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 characterization of three newly isolated CRF07_BC near full-length genomes in China

Though HIV-1 CRF07_BC rapidly spread in China, there have been few reports about this subtype since its first genetic characterization nearly 10 years ago. It was urgent and necessary to know the current gene variation of circulating CRF07_BC strains. Xinjiang was the main region for the CRF07_BC epidemic and also an ideal region for research on the viral gene evolution. The strains of Ulumuqi and Yili in Xinjiang were isolated, cloned, and sequenced in this study. Analyses of phylogenetic, potential CTL epitopes and N-glycosites were preformed simultaneously. New CRF07_BC isolates showed higher genetic diversity and more potential N-glycosites than old isolates. It was interesting that although the env and nef genes are highly variable, highly conserved potential CTL epitopes and N-glycosites were found in deduced gp120 V3 and Nef product of all CRF07_BC isolates. The analysis of the sequences provides some valuable information on the investigation of the epidemiology and on vaccine development.     

Construction and characterization of an infectious molecular clone of HIV type 1 CRF07_BC

HIV-1 CRF07_BC constitutes a large fraction of HIV-1 strains circulating in China and is responsible for the rapidly expanding epidemic across the country. Little is known about the biological characteristics of the CRF07_BC viruses, particularly the propensity by which this circulating recombinant form of HIV replicates within an infected individual. In this study, a near-full-length genome of a CRF07_BC virus from an injecting drug user (IDU) from the Xingjiang region in China was cloned and rescued by the functional 5' LTR (long terminal repeat region) and 3' LTR from pNL4-3 to produce a chimeric infectious molecular clone (NLXJDC6441X2). When cultured in interleukin-2-stimulated peripheral blood mononuclear cells (PBMCs) and the Ghost.R5 cell line, NLXJDC6441X2 produced a lower replication yield than expected. More study is needed to explore the key role that leads to the poor infectious activity of NLXJDC6441X2. This study has produced the first isolation of the HIV-1 CRF07_BC DNA clone and has provided a versatile molecular model for research focusing on the biological properties of this subtype.     

Molecular epidemiology of HIV-1 infection and full-length genomic analysis of circulating recombinant form 07_BC strains from injection drug users in Taiwan

BACKGROUND: Previously, we reported that there was an outbreak of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan in 2004. The objectives of the present study were to conduct a molecular epidemiological analysis and to characterize the full-length genome of the Taiwanese CRF07_BC. METHODS: Three hundred and fifty-eight patients with HIV-1/AIDS from hospitals and 133 HIV-1-infected inmates from detention centers were recruited. DNA sequencing and phylogenetic analysis were conducted to determine subtypes and evolutionary relationships. Recombination breakpoints of 2 full-length CRF07_BC strains were elucidated using a bootscanning method. RESULTS: Of 206 HIV-1-infected patients who received a diagnosis in 2004, 44.7% were infected with subtype B, 53.4% with CRF07_BC, and 1.5% with CRF01_AE. Ninety-eight percent (109/111) of IDUs were infected with CRF07_BC. Deletions of 7-11 amino acids in both p6(gag) and p6(pol) proteins were noted among the Taiwanese CRF07_BC strains. The CRF_07BC strains belonged to 2 phylogenetic clusters, and the first cluster contained only CRF07_BC strains from the southern part of Taiwan. CONCLUSIONS: The Taiwanese CRF07_BC strains had 97% full-length sequence homology with the prototype from mainland China. CRF07_BC was first introduced into the southern region in 2002 and then spread to other regions in Taiwan in 2004.     

