In China, home to 1/5 of the world's population, HIV has spread to all 31 provinces, regions, and municipalities 1 and is currently moving into new groups of the population. 2 Epidemiologic surveillance studies in China reveal that transmission of HIV-1 by injecting drug users (IDUs) is the predominant route of HIV transmission, with prevalence rates >70% among this group of patients, in some border communities in Xingjiang, Yunnan, and Guangxi provinces. 3 Heterosexual transmission has been on the increase in recent years, as has been shown with increased prevalence in patients with sexually transmitted diseases. 4 HIV infection by HIV-contaminated blood and blood products has been reported in different parts of China. 4
Shanghai, located in the east of China, is one of the largest cities with 16.74 million inhabitants and receiving about 4 million migrants annually, some of who come with HIV infection, thus increasing the HIV-1 epidemic in this city. Shanghai has been ranked the 9th for its HIV-1 epidemic in China. 4 The main modes of HIV-1 transmission in this city are reported to be by IDUs (35%), sexual contacts (33%), and the use of contaminated blood products (20%). 5,6 While HIV-1 subtype information in Shanghai is sparse, several HIV-1 subtypes (A, B, B′, C, D, F, and G), 3 circulating recombinant forms (CRFs) (CRF01_AE, CRF07_BC, and CRF08_BC) as well as HIV-2 have been identified in different parts of China. 7,8 Information on the HIV-1 subtype distribution in different risk groups in China is sparse. Although some heterosexuals in Shanghai were found to be infected with HIV-1 subtype C, little is known on the HIV-1 genetic diversity among different risk groups in Shanghai. To track the HIV-1 genetic distribution in this city, the subtypes infecting different risk groups must be studied.
Amino acid substitutions that lead to drug resistance in patients during treatment with protease inhibitors (PIs) have been extensively characterized into primary (major) and secondary (minor) mutations. 9–11 Primary mutations significantly decreases sensitivity to ≥1 PI drugs, 9,10 while secondary mutations may not result in a significant decrease in sensitivity but are associated with an increase in viral fitness (replication capacity). 9,12 Thus, the appearance of a primary mutation in a genome already containing secondary mutations could influence the speed at which highly resistant viruses are selected during therapy. Information on PI resistance–associated mutations in HIV-1-infected patients in China is lacking. To establish successful treatment strategies for HIV-1 patients in China, there is the need to characterize the natural polymorphisms associated with PI resistance in drug-naive HIV-1-infected individuals. The present study has examined the genetic diversity and natural polymorphisms associated with PI resistance of HIV-1 strains infecting residents of Shanghai, China.
MATERIALS AND METHODS
Study Subjects and Specimens
Blood samples from different risk groups who live in Shanghai were collected between 1999 and 2001, and the plasma samples were stored at −20°C. All plasma samples were screened for antibodies to HIV-1 by HIV-1/HIV-2 enzyme-linked immunosorbent assay (Vironostika, Organon Teknika, Oss, The Netherlands) and confirmed by Western blot HIV 2.2 (Genelab Diagnostics, Singapore). Virus loads were measured by NASBA assay (NASBA HIV-1 QT, Organon Teknika) or bDNA assay (Quantiplex HIV-RNA, Chiron Diagnostics) according to manufacturers' instructions. The 40 samples analyzed were from 30 men, aged 12–60 years (average: 30 years), and 10 women, aged 3–60 years (average: 30 years). Of the 40 samples studied, 19 were from patients who acquired their infections through sexual contacts (17 heterosexuals and 2 homosexuals), 17 patients through contaminated blood products (hemophiliacs), 2 from children born to HIV-1-infected mothers, and 2 from IDUs. Of the 40 subjects studied, 8 patients were symptomatic and 32 were asymptomatic. All the samples analyzed were from drug-naive patients except for 3 patients (2 infected through heterosexual transmission and 1 through blood transfusion) who had received indinavir treatment 2 to 3 months prior to blood collection. Informed consents were provided after assuring anonymity. The samples analyzed are listed in Table 1 and were assigned names according to UNAIDS nomenclature denoting the year of sampling, the country of origin (2-letter code), and the patient number. 13
RNA Extraction and RT-PCR Amplification
RNA extraction from plasma was performed as previously described. 14 Briefly, 100 μL plasma, 900 μL lysis buffer (120 g GuSCN, 100 mL of 0.1 M Tris-HCL, pH 6.4, 22 mL of 0.2 M ethylene diamine tetra-acetic acid [EDTA], pH 8.0, and 2.6g Triton X-100) and 40 μL silica (SiO2) were homogenized in an Eppendorf tube and shaken for 10 minutes at RT, followed by washing twice with 1 mL of washing buffer (120 g GuSCN, 100 mL of 0.1 M Tris-HCL, pH 6.4), twice with 70% ethanol, and once with acetone. RNA was eluted from silica with 0.05 M Tris-EDTA buffer, pH 7.4. Each wash step mentioned above was carried out by a short vortex and centrifugation.
