Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon,Portugal

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.     

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.     

武汉市HIV-1相关基因序列分析及亚型分布

目的研究武汉市HIV感染人群中的HIV毒株的亚型分布特点和流行规律。方法采集武汉市60名已被确认为HIV-1感染者的抗凝全血样品,提取前病毒DNA,用巢式聚合酶链反应方法（nested-PCR）扩增病毒膜蛋白env基因的C2-V5区及gag基因的部分区段,对PCR纯化产物直接测序,并应用GCG软件对序列进行分析。结果通过PCR扩增得到60份样品的结果,其中env基因序列45份、gag基因序列52份。依据env和gag区基因序列,与HIV-1各个亚型国际参考株比较,通过系统进化分析,最后确定武汉市样品分属5个亚型,分别为HIV-1B亚型中的泰国B（B’）亚型32份,流行重组型CRF07-BC12份,流行重组型CRF01-AE9份,A亚型1份和C亚型6份。结论武汉市存在多种HIV-1亚型,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略。     

Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.     

Genetic subtypes of HIV type 1 viruses circulating in the Czech Republic

We analyzed the genetic diversity of HIV-1 strains circulating in the Czech Republic. Phylogenetic analysis of the env and gag gene sequence fragments from 39 isolates revealed that the majority of these strains (32 of 39, 82%) were of subtype B; other genetic subtypes identified were A, C, F, and recombinant circulating form CRF01_AE. The isolates that did not cluster with subtype B originated almost exclusively from a heterosexual route of transmission. The molecular epidemiological data are suggestive of multiple entry of HIV-1 infection into the Czech Republic and show that the genetic pattern of the HIV-1 strains circulating in this country corresponds to that found in other European countries.     

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi Arabia: high level of genetic diversity found

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.     

Subtypes A,C, G,and recombinant HIV type 1 are circulating in Bangladesh

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.     

Highly divergent HIV type 1 group M sequences evident in Karonga District,Malawi in early 1980s

Six divergent HIV-1 partial env and gag genome sequences have been characterized in five subjects in Malawi, from whom blood spot samples were collected between 1982 and 1989, at the time that the AIDS epidemic there was starting. These sequences could not be classified with any of the recognized subtypes or circulating recombinant forms of HIV-1. They showed no consistent and/or supported associations with other subtypes by either env or gag gene phylogenetic analysis. Their genetic distances from defined subtypes suggest that they may be diverse subsubtype C viruses or, alternatively, that they may have mosaic genomes. Bootscanning analyses are consistent with their being mosaic viruses. These sequences highlight early HIV-1 diversity in a population otherwise dominated by subtype C.     

Near full-length genomes of 15 HIV type 1 group O isolates

Human immunodeficiency virus type 1 (HIV-1) is classified into three distinct groups; M (major), N (non-M/non-O), and O (outlier). Group M strains are further subclassified into subtypes, subsubtypes, and circulating recombinant forms (CRF). While the level of genetic diversity within group O is similar to that between group M subtypes, group O has not been classified into subtypes. A previous study, based on the phylogenetic analyses of the gag p24, pol p32, and env gp160 sequences from 39 group O isolates, laid the foundation for the classification of group O subtypes. Five phylogenetic clusters, I-V, were identified that have characteristics analogous to group M subtypes. However, a complete phylogenetic analysis and classification of group O requires the availability of at least two full-length and one partial genomes for each group O phylogenetic cluster. In this study, 15 group O isolates were selected for full genome sequencing. Phylogenetic analysis of the 15 sequences with eight additional group O genomes supports the classification of three group O subtypes (I-III) and the potential existence of one CRF (IV) and at least one additional subtype (V). The group O subtypes are equidistant to each other and lack subsegments of other subtypes. The intra- and intersubtype genetic distances for group O are similar in magnitude to the corresponding distances for group M subtypes. Intersubtype recombination was identified in three of the 23 (13%) group O genomes. Formal classification of group O subtypes should be forthcoming pending the analysis of additional group O genomes and agreement of the HIV nomenclature committee.     

