High Prevalence of HIV Type 1 Subtype B' Among Heterosexuals in Western Hubei,Central China: Bridging the Epidemic into the General Population

N ational molecular epidemiologic surveys in China conducted in 1996–1998 and 2002–2004 detected a wide variety of HIV-1 strains, including subtype A, B, B' (Thailand variant of subtype B), C, D, F, G, J, CRF01_AE, CRF02_AG, CRF07_BC, and CRF08_BC in China. ^1,2 Among them, HIV-1 subtypes B and B' (Thailand variant of subtype B), ³ and three circulating recombinant forms (CRFs) (CRF07_BC, CRF08_BC, and CRF01_AE) are the most prevalent strains in China. ^1,2 CRF07_BC ⁴ and CRF08_BC ⁵ are distributed widely among injecting drug users (IDUs), while CRF01_AE is prevalent among heterosexuals in China. ^{6 –8} HIV-1 subtype B and CRF01_AE are two major strains found among men who have sex with men in Beijing. ⁹ In contrast, HIV-1 subtype B' is responsible for the epidemics among IDUs in western Yunnan ¹⁰ and former plasma donors (FPDs) ¹¹ in Central China. ^12,13 An estimated 250,000 FPDs were thought to be infected as a result of unhygienic plasma collection from the early to mid 1990s. The most affected regions include Henan, Anhui, Hubei, and Shandong in Central China. ¹⁴

We conducted the molecular epidemiologic study in western Hubei Province in Central China, one of the area in which a large number of FPDs were infected in the early to mid 1990s; we found that HIV-1 subtype B' predominates among FPDs as well as heterosexuals, suggesting that the local epidemic of subtype B' among FPDs is likely responsible for the outbreak among heterosexuals in this particular region in Central China.

Study subjects (n = 62) were recruited between July 2007 and March 2008 in Enshi prefecture in western Hubei Province (Fig. 1, inset). The study was approved by the Committee for Human Research in the Center of Diseases Control (CDC) of Enshi prefecture. The patients' clinical and epidemiologic backgrounds were collected through an interview by the CDC officers. Study subjects (n = 62) include 36 heterosexuals (13 males, 23 females), 31 FPDs (26 males, 5 females), one IDU (female), and one mother-to-child transmission (MCT) case (male, 6 years old). Subjects consisted of 39 adult males with an age range of 29 to 60 years old (mean: 40.1 ± 8.6 years old) and 29 females with an age range of 22 to 52 years old (mean: 36.1 ± 8.6 years old). All 62 specimens were serologically determined as HIV-1 positive. No HIV-2 infections were detected.

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FIG. 1.

Neighbor-joining tree of HIV-1 sequences from western Hubei, Central China based on the 1.9-kb HIV-1 gag-pol region (HXB2: 682–2611 nt). An HIV-1 CRF01_AE (90TH.CM240) sequence was used as an outgroup. Only part of the tree relevant for the present study is shown for simplicity. Bootstrap values greater than 70 are shown at the corresponding nodes. Because of the shortness of the nucleotide sequences, some strains cannot be included in this 1.9-kb tree, including one subtype B'-infected female intravenous drug user (IDU) (ES58) and two CRF01_AE-infected heterosexuals (ES06 and ES68). ES65 (from a female heterosexual) is a URF comprised of subtype B' with a short segment of subtype C (see the text). Closed square (male former plasma donor, FPD); open square (female FPD); closed circle (male heterosexual); open circle (female heterosexual); closed triangle (mother-to-infant transmission, MCT). Inset, map of China on which the study site (the Enshi district in Hubei province) is indicated.

