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Currently, HIV-1 CRF63_02A6 is the prevalent genetic variant of the HIV-infected subjects in the major part of the Siberian Federal District (Russia). The HIV-1 CRF63_02A6 R5-tropic pT11.17 and X4-tropic pMtBs.18 infectious molecular clones (IMCs) were constructed using the virus isolates recovered in 2015 and 2017 of male HIV-infected Russian residents (from Tomsk and Novosibirsk, respectively). Near full-length proviral HIV-1 sequences (9,644 and 9,748 bp) were subcloned in pBluescript II KS(–). The CRF63_02A6 IMC virions were obtained by transfecting HEK293T cells with the constructed plasmids and demonstrated a stable growth in peripheral blood mononuclear cell culture (p24 concentration increased >1,000-fold and the virus protein accumulation in culture liquid exceeded 100,000 pg/mL). The tropism of CRF63_02A6 IMCs was determined genotypically (using Geno2pheno) and phenotypically by cultivating the IMC virions in MT-2, U87-CD4-CCR5, and U87-CD4-CXCR4 cell cultures. The obtained HIV-1 CRF63_02A6 IMCs may be useful in basic and applied research.

We report here two novel HIV-1 recombinant forms (CRF01_AE/B) isolated from two HIV-positive male subjects infected through homosexual contact in Beijing, China. Recombination contributes substantially to the genetic diversity of HIV-1, and is likely to occur in populations in which multiple subtypes circulate. Molecular epidemiological studies showed that subtype B, CRF01_AE, and CRF07_BC are currently cocirculating in parallel among men who have sex with men (MSM) in China, providing the opportunity for the emergence of new recombinants. Phylogenetic analysis of near full-length genome (NFLG) sequences showed that the unique recombinant forms (URFs) were composed of gene regions from CRF01_AE and subtype B. The CRF01_AE region of the recombinants clustered together with a previously described cluster 4 lineage of CRF01_AE. The B regions of both the recombinants clustered within the B strains. The two recombinants were quite similar with six breakpoints in common. These data highlight the importance of continuous surveillance of the dynamic change of HIV-1 subtypes and new recombinants among the MSM population.

We report two different unique HIV-1 recombinant viruses from two HIV-positive men who have sex with men (MSM) in Beijing, China. Phylogenetic analysis of near full-length genomes (NFLG) showed that the unique recombinant forms (URFs) were comprised of gene regions from two circulating recombinant forms, CRF01_AE and CRF07_BC, both common in China. The parental CRF01_AE region of the recombinants clustered together with a previously described cluster 4 lineage of CRF01_AE. The CRF07_BC regions of both the recombinants clustered within the CRF07_BC radiation, but were distinct from other CRF07_BC reference sequences. The two recombinant forms had two breakpoints in common. The emergence of the two URFs indicates the ongoing generation of recombinant viruses involving CRF01_AE and CRF07_BC, and may provide insight into our understanding of the dynamics and complexity of the HIV-1 epidemic in China.

HIV isolates from South Africa are predominantly subtype C. Sporadic isolation of non-C strains has been reported mainly in cosmopolitan cities. HIV isolate j51 was recovered from a rural South African heterosexual female aged 51 years. Near full length amplification of the genome was attempted using PCR with primers targeting overlapping segments of the HIV genome. Analysis of 5593 bp (gag to vpu) at a bootstrap value greater than 70% found that all but the vpu gene was HIV-1 subtype A1. The vpu gene was assigned HIV-1 subtype C. The recombination breaking point was estimated at position 6035+/− 15 bp with reference to the beginning of the HXB2 reference strain. Isolate j51 revealed a unique genome constellation to previously reported recombinant strains with parental A/C backbones from South Africa though a common recombination with subtype C within the vpu gene. Identification of recombinant strains supports continued surveillance of HIV genetic diversity.

The on-going generation of HIV-1 intersubtype recombination has led to new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in Asia. In this study, we evaluated whether previously reported URFs were actually CRFs. All available complete or near full-length HIV-1 URF sequences from Asia were retrieved from the HIV Los Alamos National Laboratory Sequence database, and phylogenetic, transmission cluster, and bootscan analyses were performed using MEGA 6.0, Cluster Picker 1.2.1, and SimPlot3.5.1. According to the criterion of new CRFs, two new HIV-1 CRFs (CRF87_cpx and CRF88_BC) were identified from these available URFs. CRF87_cpx comprised HIV-1 subtypes B, C, and CRF01_AE, and CRF88_BC comprised subtypes B and C. HIV Blast and bootscan analysis revealed that besides the three representative strains, there were two additional CRF87_cpx strains. Furthermore, we defined seven dominant URFs (dURF01-dURF07), each of which contained two strains sharing same recombination map and can be used as sequence references to facilitate the finding of new potential CRFs in future. These results will benefit the molecular epidemiological investigation of HIV-1 in Asia.

The tight bottleneck during HIV-1 transmission generally results in only a single virus variant being transmitted. Investigation of the HIV-1 envelope glycoprotein (Env) can identify vulnerabilities of transmitting viruses that can be targeted by vaccines designed to elicit protection against global HIV-1. This study generated an HIV-1 subtype C consensus transmitted and early founder virus Env (EnvFVC) after detailed sequence analysis of 1,894 env genes obtained from 80 acutely infected individuals from South Africa, Malawi, and Zambia. The inferred EnvFVC sequence incorporates characteristics of transmitted and early founder viruses and results in the expression of a functional and conformationally intact Env. Overall, the “subtype-based” or “region-based” EnvFVC described here can be used in the development of a useful immunogen for novel vaccine design.

