Detection of pretreatment minority HIV-1 reverse transcriptase inhibitor-resistant variants by ultra-deep sequencing has a limited impact on virological outcomes.

OBJECTIVES: Ultra-deep sequencing (UDS) is a powerful tool for exploring the impact on virological outcome of minority variants with low frequencies, some even <1% of the virus population. Here, we compared HIV-1 minority variants at baseline, through plasma RNA and PBMC DNA analyses, and the dominant variants at the virological failure (VF) point, to evaluate the impact of minority drug-resistant variants (MDRVs) on virological outcomes. METHODS: Single-molecule real-time sequencing (SMRTS) was performed on baseline RNA and DNA. The Stanford HIV-1 drug resistance database was used for the identification and evaluation of drug resistance-associated mutations (DRAMs). RESULTS: We classified 50 patients into virological success (VS) and VF groups. We found that the rates of reverse transcriptase inhibitor (RTI) DRAMs determined by SMRTS did not differ significantly within or between groups, whether based on RNA or DNA analyses. There was no significant difference in the level of resistance to specific drugs between groups, in either DNA or RNA analyses, except for the DNA-based analysis of lamivudine, for which there was a trend towards a higher prevalence of intermediate/high-level resistance in the VF group. The RNA MDRVs corresponded to DNA MDRVs, except for M100I and Y188H. Sequencing from DNA appeared to be more sensitive than from RNA to detect MDRVs. CONCLUSIONS: Detection of pretreatment minority HIV-1 RTI-resistant variants by UDS showed that MDRVs at baseline were not significantly associated with virological outcome. However, HIV-1 DNA sequencing by UDS was useful for detecting pretreatment drug resistance mutations in patients, potentially affecting virological responses, suggesting a potential clinical relevance for ultra-deep DNA sequencing.

CRF07_BC is associated with slow HIV disease progression in Chinese patients.

HIV subtypes convey important epidemiological information and possibly influence the rate of disease progression. In this study, HIV disease progression in patients infected with CRF01_AE, CRF07_BC, and subtype B was compared in the largest HIV molecular epidemiology study ever done in China. A national data set of HIV pol sequences was assembled by pooling sequences from public databases and the Beijing HIV laboratory network. Logistic regression was used to assess factors associated with the risk of AIDS at diagnosis ([AIDSAD], defined as a CD4 count < 200 cells/µL) in patients with HIV subtype B, CRF01_AE, and CRF07_BC. Of the 20,663 sequences, 9,156 (44.3%) were CRF01_AE. CRF07_BC was responsible for 28.3% of infections, followed by B (13.9%). In multivariable analysis, the risk of AIDSAD differed significantly according to HIV subtype (OR for CRF07_BC vs. B: 0.46, 95% CI 0.39─0.53), age (OR for ≥ 65 years vs. < 18 years: 4.3 95% CI 1.81─11.8), and transmission risk groups (OR for men who have sex with men vs. heterosexuals: 0.67 95% CI 0.6─0.75). These findings suggest that HIV diversity in China is constantly evolving and gaining in complexity. CRF07_BC is less pathogenic than subtype B, while CRF01_AE is as pathogenic as B.

Phylogenetic and temporal dynamics of human immunodeficiency virus type 1B in China: four types of B strains circulate in China.

To investigate the origin and evolutionary history of the spread of HIV-1 subtype B in China, a total of 409 sequences of pol gene sampled from 1994 to 2012 in 29 provinces across China was subjected to phylogenetic and Bayesian molecular clock analyses. The study reveals that subtype B strains in China are genetically diverse and can be classified into four distinct subgroups, namely B' (Thai-B), BJ-B (Beijing-B), Pan-B (Pandemic-B), and TW-B (Taiwan-B), according to the origin of the sequences. The BJ-B and TW-B are reported for the first time. Phylogeographic analysis reveals that B' exhibits a nationwide, transprovincial distribution, and is found in 21 provinces in China in this study, whereas the Pan-B, BJ-B, and TW-B lineages are restricted to particular regions. From the same common ancestor of B', there arise two subclusters in which sequences from Yunnan occupy the basal position. The times of the most recent common ancestors (tMRCAs) of B' and BJ-B are estimated to be 1983.6 (1975.9-1990.3) and 1995.3 (1989.6-2000.3), respectively. The skyline plot profile reveals an exponential decrease in median number of effective infections of subtype B in China from 1994 to 2009. The existence of four types of B clades also indicates distinct transmission networks of subtype B, originating from different introduction events at different time points. The data presented here offer a new perspective on the epidemic of HIV-1 subtype B in China.

Phylogenetic and temporal dynamics of human immunodeficiency virus type 1 CRF01_AE in China.

