Reverse Transcriptase Region Research Articles

Infectivity of Homologous Recombinant HIV-1 Pseudo-virus with Reverse Transcriptase Inhibitor-related Mutations from Highly Active Antiretroviral Therapy Experienced Patients

ObjectivesIn this study, the viral fitness of pseudo-viruses with a drug-resistant site in the reverse transcriptase (RT) region of the genome was investigated. The pseudo-viruses were derived from highly active antiretroviral therapy (HAART)-experienced HIV/AIDS patients. MethodsHIV-1 RNA was extracted from the plasma of HAART-experienced (KRB9149, KRB7021, KRC1097) and HAART-naïve (KRC5180, KRC5123) HIV-1 patients. The RT gene from the extracted viral RNA was amplified and the polymerase chain reaction product was cloned from the pHXB2Δ2-261 RT vector. C8166 and TZM-bl cell lines were used as the HIV-1 replication capacity measurement system. To quantify the infectivity of homologous recombinant HIV-1, the infectivity derived from each pseudo-virus was compared with the infectivity of the reference strain HXB2. ResultsPatient-derived HIV-1 was cotransfected into C8166 cells and the expression level of the p24 antigen was measured. The expression was high in the HIV-1 isolates from patients KRC5180 and KRB9149 and low in patients KRB7021, KRC5123, and KRC1097, when compared with the reference strain. The infectivity of the pseudo-virus measured in TZM-bl cells decreased in the order, reference strain HXB2 > KRC5180 > KRC5123 > KRB9149 > KRB7021 > KRC1097. ConclusionIn this study, HIV-1 infectivity of the drug-resistant strain isolated from HAART-experienced patients with HIV/AIDS was found to be lower than the infectivity of the reference strain HXB2. This study provides useful data for the phenotypic susceptibility assay in HAART-experienced patients infected with HIV-1.

Evolutionary pattern of full hepatitis B virus genome during sequential nucleos(t)ide analog therapy

The evolutionary and mutational pattern of full hepatitis B virus (HBV) quasispecies during sequential nucleos(t)ide analog (NUC) therapy remains unclear. In this study, full-length HBV clones were generated from serial serum samples of five chronic hepatitis B patients who received sequential NUC therapies (treated patients) and two untreated patients with acute flares. The evolutionary and mutational patterns of full HBV quasispecies were studied. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence all decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed a distinct change in divergence during adefovir–telbivudine sequential therapies. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at ALT flare. Four of the treated patients presented amino acid changes in the “a” determinant during NUC therapy. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in reverse transcriptase (RT) region. Co-variations in the core promoter, the preC region and in the known epitopes of the preS gene accompanied by RT mutations, were common. In untreated patients, most of these co-variations located in the preC/C gene. In conclusion, the distribution of genetic variability of HBV shows remarkably different patterns between the treated and untreated subjects and the quasispecies divergence of different regions of HBV may vary remarkably even within a single host.

Copia Retrotransposon in the Zaprionus Genus: Another Case of Transposable Element Sharing with the Drosophila melanogaster Subgroup

Copia is a retrotransposon that appears to be distributed widely among the Drosophilidae subfamily. Evolutionary analyses of regulatory regions have indicated that the Copia retrotransposon evolved through both positive and purifying selection, and that horizontal transfer (HT) could also explain its patchy distribution of the among the subfamilies of the melanogaster subgroup. Additionally, Copia elements could also have transferred between melanogaster subgroup and other species of Drosophilidae-D. willistoni and Z. tuberculatus. In this study, we surveyed seven species of the Zaprionus genus by sequencing the LTR-ULR and reverse transcriptase regions, and by using RT-PCR in order to understand the distribution and evolutionary history of Copia in the Zaprionus genus. The Copia element was detected, and was transcriptionally active, in all species investigated. Structural and selection analysis revealed Zaprionus elements to be closely related to the most ancient subfamily of the melanogaster subgroup, and they seem to be evolving mainly under relaxed purifying selection. Taken together, these results allowed us to classify the Zaprionus sequences as a new subfamily-ZapCopia, a member of the Copia retrotransposon family of the melanogaster subgroup. These findings indicate that the Copia retrotransposon is an ancient component of the genomes of the Zaprionus species and broaden our understanding of the diversity of retrotransposons in the Zaprionus genus.