EDTA-treated whole blood specimens were collected and peripheral blood mononuclear cells (PBMCs) were separated on Ficoll-Hypaque density gradient centrifugation (Amersham Biosciences AB, Uppsala, Sweden). Proviral DNA was isolated from PBMCs with guanidine detergent (Invitrogen, Carlsbad, CA). Plasma HIV-1 RNAs were extracted by the QIAamp RNA Blood Mini Kit, Qiagen, Hilden, Germany. The HIV-1 nucleotide sequences of the gag-pol regions were determined either by proviral DNA or plasma HIV-1 RNA. ¹⁵ The polymerase chain reaction (PCR) primers used for this study were as follows. For the 3.0-kb HIV-1 gag-RT region (HXB2: 760–3825 nt), the first PCR primers are 3K1L (5′-GTAACTAGAGATCCCTCAGACCCATTTAGT-3′) and 3K1R (5′-ACATTTATCACAGCTGGCTACTATTTCTTTT-3′) and the second PCR primers are 3K2L (5′-CAGTATTAAGCGGGGGACAATTAGATAGAT-3′) and 3K2R (5′-ATAATTTCACTAAGGGAGGGGTATTGATAA-3′). For the 1.9-kb gag (p17)-protease region (HXB2: 682–2611 nt), the first PCR primers are 172A (5′-ATCTCTAGCAGTGGCGCCCGAACAG-3′) and 505B (5′-ACTCTTGCTTTATGGCTGGGTCC-3′) and the second PCR primers are 174A (5′-CTCTCGACGCAGGACTCGGCTTGCT-3′) and 506B (5′-CCTGACATGCTGTCATCATTTCTTCTA-3′). For the 0.89-kb protease-RT genes (HXB2: 1726–2611 nt), the first PCR primers are 507A (5′-AAGGAACCCTTTAGAGACTATGTAGA-3′) and 503B (5′-TATGGATTTTCAGGCCCAATTTTTG-3′) and the second PCR primers are 508A (5′-GTAAAAAATTGGATGACAGAAACCTTG-3′) and 504B (5′-ACTTTTGGGCCATCCATTCC-3′). PCR products were subjected to DNA sequencing after TA cloning using an automated ABI 3730 DNA sequencer (Applied Biosystems Inc. USA).

Of 62 specimens, a 3.0-kb HIV-1 gag (p17)-RT region (HXB2: 760–3825 nt) from 33 samples, and a shorter fragment of either the 1.9-kb region covering gag (p17)-protease (HXB2: 682–2611) from 40 samples, or a 0.89-kb region covering protease-RT genes (HXB2: 1726–2611 nt) from 62 samples, were successfully amplified and sequenced. All the nucleotide sequences were screened using the BLAST program (National Center For Biotechnology Information, USA) to search for sequence similarities to previously reported sequences in the databases and to eliminate potential laboratory errors. These nucleotide sequences were aligned with reference sequences (http://www.hiv.lanl.gov/content/hivdb/) using the Clustal X 1.83 program. ¹⁶ Phylogenetic analyses were performed by the neighbor-joining method ¹⁷ based on the Kimura two-parameter distance matrix. ¹⁸ The reliability of the topology of the tree was evaluated by bootstrap analyses with 1000 replicates. The analysis was implemented in the MEGA 4.0 program. ¹⁹ Bootscanning analysis was used to assess the recombination breakpoints. ²⁰ The GenBank accession numbers of the nucleotide sequences reported in the present study are HQ197984–HQ198045.

Figure 1 shows the neighbor-joining tree based on the 1.9-kb gag (p17)-protease regions (HXB2:682–2611). The distribution of HIV-1 genotypes based on the 0.89-kb protease region (HXB2: 1726–2611) in western Hubei (n = 62) was as follows: HIV-1 subtype B′ (57/62, 91.9%); CRF01_AE (2/62, 3.2%); subtype B (U.S.-European subtype B) (1/62, 1.6%); CRF07_BC (1/62, 1.6%); and URF (1/62, 1.6%) (Table 1). HIV-1 subtype B′ predominated both among FPDs (29/29, 100%) and among heterosexuals (26/31, 83.8%). Subtype B′ was also detected in one MCT case (ES59) as well as one IDU case (ES58) (Table 1, Fig. 1). We also found that ES65 (female heterosexual) was placed outside of the known HIV-1 genotypes (Fig. 1). Recombination breakpoint analyses revealed that ES65 is a recombinant comprised of subtype B′ (1.2-kb long; HXB2: 1443–2611 nt) and a small segment of subtype C (0.7-kb long; HXB2: 682–1437 nt) in the 1.9-kb gag (p17)-protease region. In contrast, CRF01_AE, a prevalent strain among heterosexuals in China, ^{6 –8} was detected in only two heterosexuals (2/31, 6.5%) in that region (Table 1).

Interestingly, the profile of distribution of subtype B′ by gender shows a male dominance (male:female = 24:5) among FPDs and a female dominance (male:female = 8:18) among heterosexuals (Table 1). This may suggest that HIV-1 subtype B′ is likely being transmitted from the FPD population to the general population through the heterosexual route. Although no apparent epidemiologically linked pairs of heterosexual transmission (for instance, husband–wife) and MCT were reported by interviews, some of the subtype B′ sequences formed clusters with significant statistical supports in the tree: ES12 and 53; ES01, 03 and 05; and ES43, 44 and 49 (Fig. 1).

In conclusion, the high prevalence of subtype B′ among heterosexuals suggests that subtype B′ strains are bridging the spread from FPDs into the general population in China. ²¹ Our findings indicate an urgent need for effective measures to prevent further transmission from high-risk groups to the general population in China.