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We studied 123 samples from adult chronic HIV patients initiating HAART from various centers around a newly established clinical trial site in Pretoria. Each sample was sequenced in at least one structural gene (pol, gag, and env) or functional gene (vif, vpr, and vpu). A subset of 25 samples was subjected to near full-genome analysis. All samples were HIV-1 subtype C. Highly conserved regions within the gene sequences were observed. Overall, the gag and vif sequences showed closer similarity followed by the env, vpr, pol, and vpu. The env gene was the most difficult to sequence, resulting in only 31 sequences from 40 samples; of these, 25 were predicted to be R5 coreceptor tropic, while 6 were X4 tropic. The study asserted the predominance of HIV-1 subtype C within the catchment population.

The risk of zoonoses is a major obstacle to xenotransplantation. Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection, and its control is a prerequisite for the development of clinical xenotransplantation. The copy number of PERV varies among different breeds, and it has been suggested that the PERV integrations number is increased by inbreeding. The purpose of this study was (i) to examine the copy number of PERV in different Spanish pig breeds, Spanish wild boar and commercial cross-bred pigs from five different farms and genetic background (CCP1-CCP5) and (ii) to investigate the correlation between PERV copy number and the genetic background of the pigs in order to improve the selection of pigs for xenotransplantation. PERV copy number was determined by quantitative, real-time polymerase chain reactions. Thirty-four microsatellite markers were genotyped to describe the genetic diversity within populations (observed and expected heterozygosities, Ho and He, respectively) and the inbreeding coefficient (F). Pearson's correlation coefficient was used to determine the relationship between PERV copy number and Ho, He and F. The copy number of PERV among different pig breeds was estimated to range between three (CCP1) and 43 copies (Iberian Pig). Statistical differences were found among the studied populations concerning PERV copy number. No correlation was found between the PERV copy number and the heterozygosity (calculated at an individual level or at a population level) or the inbreeding coefficient of each population. Our data suggest that pigs inbreeding does not increase PERV copy number and support the idea that careful selection of pigs for organ donation with reduced PERV copy number will minimize the risk of retrovirus transmission to the human receptor., (© 2012 Blackwell Verlag GmbH.)

Background: The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city., Objectives: This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil., Study Design: RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced., Results: From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01)., Conclusion: The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

We investigated the distribution of HIV-1 subtypes and the prevalence of HIV-1 drug resistance among HIV-infected antiretroviral-naive patients in Guangxi, southern China. A total of 144 subjects from Liuzhou or Nanning, two cities having the most HIV-infected cases in Guangxi, were enrolled. HIV-1 pol fragments were amplified and sequenced from plasma of all patients. In all, 124 sequences were obtained, with 67 from Liuzhou and 57 from Nanning. All sequences were subtyped by phylogenetic analysis and analyzed for antiretroviral resistance using the HIVdb program. Our data showed that the sequences from Liuzhou were subtyped as CRF01&#95;AE (77.6%), CRF07&#95;BC(20.9%), and BC (1.5%), respectively, and the sequences from Nanning as CRF01&#95;AE (78.9%), CRF08&#95;BC (15.8%), B (3.5%), and C (1.8%), respectively. Of the sequences 11.9% from Liuzhou and 28.1% from Nanning harbored drug resistance-associated mutations, but there were only two sequences with mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs.

Characterization of HIV subtypes can provide a more comprehensive understanding of the epidemic within a distinct region, and when combined with notification data, may also be helpful in enhancing current HIV prevention strategies. In this study, we characterized 1056 HIV-positive individuals (948 males and 108 females) living in Victoria and whose infection was detected for the first time between 2005 and 2010 inclusive. HIV-1 strains were subtyped based on pol gene sequence. Phylogenetic analysis was performed on all non-B subtype sequences identified. Of the 1056 sequences analyzed, 825 were subtype B and 231 were non-B. Overall 6 HIV-1 subtypes, 6 circulating recombinant forms (CRFs), and 12 unique recombinant forms (URFs) were identified. Regardless of gender, the majority of individuals were infected with a subtype B virus (78%). Subtype B was dominant in males (n=806, 85%). In contrast, the majority of females were infected with non-B subtypes (n=89, 82%), in particular subtype C (n=48, 45%). Phylogenetic analysis of the non-B subtypes revealed that the majority of clustering, and thereby transmission, occurred with CRF01&#95;AE strains. Despite the relatively high numbers identified in females there was very little clustering of subtype C viruses. Subtypes C and A1 both historically associated with heterosexual transmission, and CRF01&#95;AE often associated with IVDU, were also associated with transmission within the MSM population, demonstrating the potential for non-B subtypes to expand into the MSM population. The observation of increasing numbers of females and heterosexual males infected with non-subtype B viruses, the majority imported through migration and travel to countries where there is a high prevalence of HIV, suggests a targeted public health message may be required to prevent further increases within these two groups.

Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

Background: Specific sequence changes of human immunodeficiency virus type 1 (HIV-1) in the presence of specific HLA molecules may alter the composition and processing of viral peptides, leading to immune escape. Persistence of these mutations after transmission may leave the genetic fingerprint of the transmitter's HLA profile. Here, we evaluated the associations between HLA profiles and the phylogenetic relationships of HIV sequences sampled from a cohort of recently infected individuals in San Diego, California., Methods: We identified transmission clusters within the study cohort, using phylogenetic analysis of sampled HIV pol genotypes at a genetic distance of <1.5%. We then evaluated the association of specific HLA alleles, HLA homozygosity, HLA concordance, race and ethnicity, and mutational patterns within the clustering and nonclustering groups., Results: From 350 cohort participants, we identified 162 clustering individuals and 188 nonclustering individuals. We identified trends for enrichment of particular alleles within individual clusters and evidence of viral escape within those clusters. We also found that discordance of HLA alleles was significantly associated with clustering individuals., Conclusions: Some transmission clusters demonstrate HLA enrichment, and viruses in these HLA-associated clusters often show evidence of escape to enriched alleles. Interestingly, HLA discordance was associated with clustering in our predominantly MSM population.

