Human immunodeficiency virus type I (HIV-1) is an enveloped RNA virus, belongs to the Lentivirinae of Retroviridae. Analysis of HIV-1 env genes of virus strains from different geographic regions reveals that HIV-1 can be divided into three main groups: M (major), O (outlier), and N (new).1-3 HIV-1 group M has been further subdivided into genetically equidistant clusters of HIV-1 env genes, comprising subtypes A to J.2 The first case of AIDS in China was reported in 1985. Since that time, it is estimated that approximately 840 000 individuals were infected by the virus by the end of 2003, including 80 000 cases of AIDS.4 China appears to be on the brink of a tremendous upsurge in cases of HIV-1 infection.5 the Joint United Nations Program on HIV/AIDS reported that if the epidemic were left unchecked, the number of HIV-1-infected individuals could reach 10 million or more by 2010.6 A large-scale survey on HIV molecular epidemiology for the second time showed that there were at least A, B', B, C, CRF_BC (CRF07_BC, CRF08_BC), CRF01_AE and CRF02_AG 8 subtypes in China, making China one of the countries having the most HIV subtypes. World widely, distinct virus subtype makes a disproportional distribution and HIV-1 B' is the most prevalent subtype in China. As the infected time goes by and the disease develops, due to the high-level variability of HIV-1, amino acid sequence and the construction of the virus envelope glycoprotein change, and these alterations switch the cytophilic and infected activity of virus to target cells.7-9
To investigate the prevalence, as well as the correlationship between biological characteristics and sequence variability of HIV-1 strains, we cloned the C2-V3 region of DNA fragments extracted from peripheral blood mononuclear cells (PBMCs) of 19 HIV-1 seropositive infectors, and then analyzed the gene sequence via bioinformatics.
Nineteen HIV/AIDS cases were collected from the Agriculture General Hospital (Heilongjiang Province, China). HIV-1 infection status was determined by an enzyme immunoassay and confirmed by a Western blot assay; the 19 cases were not treated with anti-HIV drugs. This study was conducted in accordance with China ethical guidelines, and all subjects gave informed consent for their participation. PBMCs were processed by Ficoll density gradient centrifugation and preserved at -80°C for further genetic characterization.
Provirus cDNA was extracted from PBMCs according the manufacturer's instructions of the QIAmp Blood Kit (Qiagen, USA) and stored at -20°C.
Nested PCR was performed to amplify the C2-V3 segment of HIV-1 env gene using outer primer pair env-m-env-n for the first round of amplification and inner primers env-c and env-g for the second round, primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The nested PCR was done under the following conditions: 1 cycle at 94°C for 5 minutes, 94°C for 1 minute, 55°C for 1 minute, 72°C for 3 minutes; 29 cycles at 94°C for 1 minute, 55°C for 1 minute, 72°C for 3 minutes; a final extension at 72°C for 10 minutes. Primer sequences are shown in Table 1.
Cloning of C2-V3 segment
DNA was purified and cloned into pMD-18T vector (Takara, China). The final product was transformed into E. coli JM109, crudely extracted and identificated plasmids by Amp and Blue/White Cloning. Sequenced positive clonings were walked on the automatic sequencer (ABI, USA); this process was accomplished by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.
Aligned the nucleotide sequence of 19 samples with CLUSTAL W (BioEdit) software,10 results were acquired and used for phylogenetic tree analysis after artificial adjustment. Reference sequence, downloaded from Los Alamos HIV Sequence Database, was used to identify the subtype of obtained sequence; Genetic distance between sequences was assessed using the software MEGA 3.1 Kimura 2-parameter,11 and the Phylogenetic tree was reestablished with Neighbor-Joining method.
The intraclass genetic diversity and interclass genetic divergence were calculated with Kimura 2 parameter model. Statistical analysis was done by Mann-Whitney U test12 and P value less than 0.05 was considered statistically significant.
Nineteen HIV/AIDS cases, 8 male and 11 female, ranged from 15-42 years with the average age of 31 years, were infected mainly by blood transfusion between 1997 and 2000. Among them, 8 patients' CD4+ T cell counts were less than 200/μl. Sixteen patients came from the same farm, and 13 were infected by blood transfusion, No. HLJ01, HLJ03, HLJ05, HLJ07, HLJ08, HLJ09 and HLJ10 were transfused by the same blood donor Sun, with blood type A. While HLJ04, HLJ11, HLJ12, HLJ15, donor Huang, with blood type O. The two donors were couples: Sun sold his blood and plasma in He'nan Province in the early 1990s and died in 2004, while Huang died in 2002. There were three couples among the 19 HIV infectors: HLJ02-HLJ17, HLJ06-HLJ11 and HLJ08-HLJ13 (details are shown in Table 2).
Phylogenetic tree analysis
Reference sequence from Los Alamos HIV Sequence Database was used to evaluate the genetic distance of C2-C3 region of env gene by Kimura 2-parameter model after sequence alignment, and rebuilt the Phylogenetic tree by Neighbor-Joining method (Fig. 1). All the acquired env gene sequence belonged to subtype B with a bootstrap value of 88%. Besides the patient HLJ14, the other sequence clustered together with subtype B strain rl42 separated in China with the bootstrap value of 73%, indicating the 19 samples were close to rl42 strains.
With a high reliability (100% and 84%), HLJ02-HLJ17 and HLJ06-HLJ11 clustered closely to each other, respectively, which meant HIV might be transmitted in each pairs; the dissemination might be caused by sex or close contact.