We report here a novel HIV-1 recombinant form (CRF01_AE/B) detected from a comprehensive HIV-1 molecular epidemiologic study among men who have sex with men (MSM) in Jilin province of northeastern China. The near full-length genome (NFLG) analyses showed that the novel HIV-1 recombinant isolate (JL.RF07) was composed of CRF01_AE cluster 5 (northeastern China origin) and subtype B (U.S. and European origin), with six recombinant breakpoints observed in the pol, vif, tat, rev, and env gene regions. To the best of our knowledge, this is the first detection of a novel HIV-1 recombinant form (CRF01_AE/B) in Jilin, which may indicate an active transmission network of HIV-1 infection among MSM in the region. Further studies of the molecular epidemiology of the HIV-1 epidemic among MSM in northeastern China are necessary to gain a fuller understanding of the transmission network and potential public health impact of HIV-1 among MSM in this region.

Recombinant forms contribute significantly to the genetic diversity of HIV-1. Here we report a novel HIV-1 recombinant form (CRF01_AE/B'/C) detected from a comprehensive HIV-1 molecular epidemiologic study among heterosexuals in Jilin province of northeastern China. Recombinant analyses of the near full-length genome (NFLG) of the novel HIV-1 recombinant isolate (JL.RF01) showed that the backbone of the genome was CRF01_AE, and three insertions of subtype B' (242, 370, and 233 bp) and C (1142, 230, and 271 bp), respectively, were inserted along the genome. Phylogenetic analyses revealed that the novel HIV-1 recombinant form (CRF01_AE/B'/C) more likely originated from Thailand subtype B' and CRF01_AE and India subtype C. We report a unique mosaic structure that is distinct to HIV-1 CRF01_AE/B'/C recombinant viruses reported to date. The emergence of this novel recombinant form (CRF01_AE/B'/C) suggests the increasing significance of heterosexual transmission contributing to the complexity of the HIV-1 epidemic in northeastern China.

We report here a novel HIV-1 circulating recombinant form (CRF62_BC) that was isolated from three epidemiologically unlinked individuals [one from an injecting drug user (IDU); two from heterosexuals] in Dehong prefecture of western Yunnan province. CRF62_BC harbored two subtype B segments in the pol and vpu-env regions in a subtype C backbone. Subregion tree analysis demonstrated that subtype B regions originated from a Thai-B (subtype B') lineage and the subtype C region was from an India C lineage. CRF62_BC is the fourth CRF composed of subtypes B' and C known to date after CRF07_BC, CRF08_BC, and CRF57_BC, which were originally found among IDUs in China. The emergence of CRF62_BC may indicate the continual generation of new recombinant strains in various high-risk populations in western Yunnan. This may complicate the development of effective vaccines to limit the HIV-1 epidemic and increase the difficulty of AIDS prevention and control in China.

We report a novel HIV-1 circulating recombinant form (CRF64_BC) that was isolated from five epidemiologically unlinked HIV-infected persons in Yunnan province. CRF64_BC was composed of subtype B and subtype C, with five short subtype B segments inserted into the subtype C backbone. Phylogenetic analysis demonstrated that the C subregion was correlated with the India C lineage, which was transmitted into China in the early 1990s. The evolutionary history of the B subregion was not as clear as the C subregion, as the short length of this region yielded poor phylogenetic results. Dehong is considered the epicenter of HIV-1 in China, and recombinant strains such as CRF07_BC and CRF08_BC, which also originated from this region, have spread widely in China. The newly emerged CRF64_BC increases the complexity of the HIV epidemic in China and complicates the development of subtype-specific tools against HIV transmission.

Elite controllers or suppressors (ES) control viral replication without antiretroviral therapy. While many ES are infected with replication-competent virus, others have evidence of infection with attenuated isolates. Here we report a case of an ES infected with an HIV-1 isolate that contained a 38-base pair deletion in nef that led to a reading frame shift and a premature stop codon. Interestingly, clones amplified from plasma or cultured from CD4+ T cells between 2006 and 2008 contained one of two separate compensatory deletions that restored the reading frame. A new insertion generated by duplication of adjacent sequences was found in isolates obtained in 2010 and this evolution was accompanied by the development of low level viremia. This article provides evidence of the evolution of an attenuated HIV-1 isolate toward greater virulence in an elite suppressor.

In recent years, the men who have sex with men (MSM) population has seen the fastest growing prevalence of HIV transmission in China. In addition, coinfection through sex and intravenous drug use is a major problem in HIV prevention and control. Recent studies have also revealed that three major viral strains (CRF07_BC, CRF01_AE, and subtype B) have been cocirculating among MSM in Sichuan, suggesting a high probability of generating new recombinants. This study reports a near full-length genome of a novel HIV-1 recombinant (MSM0720) between CRF07_BC and CRF01_AE. The analysis of MSM0720 shows that the genome is composed of at least 11 interlaced segments, including six CRF07_BC and five CRF01_AE segments, with CRF07_BC as the backbone; this is different from a previously identified CRF01_AE/07_BC recombinant strain in intravenous drug users from Jiangsu.