One-tube reverse transcriptase polymerase chain reaction (Access RT-PCR System, Promega) was performed according to the manufacturer's recommendations for amplification of gag, pol, and env genes. With outer primer pair ED5/ED12 and inner primer pair ES7/ES8 15 for env gene, and outer primer pair H1G777/HIP202 and inner primer pair H1Gag1584/g17 16 for gag gene, the HIV-1 env (C2V5) and gag (part of p24 and p7) genes were amplified by nested PCR, as previously described. 15,16 Alternatively, outer primer pair ENV1/ENV2 followed by inner primer pair ENV3/ENV4 17 was used for amplifying env gene. For protease gene region, the outer primer pairs NYUPOL6 (5′-AGGGAAGGCCAGGGAATTT-3′, 2114-2132nt, HXB2), or NYUPOL7 (5′-AGGAAATTTTCCTCAGAGCAG-3′, 2125-2144nt, HXB2) and NYUPOL8 (5′-CTTCTGTCAATGGCCATTGT-3′, 2615-2634nt, HXB2), and inner primer pair NYUPOL9 (5′-TCCTTTAACTTCCCTCAAATCACT-3′, 2241-2264nt, HXB2) and NYUPOL10 (5′-CTGGCACGGTTTCAATAGGACT-3′, 2556-2574nt, HXB2) were used to amplify 297 bp of the protease gene (corresponding to positions 2258-2552nt, HXB2). The first-round PCR was carried out by 1-tube RT-PCR in 25 μL of RT-PCR mixture containing 3 μL of RNA, 5 μL of AMV/TFL 5X buffer, 1 μM of each outer primer, 2.5 m M of each dNTP, 2.5 m M MgSO4, 0.5 U of AMV RT, and 0.5U of Tfl DNA polymerase. The amplification was carried out in a thermal cycler (MJ Research, Inc.) for first RT reaction (45 minutes at 48°C, 2 minutes at 94°C), followed by 35 cycles (1 minute at 94°C, 1 minute at 45°C, and 1 minute at 72°C), and a final extension for 7 minutes at 72°C. From the first-round PCR products, 2 μL as a template was used for the nested PCR with inner primers and the same cycling condition as the first round-PCR was used.
Determination of HIV-1 Subtypes and Recombination Forms
The PCR fragments were separated on an agarose gel and purified with the QiaQuick Spin kit (QIAGEN, Inc.) according to the manufacturer's recommendations, followed by direct sequencing on an automated DNA sequencer (373XL; Applied Biosystems, Foster City, CA). The sequences were aligned with previously reported HIV-1 strains of various subtypes from the Los Alamos database. Multiple alignments were performed automatically by CLUSTAL X 18 with minor manual adjustments. Gaps were excluded from all sequence comparisons. Kimura 2-parameter method was used for the determination of the evolutionary distance. The reliability of the branching patterns was assessed by the bootstrap analysis with 1000 replicates. Phylogenetic tree analysis was performed using neighbor-joining method implemented by TREECON (Treecon for Windows, version 1.3). 19 All the nucleotide sequences obtained were screened by the HIV-BLAST (http://hiv-web.lanl.gov/content/hiv-db/BASIC_BLAST/ basic_blast.html) to search for sequences in the databases and rule out potential laboratory errors.