Evolutionary patterns of hepatitis B virus quasispecies under different selective pressures: correlation with antiviral efficacy

ObjectiveTo investigate the evolution of hepatitis B virus (HBV) quasispecies (QS) within the reverse transcriptase (RT) region during the early stage of entecavir treatment and its impact on virological response,...

Impact of HIV-1 Group O Genetic Diversity on Genotypic Resistance Interpretation by Algorithms Designed for HIV-1 Group M

HIV-1 group O (HIV-O) is characterized by a high genetic divergence from HIV-1 group M viruses. Little is known about the therapeutic impact of this diversity. The aim of this study was to assess in a large series of samples (1) the genotypic impact of natural polymorphism of the HIV-O reverse transcriptase and protease genes; and (2) the predictive value of resistance interpretation algorithms developed for HIV-1 group M when used for highly mutated HIV-O viruses. Sixty-eight antiretroviral-naive and 9 highly antiretroviral-experienced HIV-O-infected patients were included. The viruses were sequenced and resistance-associated mutations were identified using 3 different algorithms (Agence Nationale de Recherches sur le SIDA et les hépatites virales, Rega, Stanford). All HIV-O samples naturally exhibited the A98G and V179E resistance mutations in the reverse transcriptase region; 54% of samples presented the Y181C mutation, conferring resistance to nonnucleoside reverse transcriptase inhibitors. Twelve minor resistance mutations, present in more than 75% of the protease sequences, led to the different algorithms giving discrepant results for nelfinavir and saquinavir susceptibility. A marked virological response was observed in 8 of the 9 antiretroviral-experienced patients, despite the prediction of limited activity of the combination for 5 to 8 patients according to the algorithm used. The high level of natural polymorphism in HIV-O genes, and the important discrepancies between genotypic resistance interpretation and the virological response, emphasize the need for resistance algorithm rules better adapted to HIV-O.

Variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus during lamivudine treatment

To investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads. HBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing. In HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups. HBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.

HIV-1 drug resistance genotypic profiles in children with undetectable plasma viremia during antiretroviral therapy

Treatment of HIV-1 infection with highly active antiretroviral therapy has led to sustained viral suppression in the plasma in a large number of children. However, studies have suggested that the integrated provirus in resting CD4+ T lymphocytes could be a source of reactivatable virus and maintain drug-resistant virus. We evaluated the resistance-related mutations in children receiving antiretroviral therapy with prolonged viral suppression. Thirty-two peripheral blood mononuclear cell samples from 16 children with viral loads that had been below detection limits for at least 12 months were obtained at two different time points and the DNAs sequenced. The median CD4 cell count was 1,016 cells/ mm3 (347-2,588) and 938 cells/mm3 (440-3,038) at the first and second time points, respectively. The median follow-up time was 15 months (9-27). Six (37.5%) and seven (43.75%) of the 16 patients showed at least one NRTI-associated mutation in the first and second samples, respectively. Two out of 16 (12.5%) had an NNRTI-associated mutation at the first time point and three out of 16 (18.75%) at the second. In addition, 14 out of 16 (87.5%) had at least one PI-associated mutation at both time points. Despite plasma HIV-1 RNA suppression for at least 12 months, resistance-related mutations from previous antiretroviral failures could still be detected in archival virus. Furthermore, viral evolution occurred at the reverse transcriptase region in spite of viral suppression to levels below 400 copies/mL. Persistence of archival resistant virus may be relevant when considering future treatment options.

HIV-1 mutational pathways under multidrug therapy

BackgroundGenotype-derived drug resistance profiles are a valuable asset in HIV-1 therapy decisions. Therapy decisions could be further improved, both in terms of predicting length of current therapy success and in preserving followup therapy options, through better knowledge of mutational pathways- here defined as specific locations on the viral genome which, when mutant, alter the risk that additional specific mutations arise. We limit the search to locations in the reverse transcriptase region of the HIV-1 genome which host resistance mutations to nucleoside (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors (as listed in the 2008 International AIDS Society report), or which were mutant at therapy start in 5% or more of the therapies studied.MethodsA Cox proportional hazards model was fit to each location with the hazard of a mutation at that location during therapy proportional to the presence/absence of mutations at the remaining locations at therapy start. A pathway from preexisting to occurring mutation was indicated if the covariate was both selected as important via smoothly clipped absolute deviation (a form of regularized regression) and had a small p-value. The Cox model also allowed controlling for non-genetic parameters and potential nuisance factors such as viral resistance and number of previous therapies. Results were based on 1981 therapies given to 1495 distinct patients drawn from the EuResist database.ResultsThe strongest influence on the hazard of developing NRTI resistance was having more than four previous therapies, not any one existing resistance mutation. Known NRTI resistance pathways were shown, and previously speculated inhibition between the thymidine analog pathways was evidenced. Evidence was found for a number of specific pathways between NRTI and NNRTI resistance sites. A number of common mutations were shown to increase the hazard of developing both NRTI and NNRTI resistance. Viral resistance to the therapy compounds did not materially effect the hazard of mutation in our model.ConclusionsThe accuracy of therapy outcome prediction tools may be increased by including the number of previous treatments, and by considering locations in the HIV genome which increase the hazard of developing resistance mutations.