The extent of HIV-1 diversity was examined among patients attending a rural district hospital in a coastal area of Kenya. The pol gene was sequenced in samples from 153 patients. Subtypes were designated using the REGA, SCUEAL, and jpHMM programs. The most common subtype was A1, followed by C and D; A2 and G were also detected. However, a large proportion of the samples was found to be recombinants, which clustered within the pure subtype branches. Phylogeographic analysis of Kilifi sequences compared with those from other regions of Africa showed that while many sequences were closely related to sequences from Kenya, others were most closely related to known sequences from other parts of Africa, including West Africa. Overall, these data indicate that there have been multiple introductions of HIV-1 into this small rural town and surroundings with ongoing diversity being generated by recombination.

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF&#95;BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Phycodnaviruses have a significant role in modulating the dynamics of phytoplankton, thereby influencing community structure and succession, nutrient cycles and potentially atmospheric composition because phytoplankton fix about half the carbon dioxide (CO(2)) on the planet, and some algae release dimethylsulphoniopropionate when lysed by viruses. Despite their ecological importance and widespread distribution, relatively little is known about the evolutionary history, phylogenetic relationships and phylodynamics of the Phycodnaviruses from freshwater environments. Herein we provide novel data on Phycodnaviruses from the largest river system on earth--the Amazon Basin--that were compared with samples from different aquatic systems from several places around the world. Based on phylogenetic inference using DNA polymerase (pol) sequences we show the presence of distinct populations of Phycodnaviridae. Preliminary coarse-grained phylodynamics and phylogeographic inferences revealed a complex dynamics characterized by long-term fluctuations in viral population sizes, with a remarkable worldwide reduction of the effective population around 400 thousand years before the present (KYBP), followed by a recovery near to the present time. Moreover, we present evidence for significant viral gene flow between freshwater environments, but crucially almost none between freshwater and marine environments.

Objectives: To evaluate the frequency and progression over time of the WHO-defined transmitted HIV-1 drug resistance mutations (DRMs) among antiretroviral treatment (ART)-naive HIV-1-infected patients in Cameroon., Design: We analyzed HIV-1 DRM data generated from 369 ART-naive individuals consecutively recruited between 1996 and 2007 in urban and rural areas in Cameroon., Methods: HIV-1 drug resistance genotyping was performed in the pol gene using plasma samples and surveillance DRMs were identified using the 2009 WHO-DRM list., Results: We observed in Yaounde, the capital city, an increasing prevalence of DRMs over time: 0.0% (none of 61 participants) in 1996-1999; 1.9% (one of 53 participants) in 2001; 4.1% (two of 49 participants) in 2002; and 12.3% (10 of 81 participants) in 2007. In the rural areas with more recently implemented ART programs, we found DRMs in six of 125 (4.8%) ART-naive individuals recruited in 2006-2007. DRMs identified in both areas included resistance mutations to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs) that might impair the efficacy of available first-line and second-line treatments., Conclusion: This report showed an increase in transmitted DRMs in areas where antiretroviral drugs were introduced earlier, although other factors such as natural viral polymorphisms and acquired DRMs through exposure to antiretroviral cannot be totally excluded. Further surveillances are needed to confirm this evolution and inform public health policies on adequate actions to help limit the selection and transmission of drug-resistant HIV, while scaling up access to ART in developing countries.

Significant subtype-specific differences were observed in the protease (PR) region of the HIV-1 pol gene. Most of the previous studies were restricted to subtype B, although subtype C accounts for more than 50% of HIV infections worldwide. In this study we characterized PR sequences from primary clinical isolates from protease inhibitor (PI)-naive patients in South India (n=39) as well as database-derived HIV-1 subtype C sequences from India (n=542) and globally (n=2970). All the study sequences were identified as subtype C, which is predominant in India. Drug resistance genotyping analysis identified 2.6% (1/39) prevalence of major PI resistance (I54T) and 7.7% (3/39) of minor PI resistance (L10I, T74S, and A71T). Selection of T12S, I15V, L19I, M36I, R41K, H69K, L89M, and I93L was observed both in global and Indian subtype C while the L63P mutation was selected in Indian PR sequences. Three different codon-based maximum likelihood methods agreed on four sites (12, 19, 36, and 82) under positive selection in Indian sequences.

The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008-2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classification of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis, is an exogenous, B lymphotropic retrovirus belonging to the Retroviridae family that induces persistent lymphocytosis in cattle and sheep. PCR has proven to be particularly suitable for investigating herds of cattle with a very low incidence of BLV infection and for clarifying doubtful serological results obtained by immunodiffusion or ELISA. The native Iranian and Russian cattle have a series of valuable traits that discriminate them as unique breeds that are well able to compete with western analogues. However, their gene pools have not been analyzed with molecular markers, including detection of BLV by PCR. Two pairs of primers were used: gag1 and gag2, and pol1 and pol2, which encompass 347- and 599-bp fragments of the BLV gene, respectively. Sixty-five Iranian Sistani, 120 Yaroslavl, 50 Mongolian, and 35 Black Pied cows were investigated. Among these 270 animals, we obtained 42 positive and 15 doubtful results in the first PCR. The second PCR was very effective in increasing BLV test reliability data to support detection of BLV.

The gag, pol, and env genomic regions of HIV-1 variants currently circulating in the Republic of Sakha (Yakutia) were analyzed. The results of the study indicated that in this area there were HIV-1 variants belonging to two subtypes: A (IDU-A) and B, the former being predominant in this area and in the Russian Federation. The IDU-B-East strain was first isolated from a heterosexually infected patient, which suggests that the strain is outside the risk group of injection drug users. No cases of primary infection with resistant variants were notified during the study.

Background: Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype., Methods and Results: First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines., Conclusion: LV-2xZifHIV-polFN and LV- 2xZifHIV-1FN may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action or disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.