Near full-length genomic characterization of a novel HIV type 1 CRF07_ BC/CRF08_ BC recombinant strain from Yunnan, China

Yunnan province was considered the HIV-1 epicenter of China, where many subtypes and CRFs of HIV-1 were circulating. CRF07_BC and CRF08_BC were two of the main circulating subtypes that caused more than 90% of the HIV-1 infections in intravenous drug users (IDUs) in this district. The cocirculation of these two CRFs in the same area and population predicted the emergence of new second-generation recombinants. This study presented a near full-length genomic analysis of a novel HIV-1 recombination (09YN072) involving CRF07_BC and CRF08_BC. The analyses of the sequence of 09YN072 showed that two CRF07_BC segments were inserted into the CRF08_BC backbone. The discovery of the novel recombinant strain complicates the HIV-1 epidemic in Yunnan, China, as well as the development of effective vaccines to limit the spread of HIV-1 in China.     

Amino acid conservation in the gp41 transmembrane protein and natural polymorphisms associated with enfuvirtide resistance across HIV-1 variants

Information about gp41 variability across distinct HIV-1 subtypes is scarce, and yet such knowledge would be desirable for designing new drugs targeting this viral protein. Conserved gp41 residues in viruses derived from 79 individuals infected with distinct HIV-1 subtypes (29 A, 25 B, 8 C, 3 D, 4 F, 4 G, 2 H, 1 J, 1 U, and 2 CRF06_cpx) and naive for entry inhibitors were examined. Conservation of gp41 was also examined in 908, 56, and 3 HIV-1 group M, O, and N sequences, respectively, available at the Los Alamos HIV Sequence Database. Among the 345 residues in the full gp41 protein, 36% showed up to 90% conservation in all 987 group M sequences, as did 40% of 56 group O sequences and 49% of 3 group N sequences. The HR1 region (residues 29-82) showed a higher proportion of highly conserved residues than did the HR2 region (residues 116-161) in all groups (65 vs. 34% in group M, 57 vs. 46% in group O, and 80 vs. 52% in group N). Some secondary resistance mutations to enfuvirtide were found as natural polymorphisms (A30V and Q56K/R in group M, Q56R and S138A in group O, and S138A in group N). In fact, A30V was a signature change in clade G and CRF06_cpx, whereas Q56K/R was a signature change for clades A and J, as well as for CRF04_cpx, CRF09_cpx, CRF11_cpx, and CRF13_cpx. The relative conservation of amino acids in gp41 across HIV-1 variants indirectly highlights the critical role of this protein for HIV infectivity and makes it feasible to design new entry inhibitors with activity against diverse HIV-1 variants.     

广州市男男性行为者HIV-1分子流行病学研究

目的了解广州市男男性行为人群（MSM）中,人类免疫缺陷病毒Ⅰ型（HIV-1）亚型流行状况和基因变异特征。方法从2006～2007年广州市发现的、全部19份经男男性传播感染的HIV-1阳性血清样本,应用套式聚合酶链式反应（Nested—PCR）分别扩增gag和fitly区基因,并测定、分析序列。结果被成功扩增的有17份样本,获得13份env基因序列和17份gag基因序列。经系统进化树分析,11份（64．71％）为CRF01_AE,2份（11．76％）为CRF07_BC,4份（23．53％）为B。各亚型组内基因离散率：在env区,CRF01_AE为（8．27±0．93）,CRF07_BC为（11．04±1．72）,B为（16．65±1．78）;在gag区,CRF01_AE为（5．36±0．68）,CRF07_BC为（4．29±1．14）,B为（11．75±1.35）。V3环顶端四肽分析显示：8份CRF01_AE和2份CRF07_BC V3环顶端四肽均为GPGQ;3份B亚型中2份为GPGR,1份为GWGR。根据V3环关键氨基酸推测辅助受体使用情况,结果显示：所有样本均被预测使用CCR5辅助受体。结论广州市MSM人群HIV-1感染者中,至少存在CRF01_AE、CRF07_BC 2种重组亚型和B亚型的流行,且大部分流行株可能为使用CCR5辅助受体的巨嗜细胞嗜性／NSI毒株。     

Chronology of the HIV-1 CRF07_BC expansion in East Asia

The HIV-1 epidemic among injecting drug users (IDU) in Taiwan is caused primarily by CRF07_BC infections. Evolutionary analyses, which utilize outgroup reference strains from northwestern China (Xinjiang), reveal that CRF07_BC was introduced into southern Taiwan in 1998-2001 and spread to central-northern Taiwan in 2001-2003, causing the largest HIV/AIDS epidemic in Taiwan. The separate introduction of CRF07_BC into Xinjiang occurred in 1992-1995. This study illustrates the temporal dynamics of CRF07_BC spread among IDU across east Asia.     