Evaluation of the performance of a DNA microarray method for detecting HBV mutations conferring antiviral resistance to lamivudine and adefovir

Objective To evaluate the performance of a DNA microarray method for detecting HBV antiviral drug-resistant mutations. Methods Two hundred and twenty four serum samples from patients with CHB were tested in parallel by DNA microarray and direct sequencing for the mutations within the HBV reverse transcriptase (rt) region, which included rtL180, rtA181, rtM204 and rtN236. Samples with discrepant results were retested by clonal sequencing. Results Complete concordance between DNA microarray and direct sequencing results was observed in 214 out of 224 samples (95. 5% ). The presence of mixed viral populations in the other 10 samples detected by DNA microarray but not by direct sequencing was confirmed by clonal analysis. The DNA microarray could detect minor viral populations which constituted 5.0%-15. 0% of the total viral load. Conclusion DNA microarray is highly consistent with direct sequencing in detecting HBV mutations conferring drug resistance and more sensitive in detecting mixed mutant and wild-type sequences than direct sequencing, which makes it a useful tool for early detection of drug resistance early. Key words: Hepatitis B,chronic; Hepatitis B virus; Oligonucleotide array sequence analysis; Drug resistance, viral; Sequence analysis, DNA

Feasibility of using dried blood spots to detect HIV drug resistance genotyping

This study aimed at exploring the feasibility of using dried blood spots (DBS) to detect HIV drug resistance genotyping in China by comparing the results of drug resistance from DBS, plasma and whole blood samples. Blood samples were collected from 39 AIDS patients from Anhui (10), Yunnan (13), Hunan (6) and Xinjiang (10) provinces and autonomous regions. The HIV strains that infected these patients covered all the major HIV-1 subtypes prevailing in China (B, CRF01_AE, CRF07_BC). HIV drug resistance genotyping assay was performed on DBS as well as on the whole blood and plasma samples from the same patients simultaneously by using an in-house nest RT-PCR method. Drug resistance levels were determined based on Stanford University HIV drug resistance database, and the results from these three types of samples were compared. The percentages of successful amplification of protease and reverse transcriptase regions in the pol gene were 95% (37/39) from DBS, 92% (36/39) from whole blood and 100% (39/39) from plasma samples. The sequences from the three types of samples showed more than 99% identity.86% (31/36) of the DBS samples had the same set of drug resistance mutations as those which were detected from plasma samples. The differences probably resulted from mixed bases. There was no major difference in detecting HIV drug resistance genotyping among DBS, plasma and whole blood samples. Therefore, DBS is useful for detection of HIV drug resistance genotyping and is particularly valuable in developing countries like China, especially in remote rural regions.

Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA

To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

Profile of HBV antiviral resistance mutations with distinct evolutionary pathways against nucleoside/ nucleotide analogue treatment among Chinese chronic hepatitis B patients