Objective: To investigate whether time-measured phylogenetic analysis of longitudinal viral sequences can establish the direction and timing of HIV-1 transmission in an epidemiologically linked transmission cluster of three homosexual men., Design: An HIV-1-infected homosexual man (patient 1) and his long-term HIV-negative partner (patient 2) engaged in a triangular relationship with an additional partner (patient 3). On the basis of phylogenetic analysis of gag sequences, patient 3 was previously identified as the source for superinfection of patient 1 but the source of HIV-1 infection of patient 2, who seroconverted during the triangular relationship, remained unclear. Here, we set out to analyze newly obtained gag, pol and env sequences from all three patients to fully elucidate the transmission history in this epidemiologically linked cluster., Methods: Bayesian Markov Chain Monte Carlo (MCMC) phylogenetic analyses incorporating a relaxed clock model and a flexible Bayesian skyride tree prior were applied to the longitudinally obtained gag, pol and env sequences from all three patients., Results: Our time-measured evolutionary reconstructions convincingly supported transmission of HIV-1 from the new partner patient 3 to both patients 1 and 2. In addition, estimates of viral divergence times assisted in narrowing down the transmission intervals delineated by seroconversion estimates., Conclusion: Our analysis implies that Bayesian MCMC phylogenetic reconstruction incorporating temporal information can indeed reveal the direction of multiple HIV-1 transmission events in an epidemiologically linked cluster and provide more detail on the timing of transmission.

Aim: Study of circulating 02&#95;AG recombinant form HIV-1 isolates that have been rapidly spreading in Novosibirsk region during 3 recent years., Materials and Methods: WHO protocol for primary HIV isolation was used, automatic sequencer was used for genetic characterization of isolates. Virus specific RNA were isolated and env HIV-1 region DNA fragments were processed. Phylogenetic analysis was also performed., Results: CRF&#95;02AG HIV-1 isolated from peripheral blood of HIV-1 positive patients belonged to CCR5 tropic viruses and had various reproduction characteristics. Most of the HIV isolated were rapidly replicating virus variants characterized by an ability to accumulate high levels of virus protein p24 in cultural fluid. Infectivity and reproductive properties of HIV isolates were confirmed in experimental infection by using clarified cultural liquid of mononuclear cells from healthy donors. Phylogenetic analysis of CRF02&#95;AG HIV-1 variants isolated in Novosibirsk region in 2007 - 2010 showed the formation of a separate outbreak in the area caused by emergence of CRF02&#95;AG HIV-1 in human population., Conclusion: A collection of genetically and biologically characterized CRF02&#95;AG HIV-1 isolates that has not been spreading previously in Russia.

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

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.

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&#95;BC and 2 CRF08&#95;BC isolates have a structural profile identical or nearly identical to CRF07&#95;BC or CRF08&#95;BC according to sequences in the gag-pol regions. The CRF07&#95;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&#95;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.

Abstract HIV diversity reflects multifactorial evolutionary forces, but monitoring subtype prevalence may provide clues to understanding the epidemic. In the Americas HIV-1 C is present at significant levels only in the southern states of Brazil. We describe in this study the presence of the HIV-1 C pol genome in 11.6% (95 CI 6-21%) of antiretroviral-naive individuals from São Paulo, the major city of South America, and 6.8% (95 CI 4-12%) from the second metropolitan area of the State of São Paulo, Brazil. Moreover, a significant growth trend of this subtype was documented among cases failing therapy in the area. Sequences were obtained by direct nested PCR from cDNA retrotranscribed from plasma RNA. Phylogenetic and amino acid signatures support an expansion from variants previously identified in southern Brazil. The evaluation of additional genomic regions (partial gag, envelope, and/or integrase) in samples with HIV-1 C at pol showed extensive recombination with clade B, observed in 47% of ARV-naive cases. The spread of HIV-1 C locally and to other areas of South America should be monitored as it may influence the dynamics of the epidemic.

Kenya is one of the sub-Saharan African countries affected by HIV-1 infection and AIDS. We investigated HIV-1 genetic diversity in 130 individuals from Busia, Bungoma, and Kakamega in western Kenya as part of an HIV-1 vaccine feasibility study in preparation for Phase III efficacy clinical trials. After RNA extraction the partial gag (484 bp) and env (1297 bp) regions were amplified and directly sequenced. Phylogenetic analysis was done using MEGA version 4 and recombinants were identified using the jpHMM tool and phylogenetic analysis. HIV-1 sequences were amplified from 122 of the 130 samples, 118 (90.8%) from the gag region and 78 (60 %) from the env region and 74 samples (56.9%) from both the gag and env regions. Of these sequenced on both regions, 51.4% were subtype A, 9.4% subtype D, 1.4% subtype C, 4.1% subtype G, and 33.7% were discordant and thus possible recombinants, including A1/C, A1/D, A1/A2, and A2/C. The jpHMM tool indicated a further two samples with CD and BD breakpoints within the env gene and one within the gag gene (A1C). An additional sample had an A1D breakpoint in the gag gene, but the envelope was not amplified. HIV-1 subtype diversity in western Kenya should be considered in vaccines designed for clinical trials in this region and this genetic diversity should be continuously monitored.

Introduction: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed., Principal Finding: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads., Conclusion: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage.

Background: Given the elevated potential for primary or transmitted drug resistance (TDR) among newly HIV-infected individuals, there is a need for a deeper understanding of the baseline resistance patterns present in young men of color who have sex with men., Methods: Genotypic data were collected for participants aged 13-24 who were enrolled from seven sites. Univariate and bivariate methods were used to describe the prevalence of TDR and characteristics associated with TDR., Results: Of the 296 individuals participating in the substudy, 145 (49%) had baseline genotypes. The majority of the individuals were African American (65%) and gay-identified (70%). There was significant variation in genotype availability by site (p < .001). Major surveillance drug resistance mutations were present in 28 subjects (19.3%); the majority were non-nucleoside reverse transcriptase inhibitor mutations (12.4%). Subjects with TDR were less likely to have used alcohol on 1 or more days in the prior 2 weeks. Location was not associated with acquisition of TDR., Conclusions: There was a high rate of TDR in a geographically and racially diverse sample of HIV-infected young men of color who have sex with men. This represents a serious public health concern given the young age of this sample and the potential need for long-term antiretroviral therapy. These findings underscore the critical roles of both early case identification and secondary prevention., (Copyright © 2011 Society for Adolescent Health and Medicine. All rights reserved.)

