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Human immunodeficiency virus 1 (HIV-1) evolves by the accumulation of mutations and by recombination. Different subtypes cocirculating in the population of a geographical region provide the basis for the generation and distribution of inter-clade mosaic viruses. Each HIV-1 epidemic in distinct geographical regions and population groups has its own specific characteristics and dynamics. Phylogenetic analyses of nucleotide sequences and HIV genetic variation have become well-accepted as tools to analyze the HIV epidemic, while methologic research continues (4, 9). HIV transmission occurs around the globe due to unprotected sexual exposure, sharing of illegal drug injection equipment, and transfusion of infected blood products. However, illegally paid blood donors were at risk for HIV exclusively in Henan and Hubei Provinces of China for a defined period of time. Until recently, blood drawing equipment was re-used for multiple donors and pooled erythrocytes were transfused back to plasma donors only in those Provinces; this was the only route of infection identified (13, 16). We hypothesized that a higher degree of common source infections expected via this unique blood donation transmission route would be evident as decreased variation in a cross-sectional sampling of HIV sequences, relative to another nearby region lacking transmission via blood donation.
Among other genes, HIV-1 includes gag, and env genes, which code for the precursor proteins of the viral capsid and the virion envelope. The P24 protein is one of the main core protein produced by the gag gene. Sequences in the P24 region are highly conserved, and mutations of epitope sequences may result in immune escape (12). On the other hand, HIV-1 envelope proteins can differ in more than 30% of their amino acids (2). In this study, we determined nucleotide sequences in gag and env genes of HIV-1 isolates from both the Shanghai and Henan regions of China, in order to compare inter-and intra-subtype genetic diversity in the two regions.
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A total of 38 HIV-1 infected plasma specimens were included in the study. Among them, 23 were collected from the Henan Province and 15 from Shanghai. Diagnosis was made by an enzyme immunoassay screening followed by a western blot confirmation. CD4 cell counts and HIV-1 viral loads were measured as previously described (5, 14). Demographic, clinical and epidemiologic information of subjects from the Henan and Shanghai regions are contrasted in Table 1. There were no significant differences between subjects from the two regions except that most from Henan had illegally donated blood, while the major infection risks were blood product transfusions and unprotected sexual intercourse among Shanghai subjects (P < 0.001).
Table 1. Characteristics of HIV-1 infected subjects from the Henan and Shanghai regions of China
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Total viral RNA was extracted from plasma specimens by using a QIAamp Viral RNA Mini Kit (Qiagen Inc., Valentia, Cali.) as previously published (14). Viral cDNA was synthesized by the use of Moloney-murine leukemia virus reverse-transcriptase (Invitrogen, Gaithersburg, MD) at 37℃ for 30 min according to the manufacturer's instructions. The primers used for cDNA synthesis were G01 for gag and E01 for env respectively. A nested-PCR was used to amplify the gag P24 region by using G01 and G10 for the first PCR round. Two fragments were amplified in the second PCR round by using primers G20, G55, G70 and G25, respectively. The 50 μL of reaction mixture contained 25mmol/L MgCl2, 100nmol-/L dNTPs, 1.25U Pfu polymerase, 25pmol/L each primer and 10μL cDNA templet (first round) or 2 μL amplicon (second round). Both first and second rounds of the PCR reaction were run using a three-step PCR procedure which included: ⅰ) an initial denaturation at 95℃ for 3 min; ⅱ) 35 cycles of 95℃ for 30 s, 52℃ for 1 min and 72℃ for 3 min; and ⅲ) a final extension at 72℃ for 10 min. The same PCR protocol was used for env gp41 IDR amplification by using E01 and E10 as the first round primer set and E170 and E35 as the second round primer set. Sequences of the above primers were published previously (11). Final PCR products included two fragments with 680 and 480 base pairs each for the gag gene and a 540 base-pair fragment for the env gene.
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DNA fragments amplified by PCR were purified by using a QIAquick PCR Purification Kit (Qiagen Inc., Valentia, Cali.) according to the manufacturer's instructions. A reaction mixture including 200 ng of purified DNA fragment and 5 pmol/L of primer was used for cycle sequencing on an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, Cali). Two orientated sequences were assembled and analyzed through blasting HIV-1 databases (http://hivweb.lanl.gov). Nucleotide sequences were aligned by the Clustal X program (version 1.81). Phylogenetic analysis was inferred using the Phylogeny Inference Package (PHYLIP, version 3.6a3) (http://evolution.genetics.washington.edu/phylip/general.html) (1). Phylogenetic trees were constructed by the neighborjoining method using DNA distance matrices, calculated with the Kimura two-parameter model. Robustness of the tree was evaluated by bootstrap analysis on 500 replicates. Phylogenetic trees were edited using a TreeView (version 1.6.5). The sequence of the reference HxB strain was used as a standard for determining the number and sites of polymorphisms (10). Genetic distances values were obtained by a Kimura two-parameter model. Representative sequence of different subtypes and of B subtype viruses from different countries was used as reference for inference of subtype differences and within-B subtype variation, respectively.