Antiviral drug-resistant HBV mutants under a variety of treatment protocols are complex and only partly understood. Here, a population-based cross-sectional study was performed to analyse the profile of resistance mutations in distinct evolutionary pathways refractory to different nucleoside/nucleotide analogues (NAs). Serum samples of 199 chronic hepatitis B patients undergoing NA treatment from five hospitals in four northern cities of China were obtained between January 2007 and July 2009. The genotypic resistance of HBV in these samples was characterized. The full-length HBV reverse transcriptase region was amplified, sequenced and analysed with particular focus on the following NA-resistant changes: rtL80, rtI169, rtV173, rtL180, rtA181, rtT184, rtA194, rtS202, rtM204, rtN236 and rtM250. Among 199 HBV isolates, 30 (15.08%) and 169 (84.92%) were genotypes B and C, respectively, and 65 (32.66%) harboured NA-resistant mutations. The prevalence of mutations at rtM204 was 34.33% in 134 patients who had received or who had been exposed to lamivudine-based therapy. Five cases of rtN236 mutations were detected exclusively among 75 patients receiving adefovir-dipivoxil-based therapies. A total of 19 cases of multidrug resistance rtA181 mutations were observed in those with lamivudine-, adefovir-dipivoxil- or telbivudine-based treatment (186 cases), but not in those undergoing entecavir treatment (13 cases). Mutations were not found at rtI169, rtT184, rtA194 or rtS202. rtM204 mutations (27 rtM204I, 15 rtM204V and 5 rtM204I/V cases) were detected at the highest frequency among 65 mutants (72.30% [47/65]) and found to display 16 combination mutation patterns, in which rtM204I and rtM204V were significantly associated with rtL80I/V and rtL180M, respectively (P<0.01). One-third of the studied population harboured NA-resistant HBV with complicated mutation patterns. Monitoring HBV genotypic resistance mutation markers and patterns is therefore shown to be beneficial for optimizing antiviral therapies and for avoiding clinical deterioration.

Short Communication: Intermediate Prevalence of HIV Type 1 Primary Antiretroviral Resistance in Ceará State, Northeast Brazil

Brazil is a large developing country where almost all FDA-licensed antiretrovirals are made available to more than 200,000 individuals under antiretroviral treatment. General primary HIV-1 resistance in Brazil is assumed to be low, but data are scarce, especially in the Northeast region. To evaluate the prevalence of primary HIV-1 antiretroviral resistance in the state of Ceará, Brazil, a cross-sectional prospective study of antiretroviral-naive HIV-1-infected individuals was performed between May 2008 and May 2009. Genomic sequences of reverse transcriptase and protease regions of the pol gene of HIV-1 using PCR products were obtained. Mutations related to resistance to NRTI, NNRTI, and PI were evaluated according to the WHO mutation list for primary resistance surveillance, which excludes common polymorphisms. Seventy-four individuals were evaluated (50% male) with a median age 30 years; 55.4% were men who have sex with men. Median CD4(+) T lymphocyte counts were 418 and 960 cells/mm(3) and the median viral loads were 4.41 and 4.46 log(10) RNA copies/ml for individuals older and younger that 18 years, respectively. Twenty-seven percent of patients were symptomatic. Five patients (6.8%) were recently infected, as detected by the BED test. The mutations 41L, 67N, 215D, 219Q, 101E, and 103N in the RT and 32I, 46I, 54V, 82T, and 90M, in the PR were identified in 9.5% of samples, more frequently in HIV subtype B (85.1%). A significant level of primary HIV resistance was detected in urban Northeast Brazil, a region geographically distant from the more highly populated and wealthier areas of Southeast Brazil, and this emphasizes the need for monitoring resistance in the studied area.

HIV type-1 drug resistance testing on dried blood spots is feasible and reliable in patients who fail antiretroviral therapy in rural Tanzania

HIV type-1 (HIV-1) drug resistance testing is rarely available in resource-limited settings because of high costs and stringent requirements for storage and transport of plasma. Dried blood spots (DBS) can be a convenient alternative to plasma, but the use of DBS needs validation under field conditions. We assessed the performance of DBS in genotypic resistance testing of patients who failed first-line antiretroviral therapy (ART) in rural Tanzania. A total of 36 ART-experienced patients with viral loads >1,000 copies/ml (median 15,180 copies/ml [range 1,350-3,683,000]) and with various HIV-1 subtypes were selected for resistance testing. DBS were stored with desiccant at ambient temperature for a median of 29 days (range 8-89). Samples were amplified using an in-house reverse transcriptase-nested PCR method and sequenced using the ViroSeq™ assay (Abbott Molecular, Des Plaines, IL, USA). DBS-derived genotypes were compared with genotypes from plasma. Overall, 34 of 36 (94%) DBS specimens were successfully genotyped. In the protease region, of 142 polymorphisms found in plasma, 132 (93%) were also detected in DBS. In the reverse transcriptase region, of 57 clinically relevant mutations present in plasma, 51 (89%) were also detected in DBS. A total of 30 of 34 (88%) patients had identical resistance profiles to antiretroviral drugs in plasma and DBS. Genotyping was successful in the vast majority of DBS specimens stored at ambient temperature for up to 3 months, and there was high concordance between mutations found in DBS and plasma. Our study suggests that DBS can be a feasible and reliable tool to monitor HIV-1 drug resistance in patients on ART in resource-limited settings.