Background: HIV-1 non-B subtypes have recently entered Western Europe following immigration from other regions. The distribution of non-B clades and their association with demographic factors, over the entire course of the HIV-1 epidemic, have not been fully investigated in Italy., Methods: We carried out a phylogenetic analysis of HIV-1 pol sequences derived from 3670 patients followed at 50 Italian clinical centres over nearly three decades., Results: Overall, 417 patients (11.4%) carried non-B subtypes. The prevalence of non-B strains increased from 2.6% in 1980-1992 to 18.9% in 1993-2008 (P<0.0001) in a subset of 2479 subjects with a known year of diagnosis. A multivariate analysis on a subset of 1364 patients for whom relevant demographic data were available indicated that African ethnicity, heterosexual route of infection and year of diagnosis were independently associated with non-B HIV-1 infection (P ≤ 0.0001). All pure subtypes, except for clade K, and seven circulating recombinant forms were detected, accounting for 56.6 and 34.1% of the non-B infections, respectively. The F1 subtype was the most prevalent non-B clade among Europeans and was acquired heterosexually in half of this patient population. Unique recombinant forms accounted for 9.4% of the non-B sequences and showed a B/F1 recombination pattern in one-third of cases., Conclusions: The circulation of non-B clades has significantly increased in Italy in association with demographic changes. Spread of the F1 subtype and B/F recombinants appears to predominate, which may result in a redistribution of the relative proportions of the different strains, and this could lead to overlapping epidemics. Thus, the HIV-1 landscape in Italy may in future be distinct from that of the rest of Europe.

Objective: To analyze antiretroviral drug resistance and determine the genotype of human immunodeficiency virus (HIV)-1 in Saudi patients by sequencing an amplified region of the viral pol gene., Methods: This retrospective study analyzed data from plasma samples submitted for genotypic drug sensitivity monitoring. Samples were analyzed at the Special Infectious Agent Unit, King Fahd Medical Research Center of King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia from August 2004 to June 2009. The Viroseq2.5 kit (Celera/Abbott) was used with ABI Prism 3100 sequencer. All patients were Saudi nationals and were on antiretroviral therapy, some experiencing treatment failure., Results: Based on protease region (PR), genotypes of 63 samples were as follows: C:22, G:21, B:9, CRF02&#95;AG:5, D:3, A:1, F:1, and J:1. Based on reverse transcriptase region (RT), genotypes were as follows: C:23, G:24, B:9, CRF02&#95;AG: 2, D:2, A:1, and F:1. Antiretroviral susceptibility testing results were as follows: 52% of the isolates were susceptible to all 3 major classes of antiretroviral drugs used, 41% had mutations known to confer high level resistance to one or more of the nucleoside analogue reverse transcriptase inhibitors, 16% had mutations known to confer high level resistance to non-nucleoside analogues reverse transcriptase inhibitors, 13% had mutations known to confer high level resistance to one or more of the protease inhibitors (PI). Most isolates were susceptible to 2 or at least one class of antiretroviral, and only 3% of the isolates had resistance to several members of all 3 classes., Conclusion: Antiretroviral resistance is not uncommon in Saudi patients on antiretroviral therapy.

The paper presents the data of an investigation of the genetic polymorphism of the pol gene encoding viral integrase (IN) in a HIV subtype A infected population in the Lipetsk Region. The investigators analyzed 32 virus subtype A samples obtained in 2002-2007. Polymorphism at the codons associated with IN resistance to chemicals was observed in 7 virus variants. The found substitutions had a pattern of genetic polymorphism and were unassociated with resistance in 6 patients with the test subtype A population. At the same time, minor RAL resistance mutation was revealed in 1 (3.1%) virus variant while the similar mutations in the subtype G population were about 10%.

In third-world countries, dried blood samples (DBS) are a convenient alternative to plasma for monitoring viral load during HIV-1 therapy. In this study, we evaluated the feasibility of using DBS to perform HIV-1 drug resistance genotyping in a ViroSeq assay in which the protease and reverse transcriptase regions of the pol gene are analyzed. Fifty-seven antiretroviral genotypes from plasma samples were tested, and drug resistance genotypes were determined. Only 38.6% paired DBS samples were sequenced. Failure to amplify DNA from DBS samples generally correlated with plasma viral loads below log(10) 5.1. The majority of the mutations identified in plasma pol sequences were also found in their DBS counterpart, with a concordance in genotype interpretation of 96.4%. Several factors were identified that could potentially improve both the sensitivity and the quality of genotype data, such as sample storage conditions and sequence analysis. Therefore, DBS sampling is useful to determine viral load and drug resistance genotypes in HIV patients.

Objective: To examine the persistence of compartmentalized HIV drug resistance mutations (DRM) over time in the female genital tract and its effect on pol gene divergence compared to that in blood., Design: Longitudinal cohort of 22 antiretroviral-experienced women in the Emory Vaginal Ecology study., Methods: Blood and vaginal secretions were collected at serial clinic visits. DRM in the HIV reverse transcriptase and protease regions of pol were determined using population based sequencing. Kimura-2 pairwise DNA distances were calculated to measure blood and vaginal secretions divergence in the intervals between clinic visits., Results: Only eight (36%) women had compartmentalized DRM detected at 14 (31%) of their 45 clinic visits. This compartmentalized resistance was transient; 13 of 14 mutations in blood and all 12 mutations in vaginal secretions were compartmentalized for only one clinic visit. Over time, divergence of both reverse transcriptase and protease were greater in vaginal secretions than in blood. However, divergence in blood, but not in vaginal secretions, increased significantly in the presence of drug resistance or compartmentalized drug resistance., Conclusion: Compartmentalized DRM between the blood and vaginal secretions are transient in nature, and the presence of DRM does not affect pol gene divergence in the vaginal secretions. Our results provide new evidence that the genital mucosa does not support an independently evolving subpopulation of HIV-1 genomes.

