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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.

To analyze HIV-1 subtype distribution, sequence analysis was performed on serum specimens obtained in 1994 from the Rakai Health Sciences community cohort in Uganda. Portions of gag-p24 and env-gp41 were sequenced and HIV subtype was determined for 773 subjects residing in 10 community clusters in rural Uganda. Subtypes A (17%) and D (70%) were the most common strains in the population. Subtype distribution varied by geographic region with significantly more subtype A in northern community clusters compared with southern clusters (21% vs. 8%, p

Six morphine-dependent and three control macaques were infected with a mixture of SIV/SHIV. Half of the animals in the morphine group developed accelerated disease (rapid progressor) and died within 20 weeks postinfection. The evolution of the envelope gene in the brain of the rapid progressor and morphine-dependent group along with that in the control group was assessed. Six to 10 clones from the brain of each macaque were sequenced and were compared against each other as well as against a challenge virus. Analysis of the sequences revealed that the diversity and divergence of the clones were higher in the control group as compared to the morphine-dependent macaques, although this difference was not statistically significant.

The emergence of HIV drug resistance is a major obstacle to effective antiretroviral (ARV) treatments. This study examined the drug resistance profiles among South African patients virologically failing ARV therapies between 2000 and 2003, prior to the introduction of a national treatment program. Samples were obtained from 65 HIV-1 subtype C-infected patients (39 children and 26 adults) who had received at least two nucleoside reverse transcriptase inhibitors (NRTIs) and either a nonnucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI). Resistance assays were performed using the HIV-1 ViroSeq Genotyping System and mutations were defined according to the Stanford Sequence Resistance Database. Ninety-one percent of patients harbored resistance mutations; the most frequent NRTI mutations were M184V/I (37%), D67N (32%), T215Y/F (25%), K70R (21%), M41L (20%), K219Q/E (14%), and K65R (14%), reflecting the frequent use of lamuvidine and zidovudine. K103N (25%), V106M (20%), and G190A (17%) were found among patients failing nevirapine- or efavirenz-containing regimens. Of the patients who received PIs, the most common mutations were V82A/T (12%), M46I (11%), and L90M (8%). Mutations were similar among adults and children. These data indicate that HIV-1 drug resistance develops in South African subtype C-infected patients failing ARV therapy with mutations comparable to those found among patients infected with subtype B viruses.

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.

Broad HIV-1 genetic diversity in Cameroon provides a unique opportunity to monitor HIV-1 evolution and allows the detection of novel strains. We have genetically characterized the HIV-1 subtypes found in 156 samples from 90 drug-naive subjects in Yaounde, Cameroon collected from 2011 to 2013, using phylogenetic analysis of regions in gag and pol. We identified subtypes CRF02_AG (64.9%), CRF22_01A1 (7.1%), D (4.5%), F2 (3.9%), G (3.2%), CRF18_cpx (3.2%), CRF37_cpx (3.2%), CRF11_cpx (2.6%), CRF13_cpx (1.9%), A1 (1.3%), CRF01_AE (1.3%), CRF09_cpx (1.3%), A2 (0.6%), and H (0.6%). Sequence data for both the gag and pol regions were obtained from 62 subjects; for 59 of these subjects the two regions were identified as the same viral subtype while three subjects were discordant, A1/CRF02_AG (subject MDC006), CRF02_AG/F2 (subject MDC179), and a dual infection with CRF02_AG/F2 (subject MDC131). Longitudinal sequence data were obtained for 28 of these 62 subjects and confirmed the cross-sectional results. These data update subtype information for this area and highlight the necessity of such studies due to the numerous circulating subtypes, the ongoing superinfection, and the risk of emerging novel recombinant viruses.

Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies.

