Identification of a newly characterized HIV-1 BG intersubtype circulating recombinant form in Galicia, Spain, which exhibits a pseudotype-like virion structure

We recently reported the finding of phylogenetically related HIV-1 BG intersubtype recombinant and G subtype nonrecombinant viruses circulating among injecting drug users in the region of Galicia in northwestern Spain. Here, we report the characterization of near full-length genome sequences of nine of these viruses (seven BG recombinant and two of nonrecombinant G subtype), obtained from epidemiologically unlinked individuals. Bootscan analysis reveals that six recombinant viruses share an identical mosaic structure, with two intersubtype breakpoints delimiting a B subtype segment comprising most of Env gp120 and the external portion of Env gp41, with the remaining portions of the genome being of subtype G, thus mimicking a pseudotype virion structure. The seventh BG recombinant virus exhibits breakpoints in env coincident with the other BG viruses but contains additional B subtype segments in gag and pol. In phylogenetic trees of complete genomes and of the B subtype segment of env, all seven BG viruses group in a monophyletic cluster. G subtype portions of the BG viruses group uniformly with the newly derived nonrecombinant G subtype viruses of Galicia in bootscan analysis, which points to the locally circulating G subtype strain as parental of the recombinants. These results allow us to define a new HIV-1 circulating recombinant form (CRF14_BG), the first reported to originate in Western Europe.     

Identification of a novel HIV-1 circulating ADG intersubtype recombinant form (CRF19_cpx) in Cuba

Circulating recombinant forms (CRFs) represent a substantial proportion of HIV-1 isolates in the global pandemic. Characterization of HIV-1 genetic forms, including CRFs, may be relevant to studies on molecular epidemiology, recombination, superinfection, vaccine development, and antiretroviral therapy. This study analyzes near complete genomes of 4 epidemiologically unlinked viruses from Cuba, originally characterized as D/A intersubtype recombinants in pol and env segments. The genomes of 3 viruses exhibited virtually coincident mosaic structures, with multiple segments of subtypes A, D, and G and uniform phylogenetic clustering with each other along the genome. These results allow us to define a new CRF (CRF19_cpx). The 4th analyzed Cuban virus was recombinant between CRF19_cpx and CRF18_cpx (which also circulates in Cuba). CRF19_cpx exhibited homology to an AG intersubtype recombinant virus from Cameroon (CM53392) along approximately 5 kb and clustered with a subtype D virus from Gabon (G109) in gag. Four other viruses from central or west Africa were also phylogenetically related to CRF19_cpx in env fragments. These results allow us to define CRF19_cpx as a second novel CRF of African origin circulating in Cuba, to identify putative representative viruses of its parental strains, and to characterize a unique CRF18/CRF19 recombinant virus.     

Genetic analysis of HIV-1 strains in rural eastern Cameroon indicates the evolution of second-generation recombinants to circulating recombinant forms

The HIV-1 genetic diversity in most parts of Cameroon is well described and shown to be very broad. However, little is known about the composition of the HIV-1 epidemic in the rural parts of eastern Cameroon. Therefore, we investigated 25 specimens from this region for their subtypes in gag, pol, and env gene fragments. Along with genetic material of subtypes A1, C, G, CRF01_AE, CRF02_AG, and CRF11_cpx, we also identified a large number (24%, 6/25) of distinct env sequences within the subtype A radiation. CRF02_AG was the predominant genetic form in all genes studied. Half of the specimens studied were considered "pure" based on concordant subtypes in the genes studied, whereas the other half were unique recombinant forms (URFs). Except for 1 URF, all were second-generation recombinants (SGRs), 90% of which contained genetic material of CRF02_AG in at least 1 gene. Notably, we identified individuals from 3 different villages infected with CRF01_AE(gag)CRF02_AG(pol)A(env) strains, which is indicative of the evolution of this URF to a circulating recombinant form (CRF). In addition, we identified a CRF02_AG(pol)C(env) recombinant infecting a man and a woman living in the same village, suggesting horizontal transmission of this recombinant. The current study emphasizes the power of HIV-1 recombination through the generation of SGRs and the evolution of URFs into CRFs. These findings suggest that, in a region where a predominant HIV-1 strain cocirculates among several subtypes, recombination could eventually decrease the proportion of this strain over time, such as CRF02_AG in Cameroon.     

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.     