Objective: The Honduran HIV/AIDS Program began to scale up access to HIV therapy in 2002. Up to May 2008, more than 6000 patients received combination antiretroviral therapy (cART). As HIV drug resistance is the major obstacle for effective treatment, the purpose of this study was to assess the prevalence of antiretroviral drug resistance in Honduran HIV-1-infected individuals., Methods: We collected samples from 138 individuals (97 adults and 41 children) on cART with virological, immunological or clinical signs of treatment failure. HIV-1 pol sequences were obtained using an in-house method. Resistance mutations were identified according to the 2007 International AIDS Society (IAS)-USA list and predicted susceptibility to cART was scored using the ANRS algorithm., Results: Resistance mutations were detected in 112 patients (81%), 74% in adults and 98% in children. Triple-, dual- and single-class drug resistance was documented in 27%, 43% and 11% of the study subjects, respectively. Multiple logistic regression showed that resistance was independently associated with type of treatment failure [virological failure (odds ratio (OR) = 1) vs. immunological failure (OR = 0.11; 95% confidence interval (CI) 0.030-0.43) vs. clinical failure (OR = 0.037; 95% CI 0.0063-0.22)], route of transmission (OR = 42.8; 95% CI 3.73-491), and years on therapy (OR = 1.81; 95% CI 1.11-2.93)., Conclusion: The prevalence of antiretroviral resistance was high in Honduran HIV-infected patients with signs of treatment failure. A majority of study subjects showed dual- or triple-class resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Virologically defined treatment failure was a strong predictor of resistance, indicating that viral load testing is needed to correctly identify patients with treatment failure attributable to resistance.

Objective: To obtain insight in the HIV-1 transmission networks among men having sex with men (MSM) in the Netherlands., Design: A phylogenetic tree was constructed from polymerase sequences isolated from 2877 HIV-1 subtype B-infected patients monitored as part of the AIDS Therapy Evaluation in the Netherlands (ATHENA) nationwide observational cohort., Methods: For MSM with a known date of infection, the most similar sequences were selected as potential transmission pairs when they clustered with bootstrap value of at least 99%. Time from infection to onward transmission was estimated as the median time between dates of infection for each transmission pair. The source of infections with a resistant strain was traced using the entire phylogenetic tree., Results: Of sequences from 403 MSM with a known date of infection between 1987 and 2007, 175 (43%) formed 63 clusters. Median time to onward transmission was 1.4 years (interquartile range 0.6-2.7). Twenty-four (6%) MSM carried a virus with resistance-related mutations, 13 of these were in eight clusters together with sequences from 28 other patients in the entire phylogenetic tree. Six clusters contained sequences obtained from 29 men all presenting the same resistance-related mutations., Conclusion: From our selection of likely transmission pairs, we conclude that onward transmission of HIV-1 from infected MSM in the Netherlands happens both during and after primary infection. Transmission of resistant strains from the antiretroviral therapy-treated population is limited, but strains with resistance-related mutations have formed subepidemics.

Objective: To define a routine algorithm for the specific diagnosis and complete follow-up of HIV-1 group O (HIV-O) infections in Cameroun., Methods: During 18 months, samples referred to Centre Pasteur du Cameroun for HIV testing or viral monitoring were screened for HIV-O infection with an in-house serotyping assay. HIV-O viral load was quantified by real-time polymerase chain reaction in the LTR gene and resistance genotyping was performed on pol and env sequences., Results: Of the 7030 samples tested, 78 HIV-O infections (1.1%) were identified, including 7 M and O dually seroreactive samples (9%). All treatment-naive patients and 59% of the patients receiving HAART had detectable viral loads. Analysis of pol sequences from 15 treatment-naive patients revealed a high number of polymorphisms in the protease region, with natural residues implicated in genotypic resistance to tipranavir and saquinavir for HIV-1 group M according to the Agence Nationale de Recherches sur le Sida et les Hépatites virales algorithm. Six patients (40%) harbored the 181C mutation conferring natural resistance to nonnucleoside reverse transcriptase inhibitors. Among antiretroviral-treated patients, major resistance mutations described for HIV-1 group M were found., Conclusions: HIV-O prevalence remains relatively low in Cameroun. The cocirculation of groups M and O in this country leads to replicative dual infections. HIV-O-infected patients in this region can now benefit from effective and specific tools for a complete monitoring of infection. However, further studies are needed to understand long-term response to antiretrovirals of these complex variants.

Infectious HBV wild-type and mutant clones were produced in vitro with three mutations in pol (rtV173L plus rtL180M plus rtM204V) or with a single mutation in the S gene (sG145R) and inoculated in treatment-naive chimpanzees. Intravenous inoculation of these mutants in chimpanzees resulted in HBV infection; the pol mutations remained stable, whereas the sG145R mutation reverted to wild type during viraemia. Additional hepatitis B vaccine efficacy studies conducted in chimpanzees showed lack of sterilizing immunity against the pol mutant. Whether such mutants can transmit to and infect vaccinated humans requires further investigation.