To determine the presence of recombination breakpoints, 2.6-kb gag-RT sequence was amplified by RT-PCR with outer primer pair HG00 (5′-GACTAGCGGAGGCTAGAAGG-3′, 764-783nt, HXB2) and NYUPOL2 (5′-TCCGCCAATTCTAATTCTGCTTC-3, 3441-3463nt, HXB2), followed by inner primer pair HGHMA1 (5′-TGGGTGCGAGAGCGTCAATATT-3′, 791-812nt, HXB2) and NYUPOL4 (5′-TACTATGTCTGTTAGTGCTTTGG-3, 3406-3428nt, HXB2). The amplification was accomplished by long PCR protocol (Expand long template PCR system; Roche Diagnostics, Indianapolis, IN) according to the manufacturer's recommendation. Cloning of 2.6-kb PCR product was performed by TOPO TA Cloning Kit for Sequencing (Invitrogen Corp., Carlsbad, CA) and sequencing was performed by primer walking protocol. Simplot 3.2 software 20 was used to identify the recombination breakpoints. The aligned DNA sequences were translated into amino acids using GeneRunner (Generunner for Windows, http://www.generunner.com).
Fisher exact test implemented by GraphPad Prism software (San Diego, CA) was used for statistical analysis of the difference in genetic variation in PR genes between subtype B/B′ and non-B strains, and between different risk groups.
HIV-1 Genetic Subtypes and Recombinant Strains Circulating in Shanghai
Of the 40 plasma samples analyzed, viral RNA from 38 samples was successfully amplified by RT-PCR on env, gag, and pol genes, DNA sequenced, and phylogenetically analyzed. Table 1 summarizes the genetic characteristics of the samples analyzed and Figure 1 shows the phylogenetic relationship of the various sequences studied. Four group M subtypes (A, B, B′, and C), 2 CRFs (CRF01_AE and CRF08_BC), and a new intersubtype recombinant (CRF01_AEenvBgagUpol) were identified. Sixteen of the 17 samples obtained from the hemophilia patients were successfully amplified and subtype analysis revealed infection in all 16 samples with HIV-1 subtype B′ (the Thai-B cluster within subtype B) (Tables 1 and 2 and Fig. 1). Sixteen samples from the 17 patients infected by heterosexual contact were successfully amplified and subtype analysis revealed a broad HIV-1 diversity characterized by several subtypes and recombinants (Tables 1 and 2 and Fig. 1). The CRF01_AE from the heterosexual subjects cluster with the Thai CRF01_AE references while the subtype B heterosexual and homosexual sunjects clustered with subtype B references from France and the United States (Fig. 1). The 2 homosexual patients, 01CNSH5 and 01CNSH29, were infected with HIV-1 CRF01_AE and subtype B, respectively. The 2 IDU patients, 01CNSH4 and 01CNSH30, were infected with HIV-1 CRF08_BC, which is reported to be the predominant CRF among IDUs in China. 21,22 These 2 IDU sequences also clustered phylogenetically with the 2 B′C recombinant references from the Guangxi province in China (Fig. 1). The 2 cases of mother-to-child transmission in the children, 00CNSH1 and 01CNSH10, analyzed were infected with HIV-1 CRF01_AE and subtype B′, respectively (Fig. 1).
Infection due to CRFs accounted for 32% (12/38) of the infections in the samples analysis. These CRFs included 8 CRF01_AE, 3 CRF08_BC, and 1 new intersubtype recombinant (CRF01_AE/B-like). Both the CRF01_AE and CRF08_BC are known to be the predominant CRFs in other parts of China. 5,7,21–23 Because these CRFs were predominantly identified in the heterosexual risk groups, the CRF01_AE in particular is believed to have been introduced into the Chinese population from Thailand through heterosexual contacts. 23 To determine the geographic region from where the CRF08_BC would have been introduced, we characterized its recombinant breakpoint and compared it with other CRF08_BC strains previously identified in other provinces in China. For this, the sample, 01CNSH4, infected with the CRF08_BC strain, was subjected to recombination breakpoint analysis. Bootscanning plot revealed that the same recombination breakpoints in the 2.6-kb gag-RT region were shared with virus 97CNGX6F, a CRF08_BC from Guangxi province, and virus WS002, a CRF08_BC from Yunnan province, both in the southwest of China (Fig. 2). 21,22,24 These breakpoints occurred at 4 positions in the gag-RT region (around 1423nt, 1468nt, 2895nt, and 3195nt, HIV-1 HXB2). Additionally, 1 patient (01CNSH7), infected by heterosexual contact, was identified with a new intersubtype recombinant strain (CRF01_AEenv BgagUpol). Bootscanning analysis revealed a B subtype in the 2.6-kb gag-RT region but with no recombination breakpoints with other reference subtypes (data not shown). Further analysis of 01CNSH7 by full-length genome sequencing is currently being performed to fully characterize this infecting strain.