HIV-1 infection is characterized by genetic diversity, with multiple subtypes and recombinant variants circulating, particularly in sub-Saharan Africa. During the Rwandan genocide, many women experienced multiple rapes and some became HIV-1 infected. We studied plasma and peripheral blood mononuclear cells (PBMCs) from 30 infected women comprising two exposure groups: those with numerous contacts, raped multiple times, and women with one lifetime sexual partner and no history of rape. Population-based sequences from gag, pol, and env genes were analyzed to determine HIV-1 subtypes and intersubtype recombination. Individual plasma-derived variants from 12 women were also analyzed. Subtype A was found in 24/30 (80%), intersubtype recombination (AC and AD) in 4/30 (13%), and subtypes C and D in 1/30 each. In two subjects, the pattern of HIV-1 recombination differed between plasma and PBMC-derived sequences. Intersubtype recombination was common, although there were no significant differences in subtype or recombination rates between exposure groups.

The Korean subclade of subtype B (KSB) is the most prevalent HIV-1 strain found in Korea. To date, only two near full-length HIV-1 sequences from Korean patients have been reported. Here, we analyzed a total of 24 near full-length genomes of HIV-1 strains that were isolated from 17 antiretroviral therapy (ART)-naive patients and four ART-exposed patients. Proviral DNA from peripheral blood mononuclear cells was PCR amplified and directly sequenced. Phylogenetic analyses were used to classify viruses from 19 patients as KSB, from one patient as subtype B, from one patient as subtype D, and three viruses from one patient as CRF02_AG. All KSB viruses demonstrated TAAAA instead of TATAA at the TATA box in the LTR. Of the 19 KSB patients, their sequence identities at the nucleotide level ranged from 89.8% to 97.1% from the lowest env gene to the highest pol gene. Other than the CRF02_AG viruses, no recombination events were noted in any of the 19 KSB patients, which is consistent with our previous studies on the pol, vif, and nef genes. Except for one strain, all of the strains were classified as non-syncytium-inducing strains. This is the first report to describe near full-length KSB.

To characterize human immunodeficiency virus (HIV-1) strains circulating in the Northern region of Colombia in South America, sequences of the viral envelope C2V3C3 region were obtained from patients with different high-risk practices. Close to 60% of the sequences were predicted to belong to macrophage-tropic viruses, according to the positions of acidic amino acids and putative N-linked glycosylation sites. This is in agreement with the fact that most of the patients were recently diagnosed individuals. Phylogenic analysis then allowed assignment of all 35 samples to subtype B viruses. This same subtype was found in previous studies carried out in other Colombian regions. This study thus expands previous analyses with previously missing data from the Northern region of the country. The number and the length of the sequences examined also help to provide a clearer picture of the prevailing situation of the present HIV epidemics in this country.

Multidrug-resistant (MDR) HIV-1 presents a challenge to the efficacy of antiretroviral therapy (ART). To examine mechanisms leading to MDR variants in infected individuals, we studied recombination between single viral genomes from the genital tract and plasma of a woman initiating ART. We determined HIV-1 RNA sequences and drug resistance profiles of 159 unique viral variants obtained before ART and semiannually for 4 years thereafter. Soon after initiating zidovudine, lamivudine, and nevirapine, resistant variants and intrapatient HIV-1 recombinants were detected in both compartments; the recombinants had inherited genetic material from both genital and plasma-derived viruses. Twenty-three unique recombinants were documented during 4 years of therapy, comprising ∼22% of variants. Most recombinant genomes displayed similar breakpoints and clustered phylogenetically, suggesting evolution from common ancestors. Longitudinal analysis demonstrated that MDR recombinants were common and persistent, demonstrating that recombination, in addition to point mutation, can contribute to the evolution of MDR HIV-1 in viremic individuals.

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.

We studied drug resistance mutations (DRMs) in 2623 pol sequences. Out of 94,828 amino acid substitutions that were detected, 8749 corresponded to nucleoside reverse transcriptase inhibitor (NRTI), 3765 to nonnucleoside reverse transcriptase inhibitor (NNRTI), and 7141 to protease inhibitor (PI) resistance-associated mutations. The most common DRMs were L10I, I54V, L90M, V82A, A71V, L10V, M46I, M184V, M41L, T215Y, D67N, L210W, K70R, N348I, V118I, K103N, Y181C, G190A, K101E, V108I, L100I, V90I, K101Q, and A98G. As expected, DRMs frequencies depended on viral genotype. The amounts of NRTI and PI resistance mutations among B and BF sequences from children were higher than among sequences from adults. The frequencies of PI and NRTI resistance mutations among B and BF sequences from adult men were higher than among sequences from women. Some of these observations can be explained in light of the available epidemiological information, but some cannot, indicating that further studies are needed to understand the antiretroviral resistance epidemics in Argentina. Fil: Jones, Leandro Roberto. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Moretti, Franco. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina Fil: Calvo, Andrea Yamila. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Dilernia, Dario Alberto. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Manrique, Julieta Marina. Fundación Playa Unión. Estación de Fotobiología Playa Unión; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gómez Carrillo, Manuel. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Salomon, Horacio Eduardo. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología. Centro Nacional de Referencia para el Sida; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Antiretroviral naive patients (n=49) were recruited in central western Brazil (Campo Grande City/Mato Grosso do Sul State, located across the Bolivia and Paraguay borders). HIV-1 protease (PR), reverse transcriptase (RT), and env gp41 HR1 fragments were sequenced. Genetic diversity was analyzed by REGA/phylogenetic analyses. Intersubtype recombinants were identified by SimPlot/phylogenetic trees. PR/RT resistance was analyzed by Calibrated Population Resistance/Stanford databases. T-20 resistance in gp41 was assessed by Stanford, Los Alamos, and other sources. Of HIV-1 subtypes 65.3% were B(PR)B(RT), 10.2% were C(PR)C(RT), and 8.2% were F1(PR)F1(RT). Intersubtype recombinants were 16.3%: four B/F1 and four B/C (two were "CRF31_BC-like"). The Pol-RT V75M mutation was detected in two homosexual partners; one patient had the T215S revertant mutation. T-20/gp41 resistance mutations were L44M (n=2) and V38A (n=1). The high percentage of non-B isolates (∼35%) highlights the importance of molecular surveillance studies in settings distant from the origin of the epidemic. Our data help elaborate the molecular epidemiological map of HIV-1 in Brazil.

