The HIV-1 epidemic in China has demonstrated both a complicated diversity and geographic characteristics. The second National HIV Molecular Epidemiology Survey revealed that HIV-1 subtypes A, B, C, CRF01_AE, CRF-BC and other unusual subtypes have been found in China. HIV-1 CRF_BC is the main epidemic component (50.2%), and subtype B (31.66%) and CRF01_AE (15.54%) take second and third places, respectively (http://shgy.jhgl.org/shownews.asp?newsid=893).
Heilongjiang province is located in the northeast of China, neighbored with the far eastern part of the Russian Federation. Since the first AIDS case was officially reported in 1993, the spread of HIV/AIDS in Heilongjiang province has been comparatively slow. By the end of 2006, 385 HIV-1-infected individuals with 106 AIDS cases have been reported (www.hlj.gov.cn).
To clarify the molecular epidemiology and the genetic diversity of HIV-1 in Heilongjiang province, we analyzed the sequence features of the gag, pol and env gene regions of HIV-1 isolates circulating locally.
Blood samples from 30 HIV-1-infected or AIDS cases were obtained from Heilongjiang province Center for Disease Control and Prevention, where the individuals presented from 2004 to 2006. Twenty-nine patients were from eight different regions of Heilongjiang province, whereas the location of the other patient was unknown. Genomic DNA was extracted from the peripheral blood mononuclear cells. Nested PCR assays were used for the individual amplifications of the gag (672 bp, nt 836–1507, HXB2 numbering), pol (835 bp, nt 2592–3426, HXB2 numbering) and full-length env regions. The PCR products of gag and pol were purified and sequenced directly. The full-length env gene was cloned and sequenced. The GenBank accession numbers of the HIV-1 sequences reported here are EU131787-EU131871 and AY905493-AY905497.
The sequences of gag, pol and C2-V3 (nt 6828–7372, HXB2 numbering) were compiled, aligned and adjusted with the reference sequences from Los Alamos database (http://www.hiv.lanl.gov/content/hiv-db/SUBTYPE_REF/align.html and ) using the CLUSTAL W program . The Neighbour-joining trees were made using DNAMAN version 4.0 (Lynnon BioSoft, Quebec, Canada). The reliability was estimated by 1000 bootstrap replications . The intergenetic distances of gag as well as the env region among Heilongjiang subtype B strains and mean pairwise nucleotide distances of the C2-V3 region between Heilongjiang B strains and the reference strains of other provinces were measured using the Kimura two-parameter model supplemented in MEGA 3.1 .
A total of 90 gene sequences including 30 each of gag, pol and env regions were obtained. Phylogenetic analysis based on gag, pol and C2-V3 sequences showed that 29 (96.7%) of the 30 samples had concordant subtypes in the three gene regions. Among them, 28 sequences showed subtype B (96.6%), whereas one (3.4%), CNHLJ_H06054, proved to be a intersubtype recombinant, CRF07-BC. However, one sample, CNHLJSH06059, was grouped in the same cluster with subtype CRF01_AE in the gag region and in the same cluster with subtype B in the pol and env (C2-V3) regions (Fig. 1).
The nucleotide divergence of the env region (mean, 6.9 ± 0.3%) was significantly higher than that of the gag region (mean = 4.1 ± 0.5%) among Heilongjiang B strains. The genetic distances of the C2-V3 region between Heilongjiang B strains and reference strains of Guangxi, Henan and Yunnan provinces of China were 8.3 ± 0.8%, 9.7 ± 1.0% and 15.3 ± 1.4%, respectively, suggesting the lowest divergent between Heilongjiang and Guangxi provinces.
BootScan analysis further confirmed that sample CNHLJSH06059 was a CRF01_AE/B recombinant. The recombination was structurally different from those reported previously as the pol and env genes of this recombinant were derived primarily from subtype B, and the gag region was derived from CRF01_AE. The blasting result indicated that there is the highest genetic homology in the gag region (CRF01_AE) between CNHLJSH06059 and one HIV-1 isolate from Khabarovsk of the Russian Federation, U93610 (97.1%).
In this study, for the first time, we found two recombinants, CNHLJ_H06054 (CRF07_BC) and CNHLJSH06059 (CRF01_AE/B), in Heilongjiang province. As the CRF07_BC strains were isolated first and distributed among injecting drug users in Xinjiang province and some HIV-1-infected individuals living in Heilongjiang province were originally from Xinjiang province, we believe that monitoring the migrated populations is extremely important for the control of the spread of HIV in this area. Furthermore, as there was active communication both economically as well as culturally between the two border regions, Heilongjiang province of China and Khabarovsk region of the Russian Federation, the fact that there is the highest homology between the sequences from the two regions might suggest there are potential transmission routes of HIV-1 between the two countries.
We are grateful to Professor Lu S from the University of Massachusetts Medical School for carefully reading the manuscript. This work was supported by the Natural Science Foundation of China (NSFC grant 30471608) and the Natural Science Foundation of Heilongjiang province (grant ZJY0601-01).
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