants were RT-PCR amplified from serum samples cryopreserved in San Francisco in 1978-1979. The HIV-1 subtype B env V3-V5 sequences from four homosexual men clustered phylogenetically, with a median nucleotide distance of 2.8%, reflecting a recent common origin. These early U.S. HIV-1 env variants mapped close to the phylogenetic root of the subtype B tree while env variants collected in the United States throughout the 1980s and 1990s showed, on average, increasing genetic diversity and divergence from the subtype B consensus sequence. These results indicate that the majority of HIV-1 currently circulating in the United States may be descended from an initial introduction and rapid spread during the mid- to late 1970s of subtype B viruses with limited variability (i.e., a founder effect). As expected from the starburst-shaped phylogeny of HIV-1 subtype B, contemporary U.S. strains were, on average, more closely related at the nucleic acid and amino acid levels to the earlier 1978-1979 env variants than to each other. The growing levels of HIV-1 genetic diversity, one of multiple obstacles in designing a protective vaccine, may therefore be mitigated by using epidemic founding variants as antigenic strains for protection against contemporary strains.     

Phylogenetic analysis of the Latvian HIV-1 epidemic

The Latvian HIV-1 outbreak among intravenous drug users (IDUs) in 1997-1998 involved subtype A1. To obtain a more complete picture of the Latvian HIV-1 epidemic, 315 HIV-1-infected patients diagnosed in 1990-2005 representing different transmission groups and geographic regions were phylogenetically characterized using env V3 and gag p17 sequences. Subtypes A1 and B infections were found in 76% and 22% of the patients, respectively. The subtype A1 sequences formed one large cluster, which also included sequences from other parts of the former Soviet Union (FSU), whereas most subtype B sequences formed three distinct clusters. We estimated that subtype A1 was introduced from FSU around 1997 and initially spread explosively among IDUs in Riga. A recent increase of heterosexually infected persons did not form a separate subepidemic, but had multiple interactions with the IDU epidemic. Subtype B was introduced before the collapse of the Soviet Union and primarily has spread among men who have sex with men.     

Identification of a novel clade of human immunodeficiency virus type 1 in Democratic Republic of Congo

The high genetic heterogeneity of HIV-1 in the Democratic Republic of Congo (DRC) constitutes a real challenge for the development of vaccines to counter the spread of the HIV-1 epidemic. It is important to continue to monitor the epidemic by studying the circulating strains and their impact on the overall spread. As part of the ongoing effort to study the global distribution of HIV-1 subtypes and circulating recombinant forms (CRFs), here we describe a new phylogenetic clade of HIV-1 by the analysis of two full-length sequences (83CD003 and 90CD121E12) collected from two individuals at a 7-year interval (1983 and 1990, respectively). One of the two sequences (90CD121E12) was obtained from a vertically infected, 12-month-old baby in Kimpese, rural DRC, an area with low and stable seroprevalence of HIV-1 in women attending antenatal clinics. The two sequences are genetically similar by 95% of their full genome and topologically form a distinct cluster that is equidistant from the existing subtypes (A through K) by the analysis of both the full genome and subgenomic regions. Furthermore, they share several other genetic features, including an additional pair of cysteine residues, predictive of an extra disulfide bridge, in the V4 loop of gp120. This new clade is currently rare, spreading at a slower pace than the other subtypes found in the DRC region. Pending the identification of at least one partial length sequence of the same lineage from another patient who is epidemiologically unlinked to those described here, this clade is not yet named as a subtype as per the recommendation of the nomenclature committee.     

Tracing the origin and history of the HIV-2 epidemic

In this study we date the introduction of HIV-2 into the human population and estimate the epidemic history of HIV-2 subtype A in Guinea-Bissau, the putative geographic origin of HIV-2. The evolutionary history of the simian immunodeficiency virussooty mangabey/HIV-2 lineage was reconstructed by using available database sequences with known sampling dates, and a timescale for this history was calculated by using maximum likelihood methods. The date of the most recent common ancestor of HIV-2 subtype A strains was estimated to be 1940 +/- 16 and that of B strains was estimated to be 1945 +/- 14. In addition we used coalescent theory to estimate the past population dynamics of HIV-2 subtype A in a rural population of Guinea-Bissau. Parametric and nonparametric estimates of the effective number of infections through time were obtained for an equal sample of gag, pol, and env sequences. Our estimates of the epidemic history of HIV-2 subtype A in Guinea-Bissau show a transition from constant size to rapid exponential growth around 1955-1970. Our analysis provides evidence for a zoonotic transfer of HIV-2 during the first half of the 20th century and an epidemic initiation in Guinea-Bissau that coincides with the independence war (1963-1974), suggesting that war-related changes in sociocultural patterns had a major impact on the HIV-2 epidemic.     