HIV-1 epidemics in South America are believed to have originated in part from the subtype B epidemic initiated in the Caribbean/North America region. However, circulation of BF recombinants in similar proportions was extensively reported. Information currently shows that many BF recombinants share a recombination structure similar to that found in the CRF12-BF. In the present study, analyzing a set of 405 HIV sequences, we identified the most likely origin of the BF epidemic in an early event of recombination. We found that the subtype B epidemics in South America analyzed in the present study were initiated by a founder event that occurred in the early 1970s, a few years after the introduction of these strains in the Americas. Regarding the F/BF recombinant epidemics, by analyzing a subtype F genomic segment within the viral gene gag present in the majority of the BF recombinants, we found evidence of a geographic divergence very soon after the introduction of subtype F strains in South America. Moreover, through analysis of a subtype B segment present in all the CRF12-BF-like recombination structure, we estimated the circulation of the subtype B strain that gave rise to that recombinant structure around the same time period estimated for the introduction of subtype F strains. The HIV epidemics in South America were initiated in part through a founder event driven by subtype B strains coming from the previously established epidemic in the north of the continent. A second introduction driven by subtype F strains is likely to have encountered the incipient subtype B epidemic that soon after their arrival recombined with them, originating the BF epidemic in the region. These results may explain why in South America the majority of F sequences are found as BF recombinants. © Copyright 2011, Mary Ann Liebert, Inc. Fil: Dilernia, Darío Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Jones, Leandro Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Estación de fotobiología ‘‘Playa Unión’’; Argentina Fil: Pando, María de los Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Rabinovich, Roberto Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Damilano, Gabriel Dario. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Turk, Gabriela Julia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Rubio, Andrea. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Pampuro, Sandra Esther. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Gómez Carrillo, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina Fil: Salomon, Horacio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; Argentina

Little is known about the molecular and biological properties of HIV-1 intersubtype B′/C in Beijing. To fill the gap, we sequenced and analyzed the gag–pol genes from 39 HIV-1 B′/C recombinant infectors in Beijing, China during 2007. The results show that 36 CRF07_BC and 2 CRF08_BC isolates have a structural profile identical or nearly identical to CRF07_BC or CRF08_BC according to sequences in the gag–pol regions. The CRF07_BC circulating in injecting drug users (IDUs) and heterosexuals forms a diverse phylogenetic tree and most isolates from homosexuals cluster together. However, all the B′/C recombinant strains were remarkable for their low interpatient diversity in gag–pol genes (3.1, 3.0, and 2.2% for isolates from IDUs, heterosexuals, and homosexuals, respectively). We identified I7V, E91G, N242T, and K361R in the gag gene and R290I (HXB2 positions) in the pol gene as signature amino acid substitutions characteristic of HIV-1 CRF07_BC from the Beijing lineage. In addition, one new B′/C recombinant was detected. These results may contribute to an understanding of HIV-1 in Beijing.

The crucial role of recombination in HIV-1 biology is being increasingly recognized. In vitro studies have shown that up to 30 strand-transfer events may occur per viral replication cycle. Thus, recombination may surpass mutation as a major mechanism driving HIV-1 evolution. Currently, recombinant strains comprise 37% of the full-genome HIV-1 sequence database, including sequences representing 47 Circulating Recombinant Forms (CRFs) and more than 250 different Unique Recombinant Forms (URFs). Mapping of recombination breakpoints helps establish relationships among strains that are related by descent, such as CRF07_BC and CRF08_BC in China, and sheds light on their origin and epidemic spread. Additionally, unrelated recombinants sharing common breakpoints may reflect recombination hotspots within the viral genome. Here we present a software tool, RecDraw, for the graphical representation and efficient comparison of recombinant HIV-1 structures and breakpoints. RecDraw is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/research.php?ServiceID = 5SubServiceID = 6 .