Short Communication: Analysis of Selection Pressure and Mutational Pattern of HIV Type 1 Reverse Transcriptase Region Among Treated and Nontreated Patients

Variation of the HIV-1 subtype C reverse transcriptase region (RT) resulting in response to the selective pressures of drug therapy remains poorly characterized. Here, we compared the genetic variation resulting in the presence and absence of antiretroviral drug selective pressures on HIV-1 subtype C RT among nontreated and treated patients. The nucleotide variability, nonsynonymous and synonymous ratio, and the positively selected mutations were determined by comparing the RT sequences isolated at two time points among nontreated (baseline and follow-up) and treated patients (baseline and treatment failure). Compared to the nontreated patients, the intrapatient nucleotide variability, the number of nonsynonymous and synonymous substitutions was significantly higher among the treated patients. Among the mutations positively selected, the frequency of D121Y, I135R, and Q207E increased and the frequency of mutation S48T decreased significantly during treatment failure. Further studies are essential to discover the role of these mutations during treatment in HIV-1 subtype C.

Characterization of Integrase Region Polymorphisms in HIV Type 1 CRF06_cpx Viruses in Treatment-Naive Patients in Estonia

Natural polymorphisms of HIV-1, often associated with drug resistance, are widely described in protease and reverse transcriptase regions but data on their presence in the integrase region, especially in non-B subtypes, are still very limited. We aimed to characterize naturally occurring polymorphisms in the integrase region in 104 treatment-naive and 10 treatment-experienced patients infected predominantly with HIV-1 CRF06_cpx and its recombinant with subtype A1 and/or CRF03_AB viruses. No primary drug resistance mutations against integrase inhibitors were found, but resistance-associated polymorphisms such as V72I, L74I, V201I, and T206S were seen in more than 90% of viruses. Substitutions E157Q and E157K, associated with raltegravir resistance, were found in only two CRF06_cpx strains. We conclude that similar to other HIV-1 non-B subtypes, the CRF06_cpx and its recombinants with subtype A1 and CRF03_AB are rich in integrase region natural polymorphisms, which may impact the development of resistance against integrase inhibitors.

Patients with only N236T mutation and patients with N236T+A181T mutation in HBV reverse transcriptase region: a comparison of clinical characteristics

Objective To compare the application of nucleotide drugs and the virology characteristics between patients with only N236T mutation and patients with N236T + A181T mutation in the HBV reverse transcriptase(rt) region.Methods Sera of patients with only rtN236T mutation and patients with rtN236T + rtA181T mutation were obtained from patients with chronic hepatitis B.Then the levels of sero-HBsAg,HBV DNA and ALT were determined and the HBV genotypes were analyzed.The treatment history with nucleotide drugs was retrospectively reviewed.Results The sero-HBsAg levels were not significantly different between only rtN236T mutation group and rtN236T + rtA181T mutation group(P=0.975 5),and significantly higher viral replication(P=0.001 4) and higher ALT level(P=0.003 2) were found in rtN236T + rtA181T mutation group.Moreover,compared to HBV B genotype,patients with HBV C genotype were prone to carry rtN236T + rtA181T mutation(40% vs 20.45%,P = 0.023 5) ;also we noticed that a switch from lamivudine medication to adefovir medication was liable to induce the virus mutation.Conclusion Nucleotide drug application in HBV patients with only rtN236T mutation and rtN236T + rtA181T mutation are concurrent(lamivudine switching to adefovir).However,the HBV genotype constituents and the serum virological characteristics are different between the two types of HBV patients.