HIV-1 subtypes and circulating recombinant forms (CRFs) in Northern Greece

In order to understand the genetic diversity of virus isolates associated with the human immunodeficiency virus type one (HIV-1) epidemic in Northern Greece, 51 specimens from HIV-1 infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty two (82.3%) specimens were identified as pol subtype B, three (5.9%) as A, and one (2%) as CRF01_AE, while the remaining five (9.8%) specimens appeared to be complex recombinants, belonging to the Cypriot/Greek form CRF04_cpx. The proportion of CRF04_cpx strains is larger than previously reported, suggesting that the CRF04_cpx is significantly contributing to the Greek HIV-1 epidemic.     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Genetic diversity of HIV type 1 in rural eastern Cameroon

To monitor the presence of genotypic HIV-1 variants circulating in eastern Cameroon, blood samples from 57 HIV-1-infected individuals attending 3 local health centers in the bordering rural villages with Central African Republic (CAR) were collected and analyzed phylogenetically. Out of the 40 HIV-1 strains with positive polymerase chain reaction (PCR) profile for both gag and env-C2V3,12 (30.0%) had discordant subtype or CRF designation: 2 subtype B/A (gag/env), 1 B/CRF01, 2 B/CRF02, 1 CRF01/CRF01.A, 2 CRF11/CRF01, 1 CRF13/A, 1 CRF13/CRF01, 1 CRF13/CRF11, and 1 G/U (unclassified). Twenty-eight strains (70.0%) had concordant subtypes or CRF designation between gag and env: 27 subtype A and 1 F2. Out of the remaining 17HIV-1 strains negative for PCR with the env-C2V3 primers used, 10 (58.8%) had discordant subtype or CRF, and 7 (41.2%) had concordant one based on gag/pol/env-gp41 analysis. Altogether, a high proportion (22/57, 38.6%) of the isolates were found to be recombinant strains. In addition, an emergence of new forms of HIV-1 strains, such as subtype B/A (gag/env), B/CRF01 and B/CRF02, was identified. The epidemiologic pattern of HIV-1 in eastern Cameroon, relatively low and high prevalence of CRF02 and CRF11, respectively, was more closely related to those of CAR and Chad than that of other regions of Cameroon, where CRF02 is the most predominant HIV-1 strain. These findings strongly suggest that this part of Cameroon is a potential hotspot of HIV-1 recombination, with a likelihood of an active generation of new forms of HIV-1 variants, though epidemiologic significance of new HIV-1 forms is unknown.     

北京市性传播HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的 了解北京市性传播HIV-1感染者流行毒株亚型特点和流行规律.方法 随机采集北京市2008年新确证性传播HIV感染者的抗凝全血标本100份,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增病毒gag基因,并进行序列测定和亚型分析.结果 系统进化分析确定北京市性传播HIV-1感染者流行毒株存在8个亚型或流行重组型,分别为B亚型22份,B'亚型8份,C亚型1份,CRF01_AE 38份,CRF02_AG 2份,CRF07 BC 9份,CRF08_BC 3份,疑似C/CRF01_AE重组型1份.结论 CRF01_AE和B亚型分别占45.2%和26.2%,为性传播感染者主要的亚型,应该加强我市HIV-1亚型流行情况的监测.

Abstract：

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.     

Absence of genotypic drug resistance and presence of several naturally occurring polymorphisms of human immunodeficiency virus‐1 CRF06_cpx in treatment‐naive patients in Estonia

All non‐B HIV‐1 subtypes and circulating recombinant forms (CRFs) are characterized by several polymorphisms in protease (PR) region. In addition, in recent years the increasing use of antiretroviral treatment (ART) has rapidly raised the spread of transmitted drug resistance. We aimed to determine the presence of naturally occurring polymorphisms and transmitted drug resistance mutations (DRMs) in ART naïve HIV‐1‐positive subjects in Estonia. A total of 115 drug‐naive HIV‐1‐infected subjects (mean age 27 years; 70% male; 65% infected via intravenous drug use and 34% by heterosexual contact) were enrolled. Viral genomic RNA from plasma was directly sequenced in PR, revertase (RT), and envelope (env) regions. Phylogenetic analysis of RT and env regions revealed that 89% and 3% of sequenced viruses belonged to CRF06_cpx and subtype A1, respectively, and 6% were described as unique recombinants (signed A1‐06) between CRF06_cpx and subtype A1 viruses. No primary DRMs were found in PR or RT regions indicating the absence of transmitted drug resistance. The most common polymorphisms in the PR region were K14R, M36I, H69K, and L89M seen in 96%, 100%, 99%, and 100%, respectively. The clinical relevance of these polymorphisms in terms of success of ART has to be monitored in future clinical studies. J. Med. Virol. 81:953–958, 2009. © 2009 Wiley‐Liss, Inc.     