Surveillance of HIV type 1 recent infection and molecular epidemiology among different risk behaviors between 2007 and 2009 after the HIV type 1 CRF07_BC outbreak in Taiwan

The objective of this study was to analyze recent infections and the molecular epidemiology of human immunodeficiency virus type 1 (HIV-1) among different risk groups since the outbreak of circulating recombinant form CRF07_BC among intravenous drug users (IDUs) in 2004 in Taiwan. Phylogenetic analysis was performed using the env and pol fragment sequences amplified from these specimens. The BED IgG capture incidence EIA (BED-CEIA assay) was used to determine recent infections. Among the 683 HIV-1-positive individuals enrolled between 2007 and 2009, 394 (57.7%) were subtype B, 260 (38.1%) were CRF07_BC, 26 (3.8%) were CRF01_AE, two (0.3%) were CRF08_BC, and one (0.1%) was CRF06_cpx. While the percentage of CRF07_BC decreased (58.5-17.9%, p < 0.001) from 2007 to 2009, the percentage of subtype B increased (37.6% to 74.9%, p < 0.001). A concordant decrease in the proportion of recent infections to new infections among IDUs (63.6% to 9.8%, p < 0.001), accompanied with an increase of the proportion of recent infections in MSM (men having sex with men) (22.4-67.1%, p = 0.77) and heterosexual groups (13.1- 23.2%, p = 0.852), was observed. The decrease in CRF07_BC infections and the reduction in the proportion of recent infections among IDUs reflected the success of harm reduction strategies initiated by the government in 2005.     

Molecular epidemiology of the heterosexual HIV-1 transmission in Kunming, Yunnan Province of China suggests origin from the local IDU epidemic

Molecular epidemiological investigation was conducted among injecting drug users (IDUs) (n = 11) and heterosexuals (n = 15) in Kunming, Yunnan Province of China. HIV-1 genotypes were determined based on the nucleotide sequences of 2.6-kb gag-RT region. The distribution of genotypes among IDUs was as follows: CRF07_BC (5/11) and CRF08_BC (5/11); subtype B' (1/11). Similarly, a majority of Kunming heterosexuals (14/15) were infected with CRF07_BC (4/15), CRF08_BC (6 /15), or subtype B' (4/15), known to predominate among IDUs in China. This contrasts with trends in the coastal regions of China and surrounding southeastern Asian countries, where CRF01_AE predominates among heterosexuals. The heterosexual HIV-1 epidemic in Kunming thus appears to derive from the local IDU epidemic. Of note, subtype B' was the most prevalent strain among heterosexuals before 1997, while CRF07_BC and CRF08_BC became predominant in 2002, indicating a transition of HIV-1 genotype distribution between the early and the more recent samples from Kunming heterosexuals.     

Identification of subtype B, multiple circulating recombinant forms and unique recombinants of HIV type 1 in an MSM cohort in China