Etravirine (ETR) has demonstrated efficacy in patients with multiple prior treatments with prior virological failure and resistance mutations to various families of antiretroviral drugs. Most of the evidence concerning this drug has been drawn from the DUET studies, consisting of two multicenter, randomized, double-blind clinical trials with identical designs that included 1,200 patients. These trials showed that ETR obtained a superior virological and immunological response to placebo, reducing the incidence of hospital admissions and progression to AIDS/death. The most frequent adverse effect was rash, which was generally mild to moderate and required treatment discontinuation in only 2%. There were no differences in gastrointestinal, liver or lipid toxicities compared with the placebo arm. Because of the recent development of new drugs, effective regimens are now available for multi-treated patients. The TRIO study evaluated the efficacy and tolerability of one of the regimens most widely used today (ETR/raltegravir/darunavir/r) with excellent virological and immunological response (86% of viral load < 50 copies and CD4 +108 at 48 weeks) and excellent tolerance. ETR is effective and well tolerated and is the first non-nucleoside reverse transcriptase inhibitor (NNRTI) that allows the sequential use of drugs in this family, due to its high genetic barrier compared with firstgeneration NNRTI. Moreover, its long half-life allows once daily administration in patients requiring a QD regimen., (2009 Elsevier España S.L. All rights reserved.)

A molecular epidemiological study was conducted on 104 HIV-1 strains isolated from HIV-infected children hospitalized in Children Hospital 1 in Ho Chi Minh City, Vietnam during 2004-2005. Genetic subtyping based on env C2/V3 sequences revealed that CRF01-AE was the sole circulating recombinant form found in this study. Sequence analysis of the V3 loop showed that GPGQ tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (89.5%). The findings raise great concern about HIV-infected children in Vietnam and provide up-to-date molecular epidemiological information of HIV-1 circulating in Vietnam during the study period.

Etravirine (ETR) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with a potent and broad in vitro spectrum of activity against HIV-1 and viruses with NNRTI resistances, allowing sequential use of drugs of this family. The potency, efficacy and safety of etravirine have been demonstrated in multi-treated patients, but few data are available on first-line antiretroviral therapy (ART) and the role of this drug in initial treatment phases has not been defined. The presence of primary NNRTI resistances and those acquired during first-line therapy is increasingly frequent. Due to its genetic barrier and efficacy, ETR can form part of a second-line ART regimen in patients with failure to a first-line regimen. In the initial phases, adverse effects continue to be the main reason for modifying ART. ETR has demonstrated safety and tolerability, with no central nervous system adverse effects and a good liver, lipid and gastrointestinal safety profile. As with the other NNRTIs, the most common adverse effect is rash. Because of ETR good tolerability profile, this drug can be considered when a new treatment is required due to adverse effects. Because of the characteristics of ETR the possibility of once-daily administration and dissolution in water, as well as the absence of drug-drug interactions with methadone this drug is especially attractive as a firstline therapy and in patients with poor adherence, such as intravenous drug users receiving methadone treatment., (2009 Elsevier España S.L. All rights reserved.)

The treatment of HIV-1 infection with antiretroviral drugs has greatly improved the survival of those who are infected. However, HIV-1 diversity and drug resistance are major challenges in patient management, especially in resource-poor countries. To evaluate HIV-1 genetic diversity and drug resistance-associated mutations among drug-naive patients in Kenya prior to antiretroviral therapy (ART), a genetic analysis of HIV-1 pol-RT and env-gp41 was performed on samples collected from 53 (18 males and 35 females) consenting patients between April and June 2005. The average age, baseline CD4(+) T cell counts, and viral loads were 38 (range, 24-62) years, 475 (range, 203-799) cells/mm(3), and 4.7 (range, 3.4-5.9) log(10) copies/ml, respectively. Phylogenetic analysis revealed that 40 samples (75.5%) were concordant subtypes for the two genes and 13 (24.5%) were discordant, suggesting possible recombination and/or dual infections. Prevalent subtypes included A1/A1(pol-RT/env-gp41), 31 (58.5%); D/D, 9 (16.9%); A1/C, 2 (3.8%); A1/D, 4 (7.5%); G/A1, 2 (3.8%); A1/A2, 1 (1.9%); C/A1, 2 (3.8%); D/A1, 1(1.9%); and D/A2, 1 (1.9%). Major reverse transcriptase inhibitor (RTI) resistance-associated mutations were found in four patients (7.5%). Of these patients, three had nucleoside RTI resistance mutations, such as M184V, K65R, D67N, K70R, and K219Q. Nonnucleoside RTI resistance-associated mutations K103N and Y181C were detected in three patients and one patient, respectively. Multiple drug resistance mutations were observed in this drug-naive population. With increasing numbers of patients that require treatment and the rapid upscaling of ART in Kenya, HIV-1 drug resistance testing is recommended before starting treatment in order to achieve better clinical outcomes.

Background: Drug resistance profiles of human immunodeficiency virus-1 (HIV-1) in treatment-naïve infections have been reported in developed countries. However, little is known in developing countries, including China, especially in treatment-naïve volunteer blood donors., Study Design and Methods: Fifty-two HIV-1-positive samples of blood donors were collected from 2005 to 2006 in Yunnan, China. Recent and long-term infections were distinguished by the HIV-1 subtypes B, E, and D immunoglobulin G-capture enzyme immunoassay assay. The nucleotide sequences of pol genes were amplified and sequenced. Phylogenetic tree and drug resistance analyses were performed., Results: Of 49 samples successfully analyzed, circulating strains were circulating recombinant form (CRF)08&#95;BC (51.0%), CRF07&#95;BC (24.5%), CRF01&#95;AE (20.4%), and B (4.1%). No protease inhibitors (PI) major drug resistance mutation (DRM) was detected. Six samples (12.2%) displayed seven minor PI DRMs. Nine samples (18.4%) displayed 10 nucleoside reverse transcriptase inhibitor DRMs, and DRMs to nonnucleoside reverse transcriptase inhibitors were present in one sample (2.0%). There was only one sample of the 49 (2.0%) in which the DRMs were of sufficient magnitude to result in a clinical change to drug susceptibility, but even in this sample, the clinical effect of these DRMs was predicted to be low. Significant differences were not observed between the long-term and recent infected population. Differences in DRMs were not observed between peripheral blood mononuclear cells and plasma within an individual., Conclusions: CRF&#95;BC was the dominant subtype circulating in HIV-1-infected donors in Yunnan. Prevalence of genotypic drug resistances among donors in Yunnan was low in this study. Surveillance on HIV-1 infections among blood donors should be continued in China.