There is continuous need to track genetic profiles of HIV strains circulating in different geographic settings to hasten vaccine discovery and inform public health and intervention policies. We partially sequenced the reverse transcriptase region of the HIV-1 pol gene from a total of 54 Kenyan patients aged 18–56 years who continued highly active antiretroviral treatment (HAART) for between 8 and 102 months. Subtyping was done using both the JPHMM tool and phylogenetic method. HIV-1 subtype A1 was the predominant strain in circulation, representing 57.4% and 70.4% of all isolates as determined by JPHMM and phylogenetic methods, respectively. Subtypes D (14.8%, 7.4%), C (5.6%, 9.3%), and A2 (0%, 5.6%) were determined at respective prevalence by both methods. JPHMM identified 22.2% of the isolates as recombinants. This surveillance focused on the RT gene and reaffirms the predominance of subtype A and an increasing proportion of recombinant strains in the Kenyan epidemic.

Eleven protease mutations have been associated with reduced susceptibility to darunavir. In this study of 87 HIV-1-infected patients experiencing virological failure to second-line regimens containing protease inhibitors boosted with ritonavir (viral load >1,000 HIV RNA copies/ml), we observed a low prevalence (3%) of ≥3 darunavir resistance-associated mutations, indicating that this drug may be a good option for third-line antiretroviral therapy in southern India.

We reported previously that the prevalence of drug-resistant HIV-1 among antiretroviral therapy (ART)-naive individuals in Northern Vietnam was 2.9% in 2007 and 6.2% in 2008. To investigate the continuing trend of prevalence, we collected plasma samples from 958 individuals in Hai Phong and Hanoi in 2009, extracted viral RNA from HIV-1 antibody-positive samples, and analyzed them genetically. HIV-1 antibody prevalence was 26.8% in injecting drug users (n=302), 13.4% in female sex workers (n=284), 0.5% in blood donors (n=206), and 0.6% in pregnant women (n=166). All HIV-1 strains were CRF01-AE. Nonnucleoside reverse-transcriptase inhibitor resistance mutations were found in two (2.0%) of the 102 successfully analyzed cases (one case with the Y181C and one with the K101E). No nucleoside reverse-transcriptase inhibitor resistance or protease inhibitor resistance mutations were detected. The prevalence of circulating ART-resistant HIV-1 in Northern Vietnam did not increase from 2007 to 2009, although the rate of ART coverage did increase. © 2012, Mary Ann Liebert, Inc.

The Brazilian AIDS epidemic is characterized by significant geographic contrasts: a reduction in incidence and mortality in the epicenter (southeast) and an increase in the northeast. HIV-1-transmitted drug resistance (TDR) and genetic diversity were investigated among 106 antiretroviral (ARV)-naive patients from Maranhão State, northeast. The HIV-1 protease (PR) and reverse transcriptase (RT) regions were sequenced; subtypes were assigned by REGA/phylogenetic analysis. TDR to the nucleoside/nonnucleoside reverse transcriptase inhibitor (NRTI/NNRTI) and protease inhibitor (PI) was identified by the Calibrated Population Resistance tool (Stanford). The median age was 31 years (range 18–72), with 54.7% women, 78.3% heterosexual transmission, and 17.9% men who have sex with men (MSM). Around 30% had

Injection drug use has been responsible for most HIV-1 transmission in Asia and has led to the generation and/or spreading of circulating recombinant CRF01_AE, CRF07_BC, and CRF08_BC, which are currently the predominant HIV-1 strains in China. Monitoring HIV-1 transmissions among injection drug users (IDUs) still provides critical information on HIV-1 prevalence and control. In the present study, we detected three HIV-1 sequences, BH048/057/066, from IDUs in Beihai city, Guangxi province, all of which share an extremely complex mosaic recombination pattern. CRF01_AE and CRF08_BC contributed to the rise of new recombinants, which contain CRF08_BC-derived env fragments with evidence of further evolution. Our data, combined with those of other studies, suggest that the BH048/057/066 recombinants represent early strains of a circulating recombinant form of HIV-1 in southern China. Information concerning such newly appearing variants is critical for both HIV-1 control and ongoing HIV-1 vaccine trials in Guangxi, China.

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

Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36-45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage therapy in patients failing first line regimens in Mozambique. However, further phenotypic studies are required to determine the clinical relevance of the polymorphisms detected in this study.

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.