HIV-1 CRF09_cpx circulates in the North West Province of Cameroon where CRF02_AG infections predominate and recombinant strains are common

This study describes the HIV-1 genetic diversity that currently circulates in Bamenda, the provincial capital of the North West province of Cameroon. Phylogenetic analysis of the protease (pro) gene of 20 HIV-1-seropositive individuals identified 11 (55%) CRF02_AG, one D, one F2, one J, and four (20%) unclassifiable strains. Interestingly, the remaining two (10%) samples, 02CMNYU3072 and 03CMNYU3224, originating from epidemiologically unlinked individuals, were classified as CRF09_cpx, representing the first reported cases of this complex circulating recombinant form (CRF) in Cameroon. Additional analysis of the C2V5 portion of the envelope (env) gene confirmed the CRF09_cpx identity of these isolates and classified the remaining isolates as CRF02_AG (n = 12, 63%), subtype D (n = 2, 11%), subtype F2 (n = 2, 11%), and subtype A1 (n = 1). In combination, the pro and env subtyping results revealed three (16%) isolates with discordant subtypes including J( pro )CRF02_AG( env ), CRF02_AG( pro )D( env ), and CRF02_AG( pro )F2( env ). In conclusion, this study highlights the presence of HIV-1 CRF09_cpx in Cameroon and identifies three possible intersubtype recombinants (ISRs) containing CRF02_AG in a town where CRF02_AG infections predominate, and stresses the commonness of HIV-1 recombinant strains in a region where broad genetic diversity exists.     

Identification of new CRF43_02G and CRF25_cpx in Saudi Arabia based on full genome sequence analysis of six HIV type 1 isolates

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.     

Genetic and biological properties of HIV type 1 isolates prevalent in villagers of the Cameroon equatorial rain forests and grass fields: further evidence of broad HIV type 1 genetic diversity

To understand the evolution of HIV-1, the genetic and biological characteristics of viruses that infect persons living in regions in which the virus has been evolving for several decades must be studied. Thus, we investigated teh genetic subtypes, coreceptor usage, and syncytium-inducing ability of viruses in 47 HIV-1-infected blood samples from individuals living in rural villages in the equatorial rain forest and grass field regions in Cameroon. Heteroduplex mobility analysis (HMA) of gag (part of p24 and p7) and env (C2V5) or sequence and phylogenetic analysis of gag (part of p24 and p7), pol (protease), and env (C2V5), revealed a broad HIV-1 group M genetic diversity. Subtype analysis revealed genetic evidence of seven subtypes (A, C, D, F, G, H, and J) and three circulating recombinant froms (CRFs) (CRF01_AE, CRF02_AG, and CRF11_cpx). Only 15 (32%) of the 47 samples analyzed revealed a concordant subtype in all three genes (gag, pol, and env), while discordant subtypes and CRFs were identified for the remaining 32 (68%) samples. Two patterns of HIV-1 diversity could be discerned in two provinces. While more concordant subtypes in gag, pol, and env genes were identified in villages of South province (10 of 13, 77%), the HIV-1 diversity in the West province was characterized by intersubtype recombinants (63%). Five new intersubtype recombinants were identified including Agag Jpol Genv, Ggag Upol Aenv, AGgag Jpol Aenv, Agag AGpol Henv, and Cgag AGpol AGenv. All of the 40 viruses tested used the R5 coreceptor, of which four also used the X4 coreceptor. Four viruses were able to induce syncytia in MT-2 cells, however, syncytium induction did not correlate with coreceptor usage. This study further reveals the complexity of HIV-1 infection in rural Cameron and points to the future of the global epidemic, which may be characterized by more genetically diverse viruses.     