Protease Inhibitor Resistance–Associated Mutations
The 297-bp nucleic acid sequences of the PR gene of 37 samples were translated into the corresponding 99 amino acids and analyzed for 19 mutations that are relevant to in vivo HIV-1 resistance to PIs such as saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir. 10,11 These mutations involve 19 amino acid substitutions at 8 primary and 11 secondary PI resistance–associated mutation sites. 10,11,25
One and 7 codons with primary and secondary amino acid substitutions, respectively, were identified in the 37 samples analyzed (Table 3). The 1 primary mutation occurred at position 30 (D30N) in a PI-naive hemophilia patient with HIV-1 subtype B infection (01CNSH14) from a sample obtained in 2001 (Table 1). This patient's viral sequence further revealed 4 other secondary mutations including M46I, L63P, A71T, and V77I. Overall, 7 secondary mutation sites were identified in 35 of the 37 protease sequences analyzed (95%). These 7 secondary mutations were L10V, K20R, M36I, M46I, L63P, A71V/T, and V77I. The frequencies of these substitutions were 3% (L10V), 8% (K20R), 38% (M36I), 5% (M46I), 57% (L63P), 14% (A71V/T), and 46% (V77I) (Table 4). No primary mutations were identified in the sequences from 3 indinavir-treated patients. Furthermore, the 2 samples that completely lacked PI resistance–associated mutation were from a homosexual (01CNSH29) and an IDU (01CNSH30) patient (Table 3). Because the 2 main risk groups, hemophiliacs and heterosexuals, were infected mainly with the subtype B′ or non-B subtype, respectively (Tables 1 and 2), we analyzed the natural polymorphisms in these 2 groups of viruses to reflect the patient risk groups. The PI resistance–associated mutations analysis revealed that the hemophilia group was characterized by a higher frequency of substitutions at codons V77I (93%), L63P (87%), and A71V/T (27%), compared with the heterosexual group, in which the frequencies of substitution at these codons were 19, 31, and 6%, respectively. Conversely, the frequency of substitution at codon M36I in the heterosexual group was higher (69%) than that (7%) in the hemophilia group. These differences in the PI resistance–associated substitution frequencies between the hemophilia and heterosexual groups were statistically significant at 4 codons: 36I (P = 0.0006), 63P (P = 0.0032), 71V/T (P < 0.05), and 77I (P < 0.0001) (Table 4). No statistically significant differences were found at the remaining 4 PI sites: 10V (P = 1.0), K20R (P = 0.2258), D30N (P = 0.4839), and M46I (P = 0.4839). When all the samples were categorized as subtype B/B′ and non-B subtypes, regardless of the risk groups they infect, we observed that mutations at codons L63P (90%), A71V/T (29%), and V77I (81%) were more frequent in subtype B/B′ than in non-B subtypes with mutations at codons L63P (13%), A71V/T (0%), and V77I (0%). Conversely, mutations at codons K20R and M36I were more frequent in non-B subtype sequence, with 19 and 81% than in subtype B/B′ sequences, with 0 and 5%, respectively. Of note was that the K20R mutations were only restricted to the CRF01_AE (Table 3). The differences in the frequencies of PI resistance–associated substitutions between the subtype B/B′ and non-B subtype strains were statistically significant at these codons K20R (P < 0.05), M36I (P < 0.0001), L63P (P < 0.0001), A71V/T (P < 0.05), and V77I (P < 0.0001).