Objectives: The aim of the study was to elucidate primary drug resistance and transmission of HIV-1 in acute and recent drug-naïve seroconverters in Singapore., Methods: Acute and recent HIV-1 seroconverters were enrolled in the study. The HIV-1 polymerase (pol) gene was sequenced and used for genotypic drug resistance analysis and phylogenetic analysis. HIV-1 transmission clusters were inferred from phylogenetic clustering analysis., Results: Of the 60 subjects analysed, 95% were men, and 73.3% were men who have sex with men (MSM). Six HIV-1 subtypes were identified, including CRF01&#95;AE (46.7%), subtypes B (30%), B' (15%) and G (1.7%), CRF33&#95;01B (1.7%) and CRF34&#95;01B (5%). Primary genotypic resistance was detected in only one (1.7%) subtype B variant. Thirty-one patients (51.7%) were phylogenetically clustered, of whom 90% reported having local risk exposure, compared with 59% of the patients who were not phylogenetically clustered [odds ratio (OR) 6.35, 95% confidence interval (CI) 1.65-23.95]. MSM (OR 5.63, 95% CI 1.17-27.15), high viral load (OR 4.28, 95% CI 1.37-13.36) and young age (OR 0.92, 95% CI 0.85-0.99) were independently associated with clustered individuals., Conclusions: In Singapore, HIV-1 primary resistance is insignificant; individuals with seroconversion account for about half of onward transmission among recently infected seroconverters. MSM, high viral load and young age are factors that facilitate transmission. Early detection of these individuals is of paramount importance for the prevention of HIV-1 transmission.

Objective: Low-level HIV-1 replication may occur during antiretroviral therapy (ART) that suppresses plasma HIV-1 RNA to less than 50 copies/ml (suppressive ART). Antiretroviral drugs appear less effective in macrophages and monocytes compared with lymphocytes, both in vitro and as implied in vivo by greater viral evolution observed during suppressive ART. Our objective was to examine sputum, which is rich in macrophages, for evidence of increased HIV-1 replication compared with that in the blood during suppressive ART., Design: A cross-sectional study during suppressive ART was performed, and HIV-1 DNA sequences derived from induced sputa and peripheral blood mononuclear cells were compared., Methods: Multiple sequences encoding HIV-1 reverse transcriptase, protease, and envelope were generated using single-genome sequencing. Reverse transcriptase and protease sequences were analyzed for genotypic drug resistance. The evolutionary distances of env sequences from the inferred most recent common ancestor of infection were calculated, and CXCR4 usage was predicted., Results: Nine hundred seventy bidirectional sequences from 11 individuals were analyzed. HIV-1 env and pol derived from sputa had greater frequency of drug-resistance mutations (P = 0.05), evolutionary divergence (P = 0.004), and tendency for CXCR4 usage (P = 0.1) compared with viruses derived from peripheral blood mononuclear cells., Conclusion: The greater frequency of HIV-1 drug-resistance mutations and divergence of HIV-1 env in sputa-derived viruses compared with peripheral blood mononuclear cell-derived viruses suggests greater HIV-1 replication in the respiratory tract compared with the blood. Characterization of viral evolution over time and by cell-type could identify cells that provide a sanctuary for low-level viral replication in the respiratory tract during suppressive ART.

The northeast of Gabon, central Africa is characterized by high population density and a high rate of immigration from the surrounding countries. To determine the prevalence, circulating subtypes, and antiretroviral resistance mutations of HIV-1, 810 blood samples were collected from the general population of the two main cities (Oyem and Makokou) of this region. Of these, 61 (7.5%) were found to be positive for HIV-1. Analysis of the env (gp120), pol, and gag (p24) sequences as well as phylogenetic analyses showed at least eight different viral lineages. The most prevalent strains were CRF02 recombinants, followed by subtypes A, D, and C. The remaining strains were found to be F, J, G, and also, for the first time in Gabon, the recombinant form CRF11cpx. Analysis of antiretroviral drug-resistance mutations in protease and reverse transcriptase from this untreated population showed a low level of specific mutations. These mutations were associated with subtype polymorphism rather than with resistance to antiretroviral drugs. The wide diversity and the emergence of recombinant strains are in accordance with the rapid spread of new HIV strains in the population and, thus, the dynamic evolution of the epidemic.

To determine the prevalence of viral hepatitis infection and hepatitis B virus (HBV) molecular characterization, 11,200 blood donors from citizens of Shahrekord (a city located in west of Iran) were investigated. Results showed HBsAg-positive in 1.78% of persons (n=200), anti-HDV-positive in 3% of HBsAg-positive cases (n=6) and anti-HCV-positive in 0.67% of donors (n=76). HBV phylogenetic analysis disclosed HBV genotype D, sub-genotype D1, and subtype ayw2. Amino acid mapping of the HBV pol region revealed various HBV drug-resistance mutations though donors had no antiviral therapy. In conclusion, this study demonstrated notable viral hepatitis seroprevalence rate in blood donors in west of Iran.

Background: Little is known about the detailed phylogeny relationships of CRF 02&#95;AG HIV-1 polymerase genes in Ghana. The use of the protease gene of HIV-1 for subtyping has shown conflicting results., Methods: The partial polymerase gene sequences of 25 HIV-1 strains obtained with Viroseq reagents were aligned with reference subtypes and alignments trimmed to a 300 bp protease, 661 bp and 1005 reverse transcriptase sequence alignments. Phylogenetic relationships of these alignments were determined with the Neighbour-Joining method using 1000 replicates and recombination patterns determined for the sequences with RIP 3.0 in the HIV sequence database., Results: Unlike the other alignments, the protease gene had nodes with bootstrap values < 100% for repeat control sequences. Majority of the CRF 02&#95;AG sequences from Ghana were made up of fragments of several strains of CRF 02&#95;AG/AG strains. The protease gene alone is not suitable for phylogenetic analysis., Conclusion: The polymerase genes of HIV-1 strains from Ghana are made up of recombinants of several CRF 02&#95;AG strains from Ghana, Senegal and Cameroon, but the clinical implications are unknown. Using the HIV-1 protease gene for subtyping will not infer subtypes correctly.