Reports show that up to 30% of antiretroviral drug-naive patients in Johannesburg have CXCR4-utilizing HIV-1 subtype C. We assessed whether HIV-1 subtype C-infected individuals failing highly active antiretroviral therapy (HAART) have a higher proportion of CXCR4-utilizing viruses compared to antiretroviral drug-naive patients. The V3 loop was sequenced from plasma from 100 randomly selected HAART-failing patients, and tropism was established using predictive algorithms. All patients harbored HIV-1 subtype C with at least one antiretroviral drug resistance mutation. Viral tropism prediction in individuals failing HAART revealed similar proportions (29%) of X4-utilizing viruses compared to antiretroviral drug-naive patients (30%). Findings are in contrast to reports from Durban in which 60% of HAART-failing subjects harbored X4/dual/mixed-tropic viruses. Despite differences in proportions of X4-tropism within South Africa, the high proportion of thymidine analogue mutations (TAMs) and CXCR4-utilizing HIV-1 highlights the need for intensified monitoring of HAART patients and the predicament of diminishing drug options, including CCR5 antagonists, for patients failing therapy.

We report the presence of drug resistance mutations in 7.4% (2/27) of the treatment-naive, HIV-1-infected children in Pune, India, who had HIV-1 RNA levels1,000 copies/ml. Nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations, namely A98G and K103N, were observed in two separate sequences. In addition, three study sequences displayed three separate HIV-1 protease minor resistance mutations-L10I, A71T, and T74S. These preliminary data from Pune, India, reporting the presence of HIVDR in treatment-naive, HIV-1-infected children, reinforces the need to conduct large-scale studies to assess the prevalence of primary HIVDR in the pediatric population, which in turn will aid in planning protocols and policies related to antiretroviral treatment for the pediatric population.

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.

To elucidate the prevalence of HIV-1 subtypes and transmitted drug resistance in Henan, central China, HIV-1-positive blood samples from 187 antiretroviral-naive patients were collected in our study from August 2009 to November 2010. Subtype B′ (92.0%, 172 of 187) remains the predominant HIV-1 subtype in Henan province and was prevalent in all risk populations and geographic regions. Of 98 pol sequences 67 (68.4%) harbored drug resistance mutations, and only 14 (14.3%, 14 of 98) sequences have mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs. The unexpectedly high percentage of drug resistance in Henan province is mainly due to the prevalence of minor mutations in the protease and integrase regions, especially A71T/V and L68V/I/IM/LV. In all, we detected a relatively high prevalence of drug resistance with unique mutation distributions among antiretroviral-naive patients from Henan province.

In Peru, there is a lack of information on molecular analysis in pediatric human immunodeficiency virus (HIV) infection. At present, the mother-to-child transmission rate is estimated at approximately 2–4%. The objective of this study was to assess the molecular epidemiology of HIV-1 in infected children. Children with suspected or confirmed pulmonary tuberculosis were evaluated at two public hospitals between 2002 and 2007. Whole blood samples were obtained from 90 HIV-positive children, who were confirmed to be positive by enzyme-linked immunosorbent assay and Western blot. The specimens were subjected to envelope heteroduplex mobility assay (env HMA) followed by gag and pol gene region sequence analysis. Subtype B was found in 88 (98%) of 90 children and 2 (2%) children were subtype BF recombinants. This is the first report of recombinant HIV strains in HIV-infected children in Peru. Understanding the origin, diversity, and spread of HIV strains worldwide will be necessary for the development of an effective vaccine that targets pediatric populations throughout the world.

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.

HIV-1, human T cell lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) and hepatitis C virus (HCV) are common among intravenous drug users (IDUs) and can cause chronic infections in the host. Usually, the diagnosis of such viruses employs serological assays; however, some difficulties in confirming HTLV-2 infection have been reported in high-risk populations in Brazil. We present data of an unusual case of coinfection with HIV-1, HTLV-1, HTLV-2, and HCV in a male IDU in which HTLV-2 was detected only by molecular assays. Comparative analysis of retroviruses from 2002 and 2012 showed identical HTLV-1 and HTLV-2 sequences (LTR, env, and tax), and a change in HIV-1 tropism from CXCR4 to CCR5. No mutation was detected in the hot points of the env region of the HTLV-2 isolate that justified the lack of rgp46-II-specific antibodies. These data emphasize the need for molecular assays to diagnose HTLV-2 in high-risk populations in Brazil.