Cameroon is a country in West Central Africa in which all four groups of HIV-1 (M, N, O, and P), some circulating recombinant forms (CRFs) and unique recombinant forms (URFs) are prevalent. The CRF22 was initially identified through a novel URF strain, 01CM53122, and later defined from two additional sequences; however, the genomic properties of CRF22 have never been demonstrated in detail. In this study, we describe the characterization of five CRF22_01A1 strains, 02CMLT72, 01CM1867LE, 01CM001BBY, 02CM3097MN, and 02CM1917LE, identified in Cameroon without apparent epidemiological links. A typical CRF22_01A1 strain contains five fragments that can be assigned to the CRF01_AE and subsubtype A1 radiations. Forty-eight percent of the genome is classified as CRF01_AE, spanning the entire region of the gag gene, part of the pol gene, and accessory genes as well as the beginning and the end of the env gene and nef gene. Fifty-two percent of the genome is subsubtype A1 including regions mostly in the pol, vif, and env genes. The five CRF22_01A1 viruses formed a deep branch outside the groups of CRF01_AE and displayed similar mosaic structure but were moderately different from the original strain of CRF22_01A1, 01CM53122. Further analysis of the 01CM53122 genome showed that this virus represents a diverse set of mosaic genomes from CRF22_01A1, including a 446-nt segment of 01CM53122 in the env region, but unlike other CRF22 strains, clustered with CRF01_AE rather than the A1 sequence, suggesting that the 01CM53122 strain is a recombinant of CRF22_01A1 and CRF01_AE.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated neurocognitive disorders (HAND). The role of HIV Nef and other accessory proteins in HAND pathogenesis is unclear. To determine whether HIV nef undergoes adaptive selection in brain, we cloned 100 nef sequences (n = 30 brain and n = 70 lymphoid) from four patients with AIDS and HIV-associated dementia (HAD). Normalized nonsynonymous substitutions were more frequent at the divergence of lymphoid and brain sequences, indicating stronger adaptive selection in brain compared to lymphoid tissue. Brain-specific nonsynonymous substitutions were found within an NH(3)-terminal CTL epitope, the PACS1 binding motif, or positions predicted to be important for activation of the myeloid-restricted Src family tyrosine kinase Hck. These results suggest that adaptive selection of HIV nef in brain may reflect altered requirements for efficient replication in macrophages and brain-specific immune selection pressures.

The prevalence of different HIV-1 subtypes in Spain varies by geographic region. In the present study isolates were collected from 72 newly diagnosed individuals in western Andalucia from 2004 to 2006. Viral sequences were amplified and the subtype diversity and prevalence of resistance mutations in the reverse transcriptase and protease genes were determined. The results presented here demonstrate that subtype B virus predominates in this region (88.9%), with the non-B subtypes CRF02_AG (9.7%) and B/G (1.4%) also present. Only two isolates (2.9%) carried resistance mutations in the reverse transcriptase gene and none of the isolates had major resistance mutations in the protease gene. Minor mutations in the protease gene were more prevalent with 86.1% of isolates containing at least one minor mutation. These results elucidate the subtype diversity present in this region and suggest that the transmission of highly resistant virus variants does not occur at a high frequency in this population.

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 02_AG is a major recombinant variant in different geographic areas and is predominant in West and Central Africa. Of particular interest is the increased frequency of CRF02_AG in patients living in Italy. In the present study, phylogenetic analyses were performed on gag, pol (integrase), and env (gp120 and gp41) gene sequences from 34 CRF02_AG-infected patients living in Italy. Thirty out of 34 (89.4%) patients were from western Africa, 3/34 (8.8%) were born in Italy, and 1/34 (2.9%) was from Cuba. Phylogenetic analysis revealed the presence of a well-supported clade (aLRT score>0.75) of sequences only in gp120 and gp41 trees. Evolutionary rate estimation showed a faster evolution for the gp120 gene with respect to the gag, integrase, and gp41 genes. This finding was confirmed by the analysis of interpatient variability. Intrapatient variability was greater in gp120 gene sequences; 10/19 (52.6%; p1 as compared with gag, integrase, and gp41 gene sequences with dN/dS ratios

One of the factors determining a high degree of heterogeneity in the HIV population is recombination-based variation, which leads to the emergence of the virus variants with a mosaic genome. An example is CRF63_02A1, an HIV-1 variant currently spreading in the Siberian region of Russia. To prove that this HIV-1 variant is a new circulating recombinant form that had emerged as a result of repeated recombination between CRF02_AG and subtype A, we have isolated seven full-length HIV genomes and theoretically analyzed them, that is, reconstructed the phylogenetic relationships, determined recombination breakpoints and regions, and compared them with the regions known for CRF02_AG.

Molecular surveillance is the backbone of HIV-1 vaccinology. Full-length HIV-1 sequences are useful tools that can provide a better understanding of the epidemiology in a given region. A limited number of full-length HIV-1 sequences are available from India, where >95% of the HIV infections are due to HIV-1 subtype C (HIV-1C), which is distinct from the prototype African HIV-1C. In this study, we sequenced six full-length clones isolated from three patients. Extensive phylogenetic analyses of the full-length viral sequences using bioinformatic tools identified a separate cluster of Indian strains, thus confirming the distinct phylogenetic identity of the Indian HIV-1C. Notably, the long terminal repeat (LTR) of two of the six molecular clones contained only two NF-κB binding sites. The sequences also displayed features characteristic of HIV-1C including a Tat dicysteine motif, a shortened Rev open reading frame, and a predicted CCR5 coreceptor tropism for gp120 of three of the proviral sequences.

We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach.

A prototype assay was used to genotype integrase (IN) from 120 HIV-1- infected IN inhibitor-naive adults from Argentina, Brazil, Cameroon, South Africa, Thailand, and Uganda. Subtype designations based on analysis of pol IN sequences were A (14), B (15), C (12), D (11), F (12), G (7), H (1), CRF01_AE (9), CRF02_AG (34), CRF22_01A1 (4), and CRF37_cpx (1). Ten (8.3%) of 120 samples had mutations associated with reduced susceptibility to the IN inhibitors, raltegravir and elvitegravir. Two samples had E92Q (both subtype B) and eight had E157Q (2A, 1C, 1D, 1F, 3 CRF02_AG). Some samples had other mutations selected by these drugs including T97A, and some had amino acid polymorphisms at positions associated with raltegravir and elvitegravir resistance. Mutations associated with other investigational HIV IN inhibitors were also identified. This suggests that HIV strains may vary in their natural susceptibility to HIV IN inhibitors.

