In Taiwan, the first infected case of indigenous human immunodeficiency virus type 1 (HIV-1) was not reported until 1986. By the end of April 2005, a cumulative total of 7808 HIV-1-infected individuals, of whom 2071 were AIDS cases, have been reported to the Department of Health.1 The estimated prevalence of HIV-1 infection in Taiwan is about 3/10,000 persons by the end of April 2005.1 It is worthy to note that there was a marked increase in the incidence rate in 2004 (76.6%), compared with the incidence rate of 11% to 22.2% in the last decade (1995-2003).1 From January to April of 2005, there were 1035 newly reported cases, which accounted for 68.0% of the total cases reported in 2004. Of note, the major risk factor among cases reported over the past 16 months was injection drug use (IDU), which accounted for 45.2% (1156 of 2556). In contrast, over the previous few years, sexual contact was the main route of transmission, and less than 3% of HIV-1/AIDS cases in Taiwan had a history of IDU.
The genetic heterogeneity of HIV-1 isolates is one of the major characteristics of the virus and the epidemic. Phylogenetic analyses of globally circulating viral strains have identified 3 distinct groups of HIV-1 (M, N, and O), 9 genetic subtypes (A to D, F to H, J, and K), and 19 circulating recombinant forms (CRFs) within the major group (M).2-4 CRFs have mosaic genomes with regions from different subtypes and are shown to be in circulation in the general population, based on the sequencing of at least 2 independent samples from individuals who were not linked by direct transmission.2-4 Such phylogenetic characterization of HIV-1 has provided a broad overview of the geographic distribution of HIV-1 and offers insights on how the virus has spread between regions and continents throughout the world. Data on the global genetic variation of HIV-1 has also provided important information for selecting appropriate strains for vaccine design and development5 and, ultimately, for the conduct of efficacy trials in an appropriate human population.
Each HIV epidemic in distinct geographic regions and population groups has its own specific characteristics and dynamics. The genetic characterization of HIV-1 in IDUs may be unique and different to other risk groups. Inter-subtype recombinant forms seem to be circulating among IDUs in China (B/C),6,7 North Vietnam and Southeast Asia (CRF_01AE),8 Argentina (B/F),9 and Russia (A/B).10 HIV epidemics can occur very rapidly among IDUs because the sharing of needles and other injection equipment is a very efficient way of transmitting HIV. Epidemiology and molecular studies also indicate that the recent heterosexual spread is related to an earlier IDU epidemic.11,12,13 Our current report focuses on the genetic characterization of HIV-1 strains circulating among IDUs in Taiwan. Accordingly, this study describes a recent outbreak of HIV-1 CRF07_BC, which was previously identified among IDUs in southwest China and which has now infected IDUs in Taiwan.
MATERIALS AND METHODS
Study Subjects and Blood Samples
HIV-1 screen testing was routinely checked in patients with IDU presenting at the E-Da Hospital and Kaohsiung Veterans General Hospital. Based on serology and Western blot confirmation, a total of 131 samples were identified to be positive for HIV-1 from January 2004 to April 2005. Eighty-one individuals were prison inmates and were referred from prisons. These prison inmates were arrested and imprisoned for injecting drugs (heroin) and, in some cases, selling them as well. At the time of the study, none showed symptoms of AIDS or opportunistic infections. Only 1 individual had a history of HIV-1 infection and had received antiretroviral therapy. HIV viral load was performed using the HIV-1 RNA 3.0 Assay (bDNA) test (Bayer, Germany) according to manufacturer s protocol, whereas CD4 counts were monitored by flow cytometry (BDIS, San Jose, CA). Demographic and behavioral information was obtained during the interview. The Institutional Review Board of E-Da Hospital approved the study protocol, and a prisoner advocate was part of the review board. All study participants provided written informed consent.