26例有偿献血员HIV-1基因分型与变异特征

目的分析某地区有偿献血人员中流行的人免疫缺陷病毒1型（HIV-1）gag、pol、env基因亚型及基因变异特征。方法提取HIV-1感染者外周血单核细胞（PBMC）DNA,经巢式PCR（NestedPCR）扩增gag（p17-p24）、pol（PR＊RT）、env（C2-V5）基因片段,纯化测序后用MEGA5．0等生物学软件对核苷酸序列进行分析。结果23份样本为B亚型,2份为B亚型与C亚型重组,1份为CRF01-AE与B亚型重组。PR区未发现蛋白酶抑制剂主要耐药性突变,RT区检测到核苷类逆转录酶抑制剂耐药性突变M184V和非核苷类逆转录酶抑制剂耐药性突变K101E,G190A。结论流行于该地区的HIV-1毒株以B亚型为主。大多数毒株对常规抗病毒药物仍然敏感,使用HARRT治疗方案依然有效。CXCR4型辅助受体的毒株顶端四肽多为GPGR（91．7％）,提示GPGR可能与疾病的进展有关。     

Generation of a consensus sequence from prevalent and incident HIV-1 infections in West Africa to guide AIDS vaccine development

We considered several key issues regarding the development of a DNA-based human immunodeficiency virus type 1 (HIV-1) vaccine: (1) should the candidate vaccine construct be derived from incident or prevalent HIV-1 strains; and (2) should circulating plasma virus, archived HIV-1 provirus recovered from peripheral blood mononuclear cells, or both be included? To address these questions, we collected circulating HIV-1 strains from infected individuals residing in Abidjan, C?te d'Ivoire. From a panel of 23 strains, 22 were HIV-1 subtype A in gag, 19 of which phylogenetically clustered with the recombinant HIV-1, CRF02-AG strains from West Africa. The mosaic genome of CRF02-AG was confirmed by sequencing the protease gene. A consensus gag p24 protein sequence was generated and 147 of 148 codons were identical to CRF02-AG (IbNG). Regardless of the sequence origin (RNA, provirus, incident, or prevalent), the gag p24 consensus sequences were highly representative of these distinct virologic compartments. These data suggest that the consensus sequence generated from incident and prevalent infections may provide an appropriate sequence for a DNA vaccine and is largely representative of the major circulating viral strain.     

A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene

Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naive and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naive samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36-45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.     

Dynamic analysis of genetic diversity of <Emphasis Type="Italic">gag</Emphasis> and <Emphasis Type="Italic">env</Emphasis> regions of HIV-1 CRF07_BC recombinant in intravenous drug users in Xinjiang Uvghur Autonomous Region,China

The aim of this study was to investigate the genetic variation of HIV-1 CRF07_BC, the most prevalent circulating strain in intravenous drug users (IDUs) in China. We studied the diversity in the C2-V5 region of the HIV-1 env gene and in the p17-p24 region of the HIV-1 gag gene from the same samples in 12 IDUs who were divided into two groups according to the length of infection time. Two IDUs were longitudinally monitored from the time of seroconversion for 2-2.5 years. The viral divergence from the founder strain and the viral population diversity between sequential time points were analyzed in two men. The data show that the divergence of the env gene is higher than that of gag in general, while the diversity of the gag gene is sometimes higher than that of env during the course of HIV evolution. In addition, env and gag gene diversity increased over time. The observed patterns and associations may enhance our understanding of HIV-1 evolution.     

CRF22_01A1 is involved in the emergence of new HIV-1 recombinants in Cameroon

Cameroon is a West African country where high genetic diversity of HIV-1 has been reported. The predominant CRF02_AG is involved in the emergence of more complex intersubtype recombinants. In this study, we sequenced the full-length genome of a novel unique recombinant form of HIV-1, 02CAMLT04 isolated in blood donors in urban Cameroon. Phylogenetic tree and bootscan analysis showed that 02CAMLT04 was complex and seemed to be a secondary recombinant derived from CRF02_AG and CRF22_01A1. The genomic composition of 02CAMLT04 strain showed that it is composed of 3 segments; 24% of the genome is classified as CRF02_AG, spanning most of the envelope gene. The remaining 76% of the genome is classified as CRF22_01A1. In addition, the sequence analysis of 13 full-length sequences from HIV-1-positive specimens received from Cameroon between 2002 and 2010 indicated that 5 specimens are pure CRF22_01A1 viruses, and 6 others have homology with CRF22_01A1 sequences in either gag, pol, or env region, whereas 6% of strains contain portions of CRF22_01A1. Further study demonstrated that CRF22_01A1 is a primary prevalence strain co-circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2), and CRFs (CRF01_AE or CRF02_AG). Our studies show that novel recombinants between CRF22_01A1 and other clades and recombinant forms may be emerging in Cameroon that could contribute to the future global diversity of HIV-1 in this region and worldwide.     

Independent introduction of transmissible F/D recombinant HIV-1 from Africa into Belgium and The Netherlands

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.