A novel HIV-1 circulating recombinant form (CRF) designated CRF65_cpx was recently characterized from three epidemiologically unlinked individuals infected through heterosexual contact in western Yunnan province of China. This is the first complex mosaic HIV-1 CRF, consisting of contributions from three or more different subtypes, identified in China. An additional full-length genome sequence with identical recombinant breakpoints was found among a previously reported recombinant strain from a man who had sex with a man in Anhui province of East Central China. The breakpoint analysis of the recombinants showed a complex genome organization composed of parental subtypes B' (Thailand variant of subtype B), C, and CRF01_AE, with 13 recombination breakpoints observed in almost all structure genes of HIV-1. The generation of complex recombinant forms is likely due to cocirculation of multiple lineages of HIV-1 strains in high-risk populations in western Yunnan.

We identified a novel HIV-1 circulating recombinant form (designated CRF57_BC) from a total of four patients with no obvious epidemiologic linkage in western Yunnan (Dehong prefecture) in China. Two strains (09CN.YNFL37 and 10CN.DHFL17) were identified in this study. An additional two strains (341 and 1439) were found among strains reported in a previous study. CRF57_BC was composed of subtype B and subtype C, with one subtype B segment inserted into the gag region of the subtype C backbone. Subregion tree analysis showed that the B regions originated from a Thai B lineage and the C regions were from an India C lineage. The emergence of CRF57_BC may reflect the continual generation of various forms of intersubtype recombinants in western Yunnan.

Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE is transmitted mainly by sexual activity in Guangxi, southwestern China. Other subtypes, including CRF07_BC, CRF08_BC, and subtype B, are also prevalent in this region. Cocirculation of multiple subtypes, as well as a high rate of drug use, creates favorable conditions for the emergence of recombinant viruses in Guangxi. In the present study, we identified a new HIV-1 unique recombinant form (CRF01_AE /08BC) transmitted from the infected index patient to his seronegative sexual partner. This is the first near full-length genome characterization of a CRF01_AE /08BC recombinant virus in Guangxi, and provides an important basis for future analysis on potential new recombinant transmission events.

An increasing number of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) all over the world has necessitated being vigilant about new recombinants. Since the first report of a recombinant virus with an A1/C mosaic in 1998 more and more B/C and A/C recombinant viruses are being reported from India. Here we report the identification and characterization of a unique HIV-1 A1/C recombinant circulating in Western India. Analysis of the full-length genome using RIP, SimPlot, and jpHMM@Gobics has confirmed its mosaic structure with insertion of subtype A1 in the backbone of subtype C at three positions: gag-pol (1973±15–2617±47), pol-vif (4879±37–5582±32), and gp41 (8437±106–8811±8); however, RIP and SimPlot showed one more small insertion in integrase (4343–4519). Phylogenetic analysis confirmed that the recombinant virus has an insertion of clade A1 in the backbone of subtype C, which has come from Indian subtype C.

We report here a novel HIV-1 B′/C recombinant isolate JL100091, identified from an HIV-positive female subject infected through heterosexual transmission in Jilin in 2006. The near full-length genome analyses of the novel recombinant (JL10091) showed that one subtype B′ region (3,085 bp) was inserted into the subtype C backbone, with two breakpoints observed in gag and pol genes. To our knowledge, this is the first detection of a novel HIV-1 B′/C recombinant in Jilin, which indicates ongoing transmission of networks among the heterosexual population in the region. The novel HIV-1 B′/C recombinant (JL10091) in Jilin originated from India subtype C and China subtype B′ may suggest potential transmission routes of HIV-1 in China. Further monitoring of the molecular epidemiology of the HIV-1 epidemic in Jilin will provide critical information for designing effective control and prevention measures against HIV transmission in the region.

We report a high frequency of drug resistance mutations among patients with unusual insertions or deletions at the β3–β4 hairpin-loop-coding region of HIV-1 subtype C reverse transcriptase, during failure of first-line antiretroviral therapy containing only reverse transcriptase inhibitors in Chennai, India.

The distribution of HIV-1 subtypes and genetic characterization of CRF01_AE in Guangxi, southern China were identified. The distribution of HIV-1 genotypes based on gag, pol, and partial env sequences (n=349) was as follows: CRF01_AE (66.5%), CRF08_BC (19.2%), CRF07_BC (7.2%), URF (4.6%), subtype B (1.7%), and subtype B′ (0.9%). CRF01_AE predominated in all geographic regions and risk populations and there were multiple introductions of CRF01_AE strains in Guangxi. We found a peculiar CRF01_AE monophyletic lineage distinct from other CRF01_AE viruses, and we designated it “CRF01_AE-v” for convenience. CRF01_AE-v circulating in both heterosexuals and injecting drug users (IDUs) had accounted for 39.7% of CRF01_AE. It showed a selective advantage in the Guangxi population and formed its own characteristic compared with all the CRF01_AE references. Our results suggested that CRF01_AE-v was a new variant of CRF01_AE and it might lead to a new epidemic in Guangxi.