Amplification of HIV-1 Gene Fragments by Polymerase Chain Reaction
Viral RNA was extracted from plasma using QIAamp Viral RNA Mini Kits (Qiagen). Samples were amplified using 2 rounds of reverse transcriptase polymerase chain reaction (RT-PCR) with nested primers at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. The primers used to generate a 377-nucleotide fragment spanning the C2-V3 region of the envelope gene were: for the first PCR, env A (5′-CCAATTCCCATACATTATTG, nt 6858-6877) and env E (5′-GTCCCTCATATCTCCTCCTCCAGG, nt 7655-7632); for the second PCR, env B (5′-TGCTGTTAAATGGCAGTCTAGCAG, nt 7000-7023) and env D (5′-TAGAAAAATTCCCCTCCACAATTAA, nt 7375-7350). The nucleotide positions in parentheses were based on the subtype B HIV complete genome of HXB2 (GenBank accession number K03455). All of the PCR products were purified for sequencing using the QIAquick PCR purification kit (Qiagen) according to the manufacturer s protocols. The sequencing of PCR products was performed with an automatic sequencer (PRISM automated sequencer, version 3100; ABI, Foster City, CA). The procedures for viral RNA extraction, PCR amplification, and nucleotide sequencing have been previously described.14,15 Sequences of HIV-1 protease (PR) and reverse transcriptase (RT) were obtained using the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System according to the manufacturer s recommendations (Bayer, Germany), which have been described previously.16 The system is designed to provide bidirectional sequencing of the PR-coding region (codons 10 to 99, nt 2262 to 2549, HXB2) and codons 41 to 237 (nt 2659 to 3289, HXB2) of the RT-coding region of HIV-1 pol.
Phylogenetic Analysis of HIV-1 Sequences
To eliminate potential contamination, all of the sequences obtained were first subjected to an HIV-1 BLAST search to compare them with related reference sequences in the HIV database (hyyp://hiv-web.lanl.gov/content/index).17 Nucleotide sequences were aligned with the references with the use of the ClustalX 1.81 program18 and were further adjusted manually. The genetic distance of the HIV-1 sequences analyzed was calculated using the 2-parameter model of Kimura.19 Phylogenetic trees were generated using the neighbor-joining method implemented in the ClustalX 1.81 program. The branch significance was analyzed by bootstrap with 1000 replicates. The trees were printed using the TreeView program.20 Amino acid sequence comparisons of the V3 loop were performed for all of the strains. The Recombinant Identification Program (RIP) (version 2.0; http:/hiv-web.lanl.gov/tools) was used to identify potential mosaic recombination (window size, 200; threshold for statistical significance, 90%; gap handling, STRIP; informative mode, OFF). Gaps were introduced to create the alignment, and the background subtypes sequences in the analysis were from HIV-1 subtype reference alignments (http://hiv-web.lanl.gov/content/hiv-db/SUBTYPE_REF/align.html).
Demographic Characteristics of HIV-1-infected Individuals
Of the 131 HIV-1 seropositive samples collected from our study population, all contained detectable sequences, including 105 from the C2-V3 region of env and 87 from PR and RT of pol. Of the 131 individuals, there were 116 men and 15 women, with a mean age of 33.42 years (range: 22-52 years), a mean CD4 count of 429/mm3, and a mean viral load of 5.40 × 104 (log 4.23) (Table 1). The duration of heroin use was from 6 months to 20 years, with a mean of 4.5 years. Ninety percent admitted to sharing needles, and 100% shared the solvent (water) used for injection. Twenty-three individuals had a travel history to the southwest provinces of China, including 9 to Yunnan, 7 to Fujian, 3 to Guangxi, 3 to Sichuan, 3 to Hunan, and 2 to Guangdong. Eight individuals had traveled to Southeast Asia, including 4 to Thailand, 3 to Indonesia, and 1 to Cambodia, the Philippines, and Malaysia. All 23 individuals admitted to sharing needles or apparatuses when they were abroad. Overall, 93.7% were coinfected with HCV.