Circulation of novel HIV type 1 A, B/C, and F subtypes in Argentina

The Argentine HIV-1 epidemic is considered to be represented mainly by subtype B and diverse B/F recombinants, with apparent absence of pure subtype F. In this study we describe three novel HIV-1 variants isolated from four infants born in different and distant provinces of Argentina. Partial analysis of different gene fragments spanning 18.5-40.8% of the HIV-1 complete genome revealed two subtype A HIV-1 strains in siblings, a B/C recombinant with a novel mosaic structure, and a putative subtype F. Characteristic patterns of genomic and amino acid sequences of the newly reported subtype F isolate suggest a closer genetic relationship to Argentine B/F recombinants than any other subtype F strain described so far, while the A and B/C subtypes found correspond to unusual genotypes in Argentina. Understanding the origin, diversity, and spread of HIV-1 strains worldwide will be necessary for the development of an effective vaccine approach.     

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.     

Genetic diversity and drug resistance profiles in HIV type 1- and HIV type 2-infected patients from Cape Verde Islands

Our aim was to characterize for the first time the genetic diversity of HIV in Cape Verde Islands as well as the drug resistance profiles in treated and untreated patients. Blood specimens were collected from 41 HIV-1 and 14 HIV-2 patients living in Santiago Island. Half of the patients were on antiretroviral treatment (ART). Pol and env gene sequences were obtained using in-house methods. Phylogenetic analysis was used for viral subtyping and the Stanford Algorithm was used for resistance genotyping. For HIV-1, the amplification of pol and env was possible in 27 patients (66%). HIV-1 patients were infected with subtypes G (13, 48%), B (2, 7%), F1 (2, 7%), and CRF02_AG (2, 7%), and complex recombinant forms including a new C/G variant (n=8, 30%). Drug resistance mutations were detected in the PR and RT of three (10%) treated patients. M41L and K103N transmitted drug resistance mutations were found in 2 of 17 (12%) untreated patients. All 14 HIV-2 isolates belonged to group A. The origin of 12 strains was impossible to determine whereas two strains were closely related to the historic ROD strain. In conclusion, in Cape Verde there is a long-standing HIV-2 epidemic rooted in ROD-like strains and a more recent epidemic of unknown origin. The HIV-1 epidemic is caused by multiple subtypes and complex recombinant forms. Drug resistance HIV-1 strains are present at moderate levels in both treated and untreated patients. Close surveillance in these two populations is crucial to prevent further transmission of drug-resistant strains.     

Genetic forms of HIV Type 1 in the former Soviet Union dominate the epidemic in Azerbaijan

A total of 125 strains collected in Azerbaijan between 1999 and 2002 from HIV seropositives were genetically classified. Of 84 strains classified using HMA, 91.6% were subtype A, 1.2% subtype B, and 7.1% untypeable. Of 41 strains analyzed using partial pol gene sequences, 90.2% were subtype A, 7.3% subtype B, and 2.4% CRF03_AB. Most sequenced A strains clustered with those circulating in countries of the former Soviet Union (FSU). Two of three sequenced B strains were from individuals who traveled to FSU clustering tightly with B strains from Nikolayev, Ukraine. CRF03_AB, characteristic of the 1996 epidemic in Kaliningrad, Russia, was sequenced from an individual who lived in Russia from 1995 until 2001. The HIV epidemic in Azerbaijan is concentrated in IDU and is closely connected to other such epidemics to the east based on genetics. Of the 41 sequenced strains, 95% were close genetic relatives of HIV strains in IDU networks in the FSU.     

Travel and the spread of HIV-1 genetic variants

HIV-1 comprises three groups, the main (M group), O (outlier) and N (non-M, non-O). The M group, divided into 11 subtypes, is responsible for the global HIV-1 pandemic. Recombination between M subtypes has resulted in the generation of multiple circulating recombinant forms (CRFs) consisting of mosaic lineages. Most subtypes and CRFs are represented in Africa, whereas predominance of one or a few subtypes was reported initially elsewhere. This finding reflects the African origin of the epidemic. In western countries, where the B subtype is predominant, there is a steep increase in non B-subtypes and CRFs, while new recombinants emerge worldwide. Travellers contribute to the spread of HIV-1 genetic diversity worldwide, and in the developing world migration of rural populations and civil war are additional contributing factors. The spreading of HIV-1 variants has implications for diagnostic, treatment, and vaccine development.     

Description of the first full-length HIV type 1 subtype F1 strain in Argentina: implications for the origin and dispersion of this subtype in South America

HIV-1 subtype F1 in South America is mainly found as part of diverse BF1 recombinant forms and only five full-length "pure" F1 strains from Brazil were characterized to date. In the present study we describe the first near full-length sequence of a nonrecombinant F1 HIV-1 strain from Argentina, and explore the epidemiological history of this subtype in South America. Three separate phylogenetic analyses were carried out: with all available F1 full-length sequences, with concatenated F1 sequences contained in F1 and BF1 strains, and with partial F1 env sequences derived from worldwide strains. All analyses were consistent in showing a local origin of the newly reported Argentine subtype F1 strain, and a common ancestry of the South American subtype F1 sequences, present either in pure or recombinant genomes. By coalescent analysis, the onset date of the HIV-1 subtype F1 epidemic in South America was estimated to be around the late 1970s. The results indicate for the first time that nonrecombinant F1 HIV-1 strains are present in Argentina, and suggest that the subtype F1 epidemic in South America was initiated by the introduction of a very small group of genetically related viruses during late 1970s.     

β-Chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian/HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inflammatory proteins 1α and 1β produced by circulating CD8⁺ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with β-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.