Multiple-mutation analysis revealed that only 1 sample (3%) was found to carry a primary mutation and that single, dual, triple, and quintuplet mutations accounted for 38, 35, 16, and 3%, respectively, in the samples analyzed. The mutational sites in the respective subtypes and risk groups are listed in Table 3. As observed, the frequency of mutations was not restricted to a particular subtype. For example, some subtype A, B′, C, AE, and BC strains had single mutational sites. Similarly, some subtype A and B/B′ strains like CRF01_AE had dual mutations, though the mutations were not at the same sites. More multiple mutations (≥2 mutations) were observed for subtype B/B′ (76%) than non-B subtype (31%), suggesting significant differences in the natural polymorphisms between subtype B/B′ and non-B subtype strains (P = 0.009) (Table 3).
Molecular epidemiology studies in China indicates that the most prevalent HIV-1 strains are subtype B′ (44%), followed by C (29%) and CRF01_AE (13%). 5,7Figure 3 shows the various subtype distributions in different provinces in China. 7 The B and B′ strains are widely distributed throughout eastern and southern China. Subtype C (usually CRF08_B′C) virus is concentrated in most areas of southeastern and western China, such as Yunnan, Guangxi, Gansu, Ningxia, and Xingjiang provinces. 7,26 HIV-1 CRF01_AE strains are mostly distributed in southeastern coastal China, such as Fujian and Guangdong provinces. 7,26 Our present study reveals the presence of a broad HIV-1 genetic diversity in Shanghai characterized by 4 subtypes (A, B, B′, and C) as well as CRF02_AE, CRF08_BC, and a new intersubtype recombinant, CRF01_AEenvBgagUpol (Fig. 3). The most prevalent subtype identified in this study was subtype B/B′ (47%) followed by CRF01_AE (21%). The fact that the hemophiliac patients studied were infected with subtype B′ suggests that they might have received transfusions of contaminated blood products from the same source, possibly from eastern, southern, or central China, where this subtype is prevalent and where blood products were prepared in the late 1980s through the early 1990s. 27–30 To our knowledge, the contamination during the blood collection process was the major reason for the spread of HIV among commercial plasma donors. 31 The infected blood products were then used for transfusion to hemophiliacs who now harbor the subtype B′ strains. A survey of 1500 former commercial plasma donors revealed 12.5% prevalence of HIV-1 infection, and the risk of infection in a blood donor increased with the number of donations. 29 The HIV-1 strain in these blood donors was identified as subtype B′. 32 Even though an epidemic of HIV infection among former commercial plasma donors was initially reported mainly in the Henan province, 28 similar problems with HIV infection among paid blood donors have been reported elsewhere in China. 33
The heterosexual group was characterized by a broad HIV-1 diversity with different subtypes and recombinant strains and was comprised of CRF01_AE strains (38%) followed by subtype B (19%), C (13%), A (13%), B′ (6%), BC (6%), and AE/B (6%). The viral sequences from these subjects clustered with reference sequences from Thailand, the United States, and France (Fig. 1). Like in other regions of the world, this broad HIV-1 diversity among the heterosexual group suggest that HIV-1 diversity in this city will increase in future if adequate intervention programs are not instituted to prevent further spread of disease. Epidemiologic evidence from other provinces indicates that there are signs of an increasing HIV epidemic spreading via heterosexual route in at least 3 provinces (Yunnan, Guangxi, and Guangdong) where HIV prevalence in the year 2000 had been as high as 11% among sentinel sex workers. 34 Similarly, the average HIV prevalence among patients with sexually transmitted diseases is also increasing remarkably. 4 Because it is strongly believed that heterosexual transmission of HIV will become the main transmission route in China, the result of this study suggests that the genetic diversity of HIV-1 strains circulating in this country will increase and become very complex.