To explore the epidemic history of HIV-1 CRF01_AE in China, 408 fragments of gag gene sequences of CRF01_AE sampled in 2002-2010 were determined from different geographical regions and risk populations in China. Phylogenetic analysis indicates that the CRF01_AE sequences can be grouped into four clusters, suggesting that at least four genetically independent CRF01_AE descendants are circulating in China, of which two were closely related to the isolates from Thailand and Vietnam. Cluster 1 has the most extensive distribution in China. In North China, cluster 1 and cluster 4 were mainly transmitted through homosexuality.The real substance of the recent HIV-1 epidemic in men who have sex with men(MSM) of North China is a rapid spread of CRF01_AE, or rather two distinctive natives CRF01_AE.The time of the most recent common ancestor (tMRCA) of four CRF01_AE clusters ranged from the years 1990.9 to 2003.8 in different regions of China. This is the first phylogenetic and temporal dynamics study of HIV-1 CRF01_AE in China.

Sequence analysis of the gag-pol gene of human immunodeficiency virus type 1 of intersubtype (B'/C) recombinant strain in Beijing, China.

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

[Genetic characteristics of HIV-1 CRF01_AE strains circulating in Beijing].

OBJECTIVE: To analyze the genetic characteristics of HIV-1 CRF01_AE strains prevailing in Beijing. METHODS: Plasma samples were collected from the newly diagnosed HIV-1 individuals being reported during 2006 to 2008 in Beijing. Gag gene fragments were amplified from RNA template which were extracted from plasma by RT and nested PCR methods. 105 CRF01_AE sequences were analyzed by phylogcnetic methods and characterized through calculating the genetic distance and Entropy analysis. RESULTS: There were four main sub-clusters in the phylogenetic tree. We named them as sub-clusters Homo-Max (67 sequences), Hetero (6 sequences), Mix (8 sequences) and Homo-Min (18 sequences) respectively, based on the mode of transmission. It was found that no international reference strain was closely related to the sub-cluster Homo-Max, Hetero or Homo-Min, including 91 samples. The strains in sub-cluster Mix consisting 8 cases that were closely related to the strains identified in Thailand and Vietnam. Genetic distance analysis on gag genes showed that the diversity of sub-clusters Homo-Max and Homo-Min was obviously less than that of the sub-cluster Hetero or Mix. When compared with sub-cluster Mix, there were 37, 29 and 11 significantly different nucleotides polymorphism compositions sites in sub-clusers Homo-Max Homo-Min and Hetero. CONCLUSION: This was the first report describing that four main epidemic sub-clusters were existed in CRF01_AE strains prevailing in Beijing. The virus with sub-cluster Homo-Max was the dominant strain in this region with shorter period of circulation and higher proportion seen in the HIV-infected persons. The virus in sub-cluster Mix was highly homologic with the CRF01_AE strains from Thailand and Vietnam.

[Subtype and sequence analysis of gag genes among HIV-1 strains circulating in Beijing during 2007].

To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent in Beijing during 2007 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routes, we collected the anti-conglutinated whole blood samples from HIV-1 newly infected individuals in Beijing during 2007 and separated plasma specimens from the aamples. RNAs were extracted and the gag genes from the various samples were amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag genes were performed using the MEGA2 software. Among 200 HIV-1 plasma samples,161 gag HIV-1 gene fragments were amplified and analyzed. Seven HIV-1 subtypes or circulating recombinant forms (CRFs) of HIV-1 including A1 (1 strains), B (35 strains), Thai B (19 strains), C (3 strains), CRF01_AE (49 strains), CRF07_BC (51 strains), CRF08_BC (3 strain) were identified circulating in Beijing. The gene divergences inside the subtypes were different, with 7.7%, 6.5%, 6.7%, 6.7%, 5.5%, 4.3%, 5.8%, in subtype A1, B, Thai B, C, CRF01_AE, CRF07_BC, CRF08_BC, respectively. Subtypes CRF07_BC and CRF01_AE were predominant in Beijing account for 31.7% and 30.4% among samples. Seven HIV-1 subtypes exist in Beijing and the surveillance of HIV-1 gene variation should be paid more attention.

[Association between sequence variation of Env, Gag genes from the same source and HIV-1 disease progression and host genetic polymorphism].

OBJECTIVE: To understand the relationship between the HIV-1 viral sequence variation and host factors associated with HIV-1 disease progression. METHODS: Env and gag fragments of HIV-1 were amplified with PCR, cloned and sequenced. Bioinformatics was employed to find the genetic variation, N-linked glycosylation, hypermutation etc. Host gene polymorphism was analysed by using restricted fragment length polymorphism (RFLP). RESULTS: Significant difference was found in genetic divergence between Env PCR dominant and clonal sequences (0.1 and 0.06, respectively) in non-treated group, but no significant difference was found in the HAART treated group. V3 GPGQ accounted for the most part in both treated and nontreated groups, rare V3 loop such as GPGH, GQGR and GLGR was found in treated group, V3 substitutions of I/V (position 12) and Y/H (position 21) was associated with the relatively rapid progression (RRP). Glycosylation was significantly higher in RRP than in TP for Env region, GA substitution in RRP was also significantly higher than that in TP group. SDF1-3primeA and CCR2 V64I gene frequency was higher in TP than in RRP, but the difference was not significant. CONCLUSION: Disease progression was associated with V3 AA change, glycosylation and GA substitution in env gene. SDF1-3primeA, CCR2 V64I and CX3CR1 V249I/M280T was not associated with disease progression significantly.