Comparative analysis on genetic divergence
Divided the acquired sequence into 2 groups according to HLJ16 and HLJ19 were transfused by another blood the blood type, one group had 7 samples (HLJ01, HLJ03, HLJ05, HLJ07, HLJ08, HLJ09 and HLJ10) infected by blood type A and the other had 6 samples (HLJ04, HLJ11, HLJ12, HLJ15, HLJ16 and HLJ19) infected by blood type O. The intraclass genetic diversity and interclass genetic divergence were calculated with Kimura 2 parameter model (Table 3). Mann-Whitney U test showed no significant discrepancy between the genetic distance of interclass and intraclass, indicating these two viruses belonged to the same virus family.
Sequence alignment of V3 region
The Clustal W multiple-sequence alignment program was used to align the whole nucleotide sequences of V3 region, consensus sequence (Fig. 2) was obtained by Consensus Maker, downloaded from Los Alamos National Laboratory. Multiple alignment indicated that except for the CHNHLJ16 having 34 amino acid residues, the V3 domain of all the other 18 clones had 35 amino acid residues.
The tip of V3 loop, consisting of specific tetramer (site of 15-18), was the principal neutralizing epitope of HIV-1. Tetramer presented to be various in different subtype strains. The amino acid 15, 16 and 17 were quite well conserved, mainly composed of GPG, P was replaced by Q at position 16 in the cases of CHNHLJ03 and CHNHLJ10. In CHNHLJ07, it was changed into L at the same location; R and Q often presented at position 18, however it was K in CHNHLJ01, H in CHNHLJ04. Among the 19 clones, 10 (52.63%) were GPGQ, 4 (21.53%) were GPGR, 2 were GQGR, 1 was GPGH, 1 was GPGK and 1 was GLGR, respectively.
Nest-PCR was performed with primers of conservative domains of env gene. C2-V3 region, derived from 19 HIV infectors, was cloned and sequenced. V3 amino acid sequences were analyzed via bioinformatics and phylogenetic tree.
HIV type B', the chief subtype prevalent among drug-taking people in Thailand, exists in almost every district of China. Subtype B' RL42 strain epidemic in Yunnan Province was originated from injecting drug users in Dehong city, neighboring the “Golden Triangle” (Myanmar, Laos, and Thailand).13 There are plenty of people engaging in blood selling in He'nan Province, making He'nan a severe disaster area of HIV infection. The virus subtype of blood donors with HIV-1 infected in Henan Province were HIV-1 B', close correlated with the strain epidemic in Yunnan Province.14 Another survey on virus subtype of partial cases in He'nan Province confirmed the former conclusion.15 It was supposed that subtype B' virus was transmitted into China through drug-taking people, especially in Yunnan Province, then introduced in He'nan Province via different ways. The PBD and transfusion recipients in He'nan Province harbored HIV-1 subtype B, which is similar to the virus in Thailand.16 In our study, 19 HIV/AIDS patients were mainly infected through blood; the epidemic subtype was formed owing to the movement of blood-selling people into Heilongjiang Province. Epidemiologic data show that blood donor Sun once sold his blood and plasma in He'nan Province. From the Phylogenetic tree analysis of env gene segment, we could know that all the gene sequence belongs to subtype B (bootstrap value 88%). Besides the 14th sample, the other sequences cluster together with subtype B strain rl42 separated in China with the bootstrap value of 73%. Therefore, we conclude that these 19 samples from Heilongjiang Province are close to rl42 strain. With a reliability of 100% and 84%, HLJ02 and HLJ17, HLJ06 and HLJ11 cluster closely to each other, respectively, which mean HIV might be spread in each pairs. The two pairs were couples; the dissemination might be caused by close contact.
Divided the acquired sequence into 2 groups, one group had 7 samples transfused by the same blood donor Sun, with blood type A, and the other had 6 samples transfused by another blood donor Huang, with blood type O. Analysis of the intraclass and interclass genetic divergence indicates that these two viruses have no striking discrepancy, belonging to the same virus family.
The V3 loop of the outer membrane gp120 of HIV-1 is the domain with the highest variation rate; however, it is also conservative relatively. Furthermore, the V3 loop is one of the HIV-1major antigenic epitopes17,18 and is the principal neutralizing determinant (PND). The variation of the same virus strain in different individuals is limited. To some extent, the variation of V3 region could not threat the existence of HIV, while variation out of selection pressure makes virus difficult to live. Tetramer on the tip of V3 loop is the functional and immunogenical domain, any little change of which could diminish the antibody combination significantly.19 A study on the V3 sequence of injected drug users in Yunnan Province from 1990 to 1997 showed GPGR was the main type of tetramer.20 While in 2002, the main type was GPGQ, with the proportion raised yearly, this result was in accordance with our previous hypothesis that there was a drift from GPGR to GPGQ motif on the tip of V3 loop of HIV-1 subtype B strain in China.21
However, of all the 19 clones, some are consistent with Thailand B' GPGQ (52.63%), and some are consistent with the United States and Europe subtype B GPGR (21.53%); the other tetramers are: GQGR (2 cases), GPGH (1 case), GPGK (1 case) and GLGR (1 case). It is clear that the mutations of tetramers on the tip of V3 loop of HIV envelope glycoprotein are rare in Heilongjiang Province; however, there presents some other patterns, such as GQGR, GPGH, GPGK and GLGR. These are consistent with former reports in China.15,22
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