Besides CRF01_AE strains, which are circulating in persons infected by the heterosexual route and are mostly distributed in southeastern coastal China, it has recently been reported that the other CRFs, CRF07_BC and CRF08_BC, are circulating widely among IDUs in China. 21,22,24,35 CRF07_BC has been spreading northwestward to Xinjiang province, whereas CRF08_BC spreads eastward to Guangxi province from their common origin, presumably in Yunnan, where subtype B′ and C are co-circulating. 36,37 In the present study, 3 samples, phylogenetically analyzed based on env, gag, and pol genes, clustered with CRF08_BC references. Furthermore, bootscanning analysis in the gag-RT regions revealed a similar structure profile of one 01CNSH4 strain (CRF08_BC) to the previously published sequences, 97CNGX-7F (CRF08_BC) from Guangxi province and WS002 (CRF08_BC) from Yunnan province, where HIV-1 subtype B′ and subtype C, CRF08_BC, and CRF07_BC co-circulate. 21,22,24 Taken together, these results suggest that the CRF08_BC infections could have been introduced into Shanghai from the Guangxi or Yunan province.
Four subtypes and 2 CRFs were identified among these patients; most of them were characterized by a concordant subtype sequence based on env, gag, and pol genes (Table 1). The presence of several subtypes and CRFs among the heterosexual group suggests that more intersubtype recombinants will emerge due to dual infections possibly through heterosexual contacts. Indeed, a new intersubtype recombinant, CRF01_ AEenvBgagUpol strain, was identified in a patient infected heterosexually. Similarly, discordant subtypes were also reported frequently among female commercial sex workers in Central Myanmar. 38 This new intersubtype recombinant, named CRF15_01B (subtype CRF01_AE, B), has been reported previously. 39
Our analysis of PI resistance–associated mutations among the different HIV-1 subtypes revealed 1 primary (codon 30N) and several secondary (codons 10V, 20R, 36I, 46I, 63P, 71V/T, and 77I) amino acid substitutions. It has been postulated that preexisting secondary mutations could reduce the effectiveness of PI treatment, resulting in rapid emergence of resistant phenotypes. 9,12 Since 92% of sequences were obtained from HIV-1-infected, PI-naive individuals, the high prevalence of PI resistance–associated mutations may be representative of their natural polymorphisms. Some of these natural polymorphisms in drug-naive individuals with respect to subtype B and non-B subtypes are similar to those reported in other studies. 40,41 The high frequency of PI resistance–associated mutations at 63P, 71V/T, and 77I reported in subtype B sequences in other studies 40,41 was observed among the hemophiliacs studied here, who were all infected with subtype B′ strains. Similarly, the high frequency of the 36I mutation observed among heterosexuals infected predominantly with the non-B subtypes was also reported in other studies. 40,41 These specific mutations characteristic of these different risk groups may help track the emergence of resistant phenotypes in these different populations and also indicate treatment that needs to be provided. For example, the high frequency of M36I indicates the presence of an increased risk of resistance to ritonavir if additional primary mutations such as V28A/T are acquired. Furthermore, L63P has been implicated in PI resistance. 42
Up to 95% of the PR sequences carried at least 1 PI resistance–associated amino acid substitution, and 57% carried multiple substitutions (from 2 to 5). Among the samples analyzed, 76% of subtype B/B′ and 31% of non-B strains harbored multiple (≥2) PI resistance–associated substitutions, compared with previous reports showing 23 and 27%, respectively, 41 suggesting a higher prevalence of multiple PI resistance–associated mutation in B/B′ subtype strain than in non-B strains in Shanghai. Because increasing prevalence of multidrug-resistant HIV may be crucial for current prevention and treatment strategies, 43 it is important to study whether individuals harboring strains with multiple secondary mutations may be at greater risk of virologic failure during PI therapy, as has been hypothesized. 44
In conclusion, this is the first study that has revealed the broad HIV-1 genetic diversity and PI resistance–associated mutations in Shanghai. The genetic diversity is characterized by several subtypes and CRFs in the heterosexual risk group but by a homogenous subtype in hemophiliac patients. The study also revealed multiple PI resistance–associated mutations in both the B and non-B subtypes that may have implications in treatment and the emergence of drug resistance phenotypes. Further studies are needed to examine other risk groups for the HIV-1 strains that circulate in Shanghai and in other regions of China so as to understand their implications in treatment and vaccine design.
The authors thank Dr. Susan Zolla-Pazner for critical reading of the manuscript and the staff in the Department of AIDS/STDs in the Shanghai Municipal Center for Diseases Control and Prevention for their support of this work.
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