To study HIV-1 genetic diversity among HIV-positive men who have sex with men (MSM) in China, a cohort consisting of HIV-positive MSM was established in 2005 and was monitored every 2 years. In 2007, 44 HIVpositiveMSM subjects were genotyped, and the results showed HIV-1 subtype B decreased from 77.5% to41.9%, but non-B subtypes increased rapidly represented by CRF01_AE from 3.7% to 30.2% compared to 2005.In addition, one case of CRF07_BC was first identified in this population, which mainly circulated among HIV-1-infected injection drug users (IDUs) in China. There were 11 unique recombinant forms (URFs) consisting ofa recombination of CRF01_AE with subtype B or CRF07_BC. The subtype-specific phylogenic tree analysis showed that the genetic distance within subtype B group viruses was larger than the genetic distance within the CRF01_AE group (p 0.001). Of the identified viruses in the Chinese MSM population, over 80% of subtype B viruses might originate from the United States and Brazil, and over 85% of the CRF01_AE viruses mightoriginate from Thailand. In addition, epidemic study data showed that some of the HIV-1-infected MSM had foreign sexual partners (13.6%) and heterosexual activities (43.2%). The patients infected with HIV-1 URF viruses had more heterosexual encounters (54.5%) and more sexual partners (72.7%) compared to those infected with subtype B (44.4%; 33.3%) and CRF01_AE (23.1%; 69.2%) viruses. Taken together, we suspected that the genetic complexity of HIV-1 viruses identified in Chinese MSM populations was more likely a result of multiple introductions of viruses from the general population infected with HIV-1 through IDUs or heterosexual transmission.     

123株人类免疫缺陷病毒-1重组亚型主要耐药性突变的基因屏障

目的 比较HIV-1 CRF01_AE、CRF07_BC和CRF08_BC毒株发生蛋白酶抑制剂(PI)、核苷类反转录酶抑制剂(NRTI)和非核苷类反转录酶抑制剂(NNRTI)主要耐药性突变的基因屏障,分析不同亚型耐药模式的差异.方法 纳入在广西壮族自治区南宁、柳州两市未经治疗的HIV感染者190例,采集外周静脉血,从血浆中提取HIV-1 RNA,扩增pol区并测序,用系统进化树分析序列的亚型,统计各序列每个主要突变位点由野生型密码子突变为耐药密码子所需的碱基转换和颠换的数量,根据转换与颠换发生概率约2.5:1的规律,将每个转换赋值设为1,每个颠换赋值设为2.5,计算各突变赋值之和,即为基因屏障值,采用Kruskal-Wallis检验法和Nemenyi法比较不同亚型的基因屏障差异.结果 共获得CRF01_AE、CRF07_BC和CRF08_BC毒株123株.CRF08_BC发生T/S69D的基因屏障低于CRF01_AE和CRF07_BC(χ2=107.501,P＜0.01),CRF01_AE和CRF07_BC发生V118I和L210W的基因屏障低于CRF08_BC,CRF07_BC发生V106M的基因屏障低于CRF01_AE和CRF08_BC.结论 在相同的选择压力下,CRF01_AE和CRF07_BC比CRF08_BC易发生V118I和L210W,CRF08_BC比CRF01_AE和CRF07_BC易发生T/S69D,CRF07_BC比CRF08_BC和CRF01_AE易发生V106M.     

Characterization of five nearly full-length genomes of early HIV type 1 strains in Ruili city: implications for the genesis of CRF07_BC and CRF08_BC circulating in China

To trace the genesis of HIV-1 CRF07_BC and CRF08_BC, two predominant circulating recombinants among intravenous drug users in China, a retrospective molecular epidemiological investigation (1996-1998) was conducted in Ruili city of Yunnan, where the first AIDS epidemic among IDUs was reported in 1989. Fifty-four HIV-1 env C2V3 sequences were determined and genotyped with 49 subtype B and only 5 subtype C strains. The nearly full-length genome analyses of these five env-based subtype C samples revealed that four of them were actually BC recombinants and only one was pure subtype C. The first identified nonrecombinant HIV-1 subtype C, genetically close to Indian C representatives, provided direct evidence for the hypothesis that subtype C in China was introduced from India. Interestingly, three BC recombinants with subtype B as backbones were identified; one BC recombinant that precisely shared a common subtype B segment (nef region) with CRF07_BC and CRF08_BC was described, which indicated a close evolutionary relationship to these two CRFs. The sequences undoubtedly lead us to a better understanding of the emergence of CRF07_BC and CRF08_BC.     