Phylogenetic reconstructions of transmission events from individuals with acute human immunodeficiency virus (HIV) infection are conducted to illustrate this group's heightened infectivity. Varied definitions of acute infection and assumptions about observed phylogenetic clusters may produce misleading results. We conducted a phylogenetic analysis of HIV pol sequences from 165 European patients with estimated infection dates and calculated the difference between dates within clusters. Nine phylogenetic clusters were observed. Comparison of dates within clusters revealed that only 2 could have been generated during acute infection. Previous analyses may have incorrectly assigned transmission events to the acutely HIV infected when they were more likely to have occurred during chronic infection.

Background: In resource-constrained settings, antiretroviral treatment (ART) is often continued based on clinical and CD4 responses, without virologic monitoring. ART with incomplete viral suppression was assessed in 27 subjects with subtype C HIV-1., Methods: Plasma HIV-1 RNA, drug resistance, viral tropism, and evolution in polymerase (pol) and envelope (env) genes were measured. The association between these viral parameters and CD4 cell change over time was analyzed using linear regression models., Results: Increased area under the curve of HIV-1 RNA replication was a predictor of lower CD4 cell gains (P < 0.007), while less drug resistance measured as a genotypic susceptibility score (GSS) (P = 0.065), and lower rates of evolution in pol and env genes (P = 0.08 and 0.097, respectively) measured as genetic distance were modestly associated with increasing CD4 cell counts. Evolution of pol and env were correlated (R2 = 0.48, P = 0.005), however, greater evolution was identified in env vs. pol (P < 0.05). CXCR4-usage (X4) was detected in 14/27 (52%) but no differences in CD4 cell change or plasma viremia were associated with X4-usage., Discussion: Among subtype C HIV-1 infected patients in Zimbabwe receiving incompletely suppressive ART, higher virus replication and lower CD4 cell gains were associated with drug resistance and evolution of polymerase and envelope.

Mutations in the HIV-1 genes associated with resistance to antiretroviral drugs were detected also in primary HIV infected individuals who did not receive antiretroviral treatment. Drug resistance genotyping of HIV pol gene was done by in situ DNA hybridization using a Line Probe Assay and by direct sequencing. Viral variants harbouring resistance mutations such as: M41, T69R, K70R, M184V, T215Y in the pol gene were detected in 14% of the subjects. HIV mutants resistant to NRT inhibitors were found in 10 and 20% of patients infected before and after the year 2000, respectively. Multiple drug resistant viruses (2-3 drug classes) were present in 3.5% of the mainly recently infected patients. In protease gene only minor resistant mutations were found such as L101 and A71V. These findings indicate the evolution of drug resistance showing a correlation with the time of introduction of combination therapy in our country, where more than 70% of HIV infections were by homo/bisexual transmission. This confirms the transmission of drug-resistant HIV shown by genotype testing during primary infection in therapy-naive patients and initiates serious clinical and public health consequences.

Background: The global HIV/AIDS epidemic consists of a number of regional epidemics caused by different HIV-1 subtypes prevailing in different regions., Objectives: To study changes in genetic diversity of HIV-1 strains isolated in the Czech Republic (CR) over a more than twenty-year period (1986-2007)., Study Design: HIV-1 strains isolated in CR from 1986 to 2007 were subtyped by pol gene sequencing followed by phylogenetic analysis. The role of HIV-1 subtyping in molecular epidemiology was considered., Results: Awide range of HIV-1 subtypes were found, with subtype B, into which 76.6% of 534 HIV-1 isolates were classified, being predominant during the whole study period. An increasing number of non-B subtypes A1, C, D, F1, G and some recombinant forms (CRF 01&#95;AE, CRF 02&#95;AG and CRF 06&#95;cpx) were identified after 1990., Conclusions: The absolute predominance of subtype B among HIV-1 strains in the Czech Republic ended in 1991 when different non-B subtypes had been introduced into the country. The East-West migration is responsible for the introduction of HIV-1 subtypes prevalent in Eastern European and some Asian countries. Genetic analysis of HIV-1 isolates from a given region can be helpful in tracing the course of the HIV/AIDS epidemic.

During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.

Objective: To investigate the association of repair gene POLI genetic polymorphisms with cervical carcinoma., Materials and Methods: Four hundred sixty one cervical carcinoma patients and 628 normal women were randomly selected for this study. Single nucleotide polymorphisms (SNPs) (POLI Thr706Ala) were genotyped, and the epidemiological risk factor was assessed., Results: Compared with the POLI Thr706Thr genotype, neither POLI Thr706Ala nor POLI Ala706Ala elevated or decreased the risk of cervical carcinoma or cervical squamous cell carcinoma [ORs and 95% CIs 1.25(0.97-1.61), 1.11(0.67-1.83), 1.22(0.94-1.58), 1.06(0.63-1.78), respectively]. When analyzing the risk of the POLI Thr706Ala variant genotype for cervical carcinoma in different histological types or HPV infection status, very similar risk factors were observed for the squamous cell carcinoma group or the HPV positive group to the overall carcinoma. Regardless of sexual or reproductive histories, patients with the POLI Thr706Ala or POLI Ala706Ala genotype showed a significantly increased risk of cervical carcinoma., Conclusion: The POLI Thr706Ala genotype was not associated with cervical carcinoma in a Chinese population, but we cannot exclude the possibility that the POLI polymorphism might be associated with sexual and reproductive history.