The HIV-1 epidemic in Russia has been insufficiently studied, with only 11 complete genome sequences from this country currently available, only three of which are of the locally predominant genetic form, the former Soviet Union (FSU) subtype A variant (A(FSU)). Here we analyze 10 newly derived A(FSU) near full-length genome sequences from Russia. Samples were selected based on phylogenetic clustering in protease-reverse transcriptase in two of the major A(FSU) clusters, V77I(PR) (n=6), widely circulating in Russia and other FSU countries, and A(SP1) (n=4), predominant in St. Petersburg. The phylogenetic analysis shows that the V77I(PR) genomes group in a monophyletic cluster together with 10 previously obtained A(FSU) genome sequences from Uzbekistan, Kazakhstan, Russia, and Cyprus, all bearing the V77I substitution in protease. Similarly, the four A(SP1) genomes group in a monophyletic cluster. These results therefore show that the monophyly of V77I(PR) and A(SP1) A(FSU) clusters is supported in near complete genomes. We thank the personnel at the Genomic Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, for technical assistance in sequencing. This work was funded by WHO-UNAIDS through agreement for performance of work 2011/120936-0, which was partially funded by Office of AIDS Research, National Institutes of Health, USA. Sequences have been deposited in GenBank under accessions JQ292891–JQ292900.

HIV proviral DNA integration into the host chromosome is carried out by integrase becoming an important target antiretroviral therapy. Raltegravir was the first integrase inhibitor approved for use in HIV therapy and elvitegravir is in the late phase of clinical development; both show good results in monotherapy studies and may be used worldwide for rescue therapy. In this work we analyzed 57 integrase sequences obtained from samples from drug-naive and first line regime-failing patients from Maputo, Mozambique, to evaluate the presence of natural polymorphisms and resistance mutations associated with raltegravir and elvitegravir. No major mutations conferring resistance to integrase inhibitors were found and polymorphic accessory mutations were solely observed in low frequency among subtype C sequences—L74M (3.4%), T97A (1.8%), and E157Q (1.8%)—suggesting that this new antiretroviral drug class will be effective in Mozambique providing a good perspective to the introduction of this class of drugs in that country.

The tax gene of human T-lymphotropic virus type 1 (HTLV-1) diverges among isolates according to geographic regions and has been classified into two genotypes: taxA and taxB. In Brazil, taxA is the most prevalent genotype in symptomatic and asymptomatic carriers. Few studies have been conducted in HIV-infected patients. The present study characterized the tax gene (1059 bp) in 13 Brazilian HIV-1/HTLV-1-coinfected patients from the south and southeast regions. The results confirmed the transcontinental HTLV-1 subgroup A of the Cosmopolitan subtype and showed high nucleotide similarity both among Brazilian sequences and in relation to the ATK prototype (99.5% and 99.2%, respectively). Six nucleotide substitutions were highly conserved among isolates, ranging from 76.9% to 100%: C7401T, T7914C, C7920T, C7982T, G8231A, and A8367C. The presence of the Brazilian molecular signature of genotype taxA was confirmed in all of the isolates, and they clustered into two Latin American clusters, which confirms the double introduction of HTLV-1 in Brazil.

The concept of transmission bottlenecks in HIV-1 infection is well established. Coinfections and superinfections have been increasingly documented and provide a founding cause for the expansion of viral diversity through recombination. It is still relatively unclear how HIV-1 will propagate and evolve in individuals infected with more than one viral strain. Here we report on the parallel transmission of genetically distant viral strains cocirculating in one individual over many years to a single recipient.

HTLV-1 is endemic in Brazil and HIV/HTLV-1 coinfection has been detected, mostly in the northeast region. Cosmopolitan HTLV-1a is the main subtype that circulates in Brazil. This study characterized 17 HTLV-1 isolates from HIV coinfected patients of southern (n=7) and southeastern (n=10) Brazil. HTLV-1 provirus DNA was amplified by nested PCR (env and LTR) and sequenced. Env sequences (705 bp) from 15 isolates and LTR sequences (731 bp) from 17 isolates showed 99.5% and 98.8% similarity among sequences, respectively. Comparing these sequences with ATK (HTLV-1a) and Mel5 (HTLV-1c) prototypes, similarities of 99% and 97.4%, respectively, for env and LTR with ATK, and 91.6% and 90.3% with Mel5, were detected. Phylogenetic analysis showed that all sequences belonged to the transcontinental subgroup A of the Cosmopolitan subtype, clustering in two Latin American clusters.

Recombination between HIV-1 subtypes B and F has generated several circulating and unique recombinant forms, particularly in Latin American areas. In Italy, subtype B is highly prevalent while subtype F is the most common pure non-B subtype. To investigate the recombination pattern in Italian BF recombinant viruses, we characterized full-length sequences derived from 15 adult patients, mostly Italian and infected by the heterosexual route. One of the BF mosaics was a CRF29, three sequences clustered with low bootstrap values with CRF39, CRF40, and CRF42. With the exception of the CRF29-like sequence, the other recombination patterns were unique, but two possible clusters were identified. Analysis of the gp120 V3 domain suggested a possible link with subtype F from Eastern Europe rather than from Latin America, favoring the hypothesis of local recombination between clade B and F viruses over that of import of BF recombinants from Latin America. HIV-1 subtypes B and F appear prone to generation of unique recombinants in Italy, warranting epidemiological surveillance and investigation of a possible clinical significance.