We report here a novel HIV-1 second-generation recombinant form (CRF01_AE/CRF07_BC) composed of CRF01_AE and CRF07_BC, identified among men who have sex with men (MSM) in Jilin, with four breakpoints observed in the pol, vif, and vpr genes. The CRF01_AE regions of the recombinant were clustered with the CRF01_AE lineage, which is mainly circulating among MSM in northern China, with the support of 100% bootstrap value, indicating that the parental origin of the CRF01_AE regions was from MSM, in which recombination events may be more likely to occur. To the best of our knowledge, this is the first detection of a novel HIV-1 second-generation recombinant form (CRF01AE/CRF07_BC) in Jilin, which indicates active transmission networks of HIV-1 infection among MSM in the region. Therefore, it is necessary to continue monitoring the molecular epidemiology of HIV-1 among MSM in Jilin to obtain a better understanding of the transmission and potential public health impact of HIV-1 among MSM in the region.

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.

We sought to identify the prevalent subtypes and study the genetic variation of HIV-1 circulating in HIV infections in Harbin City, China. Forty-seven samples from the env V3-V4 region were successfully sequenced and analyzed, which involved thirty-one men who have sex with men (MSM), eight heterosexuals, seven former plasma donors (FPD)/blood transfusion recipients (BT), and one injection drug user (IDU). In all, 46.8% of CRF01_AE, 40.4% of subtype B, and 12.8% of CRF07_BC were identified. CRF01_AE (64.5%) was the dominant strain in MSM, and subtype B (81.2%) was the chief strain in other infected subjects except for the MSM population. Among all the genotypes, the B subtype possesses greater diversity of the tetramer on the tip of V3 loop than CRF07_BC and CRF01_AE, in which the peculiar GWGR was commonly found. Because nationwide there is a trend toward the increasing presence of CRF01_AE, a consecutive surveillance campaign was necessary among all HIV vulnerable populations in this locality.

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.

The advent of next generation sequencing technologies is providing new insight into HIV-1 diversity and evolution, which has created the need for bioinformatics tools that could be applied to the characterization of viral quasispecies. Here we present Nautilus, a bioinformatics package for the analysis of HIV-1 targeted deep sequencing data. The DeepHaplo module determines the nucleotide base frequency and read depth at each position and computes the haplotype frequencies based on the linkage among polymorphisms in the same next generation sequence read. The Motifs module computes the frequency of the variants in the setting of their sequence context and mapping orientation, which allows for the validation of polymorphisms and haplotypes when strand bias is suspected. Both modules are accessed through a user-friendly GUI, which runs on Mac OS X (version 10.7.4 or later), and are based on Python, JAVA, and R scripts. Nautilus is available from www.hivresearch.org/research.php?ServiceID=5&SubServiceID=6.

We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3' splice site (3'ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3'ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3'ss A4c. Additionally, unusual nef RNAs using 3'ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated. We thank the personnel at the Genomic Unit of Centro Nacional de Microbiología, Instituto de Salud Carlos III, for technical assistance in sequencing and GeneMapper analyses. This work was funded by Ministerio de Economía y Competitividad (Spain), Plan Nacional de I+D+I, through grants SAF2007-61688 and SAF2010-2096. Sequences of PCR clones derived from P2149-3 DS transcripts have been deposited in GenBank under accession numbers JF808039–JF808078.

Although 2-long terminal repeat (2-LTR) circles are only a fraction of the total viral DNA in infected cells, sequence analysis of 2-LTR circles reveals critical information regarding viral DNA synthesis and the nature of actively replicating virus. It was observed that a large proportion of the 2-LTR circular molecules in the peripheral blood mononuclear cell (PBMC) DNA of infected individuals are mutated at the circle junction. The integrase inhibitor raltegravir (RAL) blocks the strand transfer step of the integration of HIV-1; as a consequence of abortive integration a significant increase of episomal 2-LTR circles is observed. Moreover, it was demonstrated that in patients treated with highly active retroviral therapy (HAART) changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated whether RAL therapy could have a differential impact on the 2-LTR circle junctional sequences in patients with different virological profiles at the time of starting RAL therapy. Sequence analysis indicates that RAL acts differently in the two populations.

