Phylogenetic analyses of globally circulating HIV strains have identified a major group (M) of 10 different sequence subtypes (A to J) (13
) exhibiting sequence variations in the envelope protein of up to 24%, in addition to group O viruses, which differ from group M viruses by more than 40% in some reading frames (22
). Although the extent of global HIV-1 variation is well defined, little is known about the biological consequences of this genetic diversity and its impact on the design of candidate vaccines.
Due to a lack of well-characterized molecular references, little information is available so far regarding the biological, pathogenic, and immunological properties of subtype C viruses. Regarding the complex situation in developing countries, where multiple subtypes of HIV-1 are known to cocirculate, extensive molecular epidemiological studies are required to identify representative local virus strains. Particularly in the light of the potential for recombination (3
), full-length viral sequences are necessary for an optimal characterization of relevant virus strains. Accordingly, this study describes the identification and phylogenetic characterization of a clade C HIV isolate representing one of the most prevalent virus variants circulating throughout China.
Clade C HIV-1 strains play a leading role both in the total number of infected people and in the high incidence of new infections, especially in South America and Asia. Currently, there is an increasing number of nonrecombinant molecular clones and a few mosaic genomes available for viruses other than B. Regarding clade C HIV-1 viruses, only a few nonrecombinant representatives and four A/C recombinants have been published so far, all of them originating from Africa, South America, or India (11
, Luo et al., Letter).
With the exception of Thailand, limited information has been available until recently on the distribution and molecular characteristics of HIV-1 strains circulating throughout Asia. The World Health Organization estimates that South and Southeast Asia have the highest rate of HIV spread and will soon become the world's largest HIV epidemic region. China has very similar social and economic conditions and direct ethnic and economic connections to these regions. Since early 1995, a rapid increase in HIV infection was clearly seen in many provinces of China. Compared with the cumulative total of 1,774 cases of HIV infection and AIDS detected from 1985 to 1994, 1,421 cases were detected in 1995 and more than 4,000 cases were detected in 1997 alone. The World Health Organization estimated that there would be more than 400,000 HIV infections in China by the end of 1997, with an estimated 6,400 cumulative deaths and 4,000 people dying of AIDS in 1997 alone. In the recent national HIV molecular epidemiology survey, it was found that the prototype B and B′-subtype Thai strains in Dehong (15
) were spread to central and eastern China by drug users and contaminated blood and plasma collection services. The subtype C strains of Yunnan were transmitted along the drug-trafficking routes to central western and northwestern China. Today, subtype C HIV-1 strains account for the majority of HIV-1 infections among IDUs in China.
In this report, we show for the first time that the prevalent HIV-1 strains transmitted among the IDUs in the northwestern provinces of China represent C/(B′) interclade mosaic strains. This study was based on genotyping the C2V3 envelope coding region amplified from proviral DNA isolated from PBMCs of more than 100 HIV-1 clade C-positive IDUs. The C2V3 nucleotide distances among the different virus isolates were in a range of 2 to 3%, indicating that the epidemic caused by the clade C HIV strains in this area is still very young (1
; Luo et al., Letter). Phylogenetic tree analysis based on the C2V3 region of a representative virus strain suggested that clade C HIV-1 strains circulating throughout China are closely related to those of Indian origin (21
; Luo et al., Letter) and distinct from clade C viruses isolated in South America and Africa (11
; Luo et al., Letter).
Detailed molecular characterization of a virtually full-length genome representing the most prevalent species of clade C HIV-1 strains circulating in China suggested several intersubtype recombination events between clade C and (B′)-Thai sequences. RIP analysis indicated a total of 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3′ end of the vpu gene, and (iii) in the nef open reading frame. This finding has been strongly supported by establishing distinct phylogenetic trees based on sequences flanking the recombination points.
The two parental (B′)-Thai and clade C HIV-1 subtypes had been reported earlier to cocirculate among IDUs in southwestern China, therefore clearly representing a potential reservoir for the observed interclade recombination (Luo et al., Letter; Ma et al., Abstract). The reason why Chinese B′ strains exclusively seem to exhibit homogeneous genome structures, whereas all so-called clade C virus strains identified in this area are interclade mosaics so far, remains unclear (1
). However, the isolated appearance of C/(B′) chimeras in the northwestern provinces of China may be suggestive of a founder virus effect.
RIP analysis and phylogenetic bootstrapping of clade C sequences obtained from various independent IDUs living along the northwestern drug trafficking route from Yunnan to the northwestern Sichuan and Xinjiang Provinces revealed almost identical recombination points for all the analyzed subtype C/(B′) strains. This suggests that the observed recombination events had already occurred before this virus started to spread. Strikingly, only recombinant C/(B′) strains seem to travel along the drug-trafficking route to the far northwestern autonomous region, whereas the B′ parental strains are preferentially found in the Southwest (1
It is noteworthy that the C/(B′) recombinants found along the northwestern drug-trafficking route differ from the very recently reported C/(B′) recombinants isolated from IDUs living in the area of Guangxi neighboring the Yunnan Province and Myanmar (26a
). The Guangxi-derived C/(B′) chimeras seem to share only part of the B′ sequence in the central portion of the RT gene whereas the breakpoints in the p17/p24 overlap region, in the p7/p6/p6* coding region, in the vpr/vpu
genes, and in the 3′ portion of the nef
gene are unique to those in the viruses found along the drug-trafficking route from Yunnan to Sichuan, Gansu, Ninxia, and Xinjiang Provinces. These data add clear evidence to previous observations that suggest two different routes of HIV subtype C/(B′) spread throughout China: one from Yunnan through the northwestern Sichuan, Gansu, Ninxia, and Xinjiang Provinces and across the border into Kasakhstan, and one spreading from Myanmar across the border into Yunnan and then through Guangxi and Hongkong to western countries. Taking these data into account, it seems as if each drug-trafficking route is associated with a different and relatively homogeneous HIV-1 recombinant, underlining the linkage between intravenous drug use, needle sharing, and HIV spread in China.
Based on these observations, it is tempting to speculate on whether interclade recombination events may confer selective advantages to the mosaic viruses. Each of the recombination events shown for 97cn54 might contribute to a more efficient transmission of the C/(B′) chimera compared to the proposed B′ parental virus. At least some of these questions may be answered by the availability of the B′ parental virus, the knowledge of breakpoints, the possibility of reconstituting replication-competent molecular clones (in process), and the existence of a wide variety of test systems allowing us to analyze distinct viral functions in vitro, in cell culture, and in appropriate small-animal models.
Finally, the high incidence of new infections in combination with the homogenous seed virus following a single and well-documented transmission route may suggest that IDUs from the northwestern and southwestern area form a potential high-risk population group for safety and efficacy vaccine trials in China. Initial analysis has demonstrated that probably about 50% of the CTL epitopes in Gag and RT are completely shared by prototype B variants and the C/(B′)-Thai interclade mosaics. These observations clearly predict a considerable cross-clade CTL reactivity, suggesting that the functionally and immunologically conserved HIV-1 proteins are strong potential candidates for future vaccine constructs.
In summary, this is to our knowledge the first report of a cloned virtual full-length C/(B′) chimeric HIV genome, which simultaneously represents one of the most prevalent subtype C virus strains from China. The reported data will be useful as a reference for future studies on the genetic diversity of HIV. Moreover, the established and carefully characterized clone may serve as a basis for the generation of subtype-specific immunological reagents and the development of candidate vaccines based on regional virus strains (27
). Finally, the homogeneous seed virus with a single transmission route within a well-characterized population group suggests that this area is a good potential site for safety and efficacy vaccine trials.