For HIV recombination to occur, the RNAs from two infecting strains within a cell must dimerize at the dimerization initiation site (DIS). We examined the sequence identity at the DIS (697-731 bp, Hxb2 numbering engine) in patients superinfected with concordant HIV-1 strains and compared them to those with discordant strains. Viral RNA in sequential plasma from four subjects superinfected with subtype-discordant and two subjects superinfected with subtype-concordant HIV-1 strains was extracted, amplified (5' LTR-early gag: 526-1200 bp, Hxb2 numbering engine), sequenced, and analyzed to determine their compatibility for dimerization in vivo. The concordant viruses infecting the two subjects exhibited identical sequences in the 35-bp-long DIS region while sequences from the discordant viruses revealed single nucleotide changes that were located in the DIS loop (715 bp), its flanking nucleotides (710 bp and 717 bp), and the DIS stem (719 bp). Evidence from in vitro experiments demonstrates that these in vivo changes identified can abolish dimerization and reduce recombination frequency. Therefore, these results revealing differences in the DIS of discordant strains versus the similarity noted for the concordant strains may contribute to the differences in the frequency of recombination in patients superinfected with such HIV-1 variants.

We assessed the viral envelope (V3-V5 region) sequence diversity from 13 HIV-1-infected Indian children from north India. All of the 13 children were found to be infected with subtype C viruses. One of the viral sequences exhibited usage of the CXCR4 coreceptor predicted by Web PSSM and Geno2pheno tools. This virus also had a longer V3 sequence with 37 amino acids, a GRGQ motif, and a methionine residue before it (AIIMS_307). A unique finding was the complete deletion of the V4 region of another virus (AIIMS_363). High sequence diversity was observed in the envelope of the HIV-1-infected Indian children.

Here we present new sequence data from HIV-1 subtype C-infected long-term survivors (LTS) from Karonga District, Malawi. Gag and env sequence data were produced from nine individuals each of whom has been HIV-1 positive for more than 20 years. We show that the three amino acid deletion in gag p17 previously described from these LTS is not real and was a result of an alignment error. We find that the use of dried blood spots for DNA-based studies is limited after storage for 20 years. We also show some unlikely amino acid changes in env C2-V3 in LTS over time and different patterns of genetic divergence among LTS. Although no clear association between mutations and survival could be shown, amino acid changes that are present in more than one LTS may, in the future, be shown to be important.

Infecting rhesus macaques (Macaca mulatta) with the simian immunodeficiency virus (SIV) is an established animal model of human immunodeficiency virus (HIV) pathogenesis. Many studies have used various derivatives of the SIVmac251 viral swarm to investigate several aspects of the disease, including transmission, progression, response to vaccination, and SIV/HIV-associated neurological disorders. However, the lack of standardization of the infecting inoculum complicates comparative analyses. We investigated the genetic diversity and phylogenetic relationships of the 1991 animal-titered SIVmac251 swarm, the peripheral blood mononuclear cell (PBMC) passaged SIVmac251, and additional SIVmac251 sequences derived over the past 20 years. Significant sequence divergence and diversity were evident among the different viral sources. This finding highlights the importance of characterizing the exact source and genetic makeup of the infecting inoculum to achieve controlled experimental conditions and enable meaningful comparisons across studies.

Cocirculation of subtype B and CRF01_AE in Southeast Asia has led to the establishment of new recombinant forms. In our previous study, we found five samples suspected of being recombinants between subtype B and CRF01_AE, and here, we analyzed near full-length sequences of two samples and compared them to known CRFs_01B, subtype B, and CRF01_AE. Five overlapped segments were amplified with nested PCR from PBMC DNA, sequenced, and analyzed for genome mosaicism. The two Indonesian samples, 07IDJKT189 and 07IDJKT194, showed genome-mosaic patterns similar to CRF33_01B references from Malaysia, with one short segment in the 3' end of the p31 integrase-coding region, which was rather more similar to subtype B than CRF01_AE, consisting of unclassified sequences. These results suggest gene-specific continuous diversification and spread of the CRF33_01B genomes in Southeast Asia.

In this preliminary study we show that in 2008, 3 years after antiretroviral therapy was introduced into the Karonga District, Malawi, a greater than expected number of drug-naive individuals have been infected with HIV-1 subtype C virus harboring major and minor drug resistance mutations (DRMs). From a sample size of 40 reverse transcriptase (RT) consensus sequences from drug-naive individuals we found five showing NRTI and four showing NNRTI mutations with one individual showing both. From 29 protease consensus sequences, again from drug-naive individuals, we found evidence of minor DRMs in three. Additional major and minor DRMs were found in clonal sequences from a number of individuals that were not present in the original consensus sequences. This clearly illustrates the importance of sequencing multiple HIV-1 variants from individuals to fully assess drug resistance.

In this study, HIV strains circulating among military personnel were characterized, in Malabo, the capital city of Equatorial Guinea. One sample was found to be HIV-2 group A while a high degree of genetic diversity was recorded in the pol region of 41 HIV-1-positive samples. CRF02_AG accounted for 53.7% of the strains, and 11 different variants were obtained in the remaining 19 samples: subtype G (n = 3), A3 (n = 2), C (n = 2), CRF26_A5U (n = 2), F2 (n = 1), CRF06 (n = 1), CRF09 (n = 1), CRF11 (n = 1), CRF22 (n = 1), and divergent subtype A (n = 1) and F (n = 1). One strain could not be classified and three were unique recombinants. Analysis of antiretroviral drug resistance mutations revealed two patients each harboring one major mutation, M46I in protease and D67N in reverse transcriptase sequences, respectively. The high genetic diversity and emerging ARV resistance mutations call for frequent surveys and appropriate monitoring of ARV considering the increasing access to ARV in the country.