Patients and specimens
Reverse transcription, PCR and sequencing
Amplicon sequencing and phylogenetic analysis
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Target gene fragments were amplified and sequenced from all specimens. Sequences of gag and gp41 IDR were used for subtype determination in comparison to reference isolates representative of the A, B, C, D, F, G, H clades, and the circulating recombinant forms CRF01 and CRF02, respectively (Fig. 1 A). Among the 38 isolates studied, 31 (81.6%) were subtype B, 1 (2.6%) was subtype A, 5 (13.2%) were CRF01_AE, and 1 (2.6%) was CRF02_AG. All 23 (100.0%) sequences from Henan and 8 of 15 (53.3%) from Shanghai were subtype B. Comparison of sequences in the gag P24 region indicated that all Henan and most Shanghai subtype B isolates clustered with a B'-Thai strain representing the most prevalent B-clade isolates in China (3). Only two subtype B isolates from Shanghai (SH06 and SH14) were close to subtype B sequences from the United States and Australia (7) (Fig. 1.B). Two recombinant viruses (CRF01_AE and CRF02_AG) were recognized in Shanghai, indicating multiple HIV-1 viruses have been circulating in the region, which is consistent with previous findings (6, 8, 15).
Figure 1. Phylogenetic analysis of the HIV-1 gag P24 coding region of 38 isolates from Henan and Shanghai by the neighbor-joining method. Genetic distances were calculated by the Kimura two-parameter model. The scale indicates the relative phylogenetic distance. Bootstrap values were generated from 500 bootstrap replicates, and values greater than 70% are labeled. A: HIV-1 subtyping analysis. Reference isolates from clades A, B, C, D, F, G, H, J, K, and recombinant forms CRF01_AE, CRF02_AG, CRF07_BC and CRF08_BC were downloaded from the HIV-1 databases (http://hiv-web.lanl.gov). The bar represents 1% genetic distance. B: Nucleotide sequences in the gag P24 coding region of subtype B isolates. The two letters, US, CN, AU, JP, KR, TW, DE, and GB, following accession numbers indicated that these isolates were originally recovered from the U.S., China, Australia, Japan, Korea, Taiwan, Germany and England, respectively. The bar represents 1% genetic distance.
Among all 31 subtype B isolates, the mean intrasubtype sequence genetic distance in the gag p24 region was 3.6%, with a range from 0.4% to 6.7%. Inter-sequence genetic distance in the gag p24 gene in the 23 Henan sequences averaged 3.2%, in comparison to an average of 3.4% for the 8 isolates from Shanghai (P > 0.05) (Table 2). In contrast, in the env gp41 IDR region, all subtype B isolates had an average intra-subtype distance of 4.4%, ranging from 2.0 to 7.6%. For the 23 isolates from Henan, the mean genetic distance in the env gp41 IDR was 3.7%, which was significantly less than the mean genetic distance in subtype B isolates from Shanghai (4.6%, P=0.038) (Table 2). When nonsubtype B isolates were included in the analysis, the mean sequence variation in Shanghai reached 9.3%, which is also significantly more diversified than in the Henan sequences (Table 2). These data demonstrate that HIV-1 isolates from Henan were solely the B subtype and relatively more conserved in env gp41 IDR region sequences than the Shanghai isolates.
Table 2. Nucleotide sequence variation between gag and env sequences from the Henan and Shanghai regions of China
This comparison of sequence variation in the env gp41 region between HIV-1 isolates recovered from Henan and Shanghai indicates that isolates from Shanghai were more heterogenous than those from Henan. This was attributable to more subtypes, as well as more sequence variation within env subtype B sequences from Shanghai. There are several potential reasons for differences in diversity in HIV sequences across groups in such cross-sectional analyses. One cause not controlled for in this report involves differences in the time since the individuals studied were infected, given the ongoing sequence change within infected persons. Other mechanisms for differences in genetic variation may include differrences in the relative replication rate/substitution rate of viruses, and the relative degree of superinfection and recombination. The difference noted here in populations with distinct transmission routes suggests that the difference in number of individuals infected independently with the same genetic variant needs to also be considered.
Subsequent studies are needed to confirm this hypothesis that sequence diversity differs in Henan versus Shanghai isolates because of the unique way in which there were common infection sources for many individuals in Henan who each received the same infecting inoculum when donating blood. Such confirmatory studies should compare viruses from subjects known to be infected for similar durations and also evaluate whether the rate of genetic change over time in individuals was similar in the different regions. If the hypothesis suggested by the data in this report is confirmed, the degree to which there are common sources of infection in a population would be established as another explanation for differences in genetic diversity of HIV among different regions or population groups.
Nucleotide sequence accession numbers. The partial gag and env gene sequences included in the study have been deposited in the GenBank sequence database under the accession numbers AY934644-AY934680.