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.

The main heroine traffic from Yunnan province to the Xinjiang Autonomous Region is believed to initiate the transmission of CRF07_BC which is the predominant strain in intravenous drug users (IDUs) in China. However, the great distances between Yunnan and Xinjiang lead to an unclear and elusive diffusion process of CRF07_BC due to the absence of an important middle site such as Sichuan province. Moreover, in recent years the rapidly increasing infection rate among IDUs in the Liangshan region of Sichuan made it necessary to characterize the genetic character of the circulating strain of Sichuan IDUs. In this study, we characterized the genetic character of seven newly isolated CRF07_BC genomes (five from Sichuan and two from Xinjiang) and analyzed the transmission linkage among strains from IDUs in different regions. By conducting Markov chain Monte Carlo (MCMC) analysis and reconstruction of neighbor-joining trees and maximum-likelihood trees, our results revealed the genetic variation and important role of Sichuan-derived CRF07_BC strains during the transmission of CRF07_BC.

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.

Zhengzhou is the capital of Henan province, where severe HIV prevalence was found in former paid plasma donors. In recent years, the HIV epidemic in men who have sex with men (MSM) increased rapidly in the city. To explore the subtype distribution and genetic characterization of HIV in MSM in Zhengzhou city, phylogenetic analysis was fulfilled based on the full-length gag, pol, and partial env gene. A total of 31 HIV-1-seropositive MSM individuals were enrolled. The full length gag, pol, and partial env gene were amplified and sequenced. Multiple subtypes, including CRF01_AE (45.2%), subtype B (38.7%), and CRF07_BC (16.1%), were identified. Close phylogenetic relationships among our strains with strains from the Henan local area, Hebei MSM population, Beijing area, and Liaoning area were found, suggesting a multiple introduction of HIV into the population. The results will provide clues for prevention and for changes in behavior in the Henan MSM population and also detailed sequence data for vacc...

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.

Four types of human T cell lymphotropic viruses (HTLV) have been described (HTLV-1 to HTLV-4) with three of them having closely related simian virus analogues named STLV-1, −2, and −3. To assess the risk of cross-species transmissions of STLVs from nonhuman primates to humans in the Democratic Republic of Congo, a total of 330 samples, derived from primate bushmeat, were collected at remote forest sites where people rely on bushmeat for subsistence. STLV prevalences and genetic diversity were estimated by PCR and sequence analysis of tax-rex and LTR fragments. Overall, 7.9% of nonhuman primate bushmeat is infected with STLVs. We documented new STLV-1 and STLV-3 variants in six out of the seven species tested and showed for the first time STLV infection in C. mona wolfi, C. ascanius whitesidei, L. aterrimus aterrimus, C. angolensis, and P. tholloni. Our results provide increasing evidence that the diversity and geographic distribution of PTLVs are much greater than previously thought.

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.

For the first time the genetic diversity among the uniformed personnel in Kinshasa, the capital city of the Democratic Republic of Congo (DRC), a country that has experienced military conflicts since 1998 and in which the global HIV-1/M pandemic started, has now been documented. A total of 94 HIV-1-positive samples, collected in 2007 in Kinshasa garrison settings from informed consenting volunteers, were genetically characterized in the pol region (protease and RT). An extensive diversity was observed, with 51% of the strains corresponding to six pure subtypes (A 23%, C 13.8%, D, G, H, J, and untypable), 15% corresponding to nine different CRFs (01, 02, 11, 13, 25, 26, 37, 43, and 45), and 34% being unique recombinants with one-third being complex mosaic viruses involving three or more different subtypes/CRFs. Only one strain harbored a single mutation, I54V, associated with drug resistance to protease inhibitors. Due to their high mobility and potential risk behavior, HIV infections in military personnel can lead to an even more complex epidemic in the DRC and to a possible increase of subtype C.