Identification and characterization of two new HIV type 1 Unique (B/C) recombinant forms in China

Recombination was most important in the generation of new viral strains and in the increase of HIV diversity. There were more and more new HIV-1 strains. Not only circulating recombinant forms (CRFs) but also unique recombinant forms (URFs) have been reported around the world. CRF07_BC and CRF08_BC were the two predominant CRFs circulated in Yunnan Province, southwest China. In the present study, we identified two new HIV Type 1 unique (B/C) recombinant gorms in Yunnan Province by nucleotide sequencing in two halves of HIV genome. Although the genomic structures of the two B/C recombinants were different from previously identified CRFs (CRF07_BC and CRF08_BC) and URFs in Yunnan Pprovince, they have several common recombination sites with CRF07_BC and CRF08_BC.     

Molecular epidemiology of HIV type 1 subtypes in Taiwan: outbreak of HIV type 1 CRF07_BC infection in intravenous drug users

In Taiwan, sexual transmission is responsible for most HIV-1 infections with two dominant subtypes, subtype B and CRF01_AE, distributing among homosexual and heterosexual groups, respectively. Recently, intravenous drug use has become an emerging route of HIV-1 transmission and contributed to a significant increase of HIV-1 infection. To characterize the HIV isolates responsible for the outbreak among intravenous drug users (IDUs), phylogenetic analysis was performed to analyze the protease/RT sequences amplified from HIV-1-infected IDUs at National Taiwan University Hospital and Taipei City STD Control Center. CRF07_BC, which is circulating in northern China, was demonstrated to account for the majority of HIV-1 infection in IDUs in the past 2 years. Although these Taiwanese CRF07_BC sequences shared the same breakpoint positions as those described in the CRF07_BC reference sequences, they formed a unique cluster in the phylogenetic tree, suggesting they originated from a founder virus. This finding was further supported by the relative low genetic diversity and unique sequence features. Our results demonstrated the emergence of CRF07_BC and its association with the HIV-1 outbreak among IDUs between 2004 and 2005 in Taiwan. This finding not only helps us to have a better understanding of the HIV evolution in Asia, but also has important implications for vaccine design in the future.     

Emergence of a New HIV Type 1 CRF01_AE Variant in Guangxi, Southern China

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

我国男男性行为人群中HIV-1病毒亚型分布及其演变

男男性行为人群(MSM)仍是我国HIV感染的高危人群,欧美B亚型、CRF01_AE和CRF07_BC亚型是其人群中流行的主要病毒亚型。近年来,MSM中HIV-1亚型的流行趋势发生改变,欧美B亚型所占比例显著下降,CRF01_AE和CRF07_BC亚型所占比例则明显增加,并且出现新的重组亚型。运用分子生物学技术分析我国男男性行为人群中HIV-1病毒亚型分布和变化趋势,可有助于阐明MSM人群中HIV-1的流行规律,追踪传染来源和确定相关危险因素,并为MSM人群精准干预提供重要的参考依据。     

河北省治疗前人群HIV-1耐药毒株分子传播网络研究

目的 了解河北省抗病毒治疗前人群HIV-1耐药毒株流行与分子传播网络情况。方法 用in-house的方法扩增HIV-1 pol区基因序列,并利用TN93模型计算pol序列基因距离,构建HIV-1分子网络图。结果 研究发现17名HIV-1感染者出现耐药突变,耐药突变率为6.16%(17/276),其中,NNRTI类耐药率为4.35%(12/276),PI类耐药率为1.09%(3/276), NRTI类耐药率为0.36%(1/276),NNRTI类和PI类双重耐药率为0.36%(1/276);多因素Logistic回归分析显示年龄和亚型是HIV-1产生耐药的影响因素,虽然HIV-1耐药突变在CRF07_BC(2.44%)亚型中的分布明显低于CRF01_AE(8.03%)和B(3.70%)亚型,然而CRF07_BC序列构建的分子传播网络最多,传播速度最快。结论 河北省治疗前HIV-1耐药突变处于较高水平, 应制定措施实时监测未治疗人群中HIV-1耐药毒株流行情况。