Genotypic resistance profile of hepatitis B virus (HBV) in a large cohort of nucleos(t)ide analogue‐experienced Chinese patients with chronic HBV infection

Summary. The study investigated the hepatitis B virus (HBV) genotypic resistance profile in 1803 nucleos(t)ide analogue (NA)‐experienced Chinese patients with chronic HBV infection. Serum HBV DNA was extracted, and the reverse transcriptase region was analysed by a high‐sensitive direct PCR sequencing and verified by clonal sequencing if necessary. Drug‐resistant mutations were detected in 560 of the 1803 patients, including 214 of 490 patients who received lamivudine (LAM), 35 of 428 patients who received adefovir (ADV), five of 18 patients who received telbivudine and 306 of 794 patients who received various sequential/combined NA therapies. ADV‐resistant mutations were detected in 36 of 381 patients who received LAM and then switched‐to ADV in contrast to one of 82 patients who received ADV add‐on LAM. Entecavir (ETV)‐resistant mutations were detected not only in LAM‐ and ETV‐treated patients but also in LAM‐treated ETV‐naïve patients. Double mutations rtM204I and rtL180M were detected more frequently in genotype C than in genotype B virus, and patients infected with this mutant had higher alanine transaminase levels than those infected with mutant containing the rtM204I substitution alone. Multidrug‐resistant HBV strains were identified in eight patients, including two novel strains with mutational patterns rtL180M + A181V + S202G + M204V + N236T and rtL180M + S202G + M204V + N236T. The results provide new information on HBV genotypic resistance profiles in a large cohort of Chinese patients with chronic HBV infection and may have important clinical implication for HBV drug resistance management in China.

Short Communication: Nucleotide Variation and Positively Selected Sites in HIV Type 1 Reverse Transcriptase Among Heterosexual Transmission Pairs

Mutations in the env gene of HIV-1 have been the primary focus in most epidemiologically related cohort studies of virus evolution and very limited studies have focused on the reverse transcriptase (RT) region, the primary target of antiretroviral therapy (ART). Hence, we measured the selection pressure and searched for the positively selected sites in the RT sequences amplified from HIV-1-infected heterosexual transmission pairs. Married couples (n = 10) who were ART naive were included in this study. Phylogenetic analysis, the measurement of synonymous and nonsynonymous ratio (dN/dS) and the interpatient nucleotide variation, was done. Phylogenetic analysis demonstrated distinct subclusters of the RT sequences from heterosexual transmission pairs and the median (IQR) nucleotide variation between the epidemiologically related transmission pairs was significantly (p < 0.001) lower [0.01% (0.01-0.02%)] compared to the epidemiologically unrelated transmission pairs [0.04% (0.03-0.04%)]. The ratio of dN/dS was <1 and codons 135, 162, 166, 207, and 211 were positively selected in >50% of the donor and recipient RT sequences. Purifying selection pressure and low nucleotide variation in the RT sequences between epidemiologically related transmission pairs highlight its essential role in HIV-1 replication. The effect of the RT positively selected mutations that persist over time following transmission between individuals needs to be studied to determine the fitness cost of the mutations in vivo, which may possibly represent good targets for inclusion in HIV-1 vaccines.

Prevalence, estimated HIV-1 incidence and viral diversity among people seeking voluntary counseling and testing services in Rio de Janeiro, Brazil

BackgroundBED-EIA HIV-1 Incidence Test (BED-CEIA) has been described as a tool to discriminate recent (RS) from long-term (LTS) seroconversion of HIV-1 infection, contributing to a better understanding of the dynamics of the HIV/AIDS epidemic over time. This study determined the prevalence, estimated incidence and HIV-1 subtype infection among individuals seeking testing in Voluntary Counseling and Testing centers (VCTs) from Rio de Janeiro, Brazil.MethodsDemographics and behavioral data were obtained from 434 individuals, diagnosed as HIV-positive among 9,008 volunteers screened from November 2004 to October 2005 in three VCTs located in the Rio de Janeiro Metropolitan area, Brazil. BED-CEIA protocol was performed to identify RS. DNA samples from RS and a subset of LTS (under a proportion of 1:2) were selected for gp120 C2-V3 and pol (protease and reverse transcriptase) regions genomic sequencing.ResultsOverall HIV-1 prevalence was 4.8%. Sixty-one of 434 seropositive individuals were classified as RS, corresponding to an incidence rate of 1.68%/year (95%CI 1.26% -2.10%). Estimated incidence between Men Who Have Sex with Men (MSM) was 11 times higher than among heterosexual men and 55% of the new cases were identified in volunteers aged 25-40 years. A similar distribution of different HIV-1 subtypes was found among RS and LTS.ConclusionsOur data suggest that prevention for MSM remains a challenge and efforts focusing on prevention targeting this population should be prioritized. No significant changes in HIV-1 subtypes were observed among the RS and LTS subgroups. One case of HIV-1 AUK (pol)/A (env) recombinant genome was detected for the first time in Brazil.