Genetic and biologic characterization of HIV type 1 subtype C isolates from south Brazil

The molecular and biological properties of HIV-1 subtype C strains from South Brazil were investigated. We sequenced gag and env fragments of viruses from 22 HIV-1-infected individuals from Porto Alegre City, which has the highest frequency of subtype C in the country. The sequences were then compared with other subtype B, C, and F strains isolated in Brazil and other countries using phylogenetic methods. Amino acid signatures were identified and correlated with phenotypic characteristics. We identified six strains with subtype C (27.3%), eight subtype B (36.4%), one subtype F (4.5%), six C/B recombinants (27.3%), and one B/F recombinant (4.5%). The Brazilian subtype C sequences formed a unique phylogenetic group and presented 6 and 18 specific amino acid signatures in gag and env, respectively. Three distinct patterns of C/B recombinants presented characteristic Brazilian amino acid substitutions. Subtype C viruses were predominantly R5 and non-syncytium-inducing, while C/B recombinants were R5/X4 and syncytium-inducing viruses. These findings suggest that subtype C viruses circulating in Brazil are the result of a unique introduction into the country. Recombination events between subtypes B and C have been occurring frequently for more than 10 years in South Brazil. Biological characterization confirms the hypothesis that subtype C is distinct from the others in the evolution of coreceptor utilization.     

Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.     

Subtype B and subtype C HIV type 1 recombinants in the northeastern state of Manipur, India

The predominant HIV-1 strain circulating in India is subtype C. However, subtype A and B strains of HIV-1 have also been reported in India. In 1999, the first A/C recombinant strain was reported from Pune in India. Intravenous drug users (IVDUs) from the northeastern region of India have a high HIV-1 seroprevalence. Studies carried out in intravenous drug users in the northeastern region of India have shown that HIV-1 subtype C is the predominant strain infecting IVDUs. Fourteen blood samples were collected from HIV-1-infected individuals from the northeastern region of India and screened by env and gag heteroduplex mobility assays (HMA). Where the env and gag HMA results from a sample yielded different subtypes, sequencing of env and gag PCR products was carried out to confirm the presence of HIV-1 recombinants. Of the 14 samples subtyped, nine samples belonged HIV-1 subtype C (gag C/env C), one to HIV-1 subtype B (gag B/env B), and the remaining were B/C recombinants (gag C/env B). This is the first report of HIV-1 B/C recombinants from India.     

HIV-1 genetic diversity and genotypic drug susceptibility in the Republic of Georgia

The genetic diversity and genotypic drug susceptibility of HIV-1 strains circulating in the Republic of Georgia, formerly part of the Soviet Union, were investigated for first time. Forty-eight HIV-positive drug-naive Georgian individuals contributed PBMC DNA between 1998 and 2003. On the basis of phylogenetic analyses of partial pol sequences, the predominant HIV-1 genetic forms were subtype A (70%), followed by subtype B (26%); both genetic forms were carried by injecting drug users and heterosexuals. There was also one subtype C (2%) and one CRF18_cpx (2%). The Georgian subtype A strains clustered with subtype A from Russia, designated A(FSU). Twelve of the subtype A strains (25%) contained the secondary protease inhibitor mutation V77I and 9 also had two other silent mutations. This "V77I haplotype" marks one particular genetic lineage of the epidemic in the former Soviet Union. Two strains (4%) carried antiretroviral (ARV) drug resistance mutations. Nearly full-length genome sequences of five Georgian strains were also completed. Two, 98GEMZ011 (subtype A) and 98GEMZ003 (subtype B), closely resembled the parental strains that recombined to create CRF03_AB. The use of these parental strains in the analysis revealed an additional segment of subtype A in CRF03_AB. Thus, the HIV-1 epidemic in Georgia was composed of a mixture of subtype A(FSU) and subtype B.     

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。     

Genetic analysis of the complete gag and env genes of HIV type 1 subtype C primary isolates from South Africa.

South Africa has one of the fastest growing HIV-1 epidemics, with an estimated 4.7 million people infected. To better understand the genetic diversity of this epidemic and its potential impact on vaccine development, we have cloned and sequenced the complete gag and env genes of 13 primary virus isolates. Phylogenetic analysis of our sequences and 69 complete env genes from the Los Alamos and GenBank databases revealed multiple subclusters within subtype C. The V3 loop region was relatively conserved in all our strains when compared with other subtypes, but the region immediately downstream was highly variable. No intersubtype recombinant forms were observed when comparing the gag and env sequences. Characterization of the complete gag and env genes enabled us to select specific strains for further vaccine development.