Molecular Epidemiology of the HIV-1 Infection
Phylogenetic analysis using the neighbor-joining method revealed that 128 samples belonged to CRF07_BC, including 102 env sequences (Fig. 1) and 84 protease (Fig. 2A) and reverse transcriptase of pol sequences (Fig. 2B). These sequences are tightly clustered with original CRF07_BC sequences identified in China irrespective of viral gene fragments used, although env and reverse transcriptase gene fragments are grouped with subtype C reference sequences, whereas protease gene fragments are grouped with subtype B reference sequences (Figs. 1 and 2). Such dichotomy in phylogeny for different gene fragments is consistent with the mosaic feature of these sequences and supports the notion that they are derivatives of original CRF07_BC sequences in China. In addition, 2 individuals (tw101 and tw145) were infected with subtype B, one of whom was a homosexual who contracted HIV-1 infection 2 years ago and received antiretroviral therapy. Sequences from these 2 individuals are quite distinct from the reference subtype B sequences, reflected by their relative long branch length in the phylogenetic trees (Fig. 1 and 2). In particular, in the protease tree, these 2 sequences are almost of equal distance to subtypes B and D sequences and are distinct from the rest of the sequences obtained from the IDUs (Fig. 2A). Lastly, one individual who harbored CRF01_AE had sexual contact with female commercial sex workers in Taiwan (Fig. 1 and 2). RIP analysis of the protease and reverse transcriptase of pol sequences revealed that our samples had similar breakpoints in the pol coding region with CRF07_BC and different from CRF08_BC (data not shown).
Characteristics of the V3 Loop Motif of CRF07_BC
Deduced amino acid sequences of the V3 loop motif of CRF07_BC are consistent with data previously generated showing little variation in V3.21 The 8-amino-acid core motif at the tip of the V3 loop of the CRF07_BC sequences was conserved with SIRGPGQT present in 99.0% (101 of 102) of the subjects. Only 1 of the CRF07_BC revealed SIRLPGHP. Two individuals infected with subtype B revealed GPGR that was the same as the typical North American subtype B strain other than Thai-B.
Our report demonstrated that the recent outbreak of HIV-1 infection among IDUs in Southern Taiwan was caused by CRF07_BC. This strain has never been reported in Taiwan in previous literature, and previous studies of the molecular epidemiology of HIV-1 revealed that the predominant subtype of HIV-1 infection is B, followed by CRF01_AE. The remaining subtypes, which included subtypes G, A, and C, constituted only about 2.3%.22-25 One study that included 18 IDUs showed that 12 were infected with the HIV-1 B subtype and 6 were infected with CRF01_AE.23 In addition, 5 of 6 IDUs with CRF01_AE had a history of sexual contact with female commercial sex workers, compared with only 1 of 12 IDUs. Therefore, sexual transmission was an important risk factor for male IDUs infected with CRF01_AE in Taiwan. Most of those infected with CRF07_BC during this outbreak were due to the sharing of needles or solvent (water) used for drug injection.
Although the full genomes of our strains were not sequenced, the phylogenetic and RIP analysis demonstrated that they resemble CRF07_BC sequences originally identified in China. A description of this CRF07_BC was first published by Su et al in 2000,26 and a patent of the CN54 sequence was reported by Rodenburg et al in 2001.27 Afterward, 2 closely related CRFs, CRF07_BC and CRF08_BC, have been more fully documented among IDUs in China.6,7,28 CRF07_BC (prototype strains, 97CN54 and 97CN001) was distributed among IDUs in Xinjiang Province in northwest China, whereas CRF08_BC (prototype strain, 97CNGX6F) was circulating widely among IDUs in Guangxi Province in southeast China. Each CRF seemed to be associated with a different overland heroin trafficking route. CRF07_BC spread northwestward to Xinjiang Province, whereas CRF08_BC spread eastward to Guangxi Province, from their common origin, presumably in Yunnan, where subtypes B' and C are cocirculating. The Yunnan Province in southwestern China is thus thought to be an epicenter of the HIV-1 epidemic in China, where the cumulative number of HIV cases would be expected to reach 10 million by 2010 at the current rate of increase of 30%. Now, the CRF07_BC has further disseminated to Taiwan through IDUs.
Twenty-three individuals of our cohort had a travel history to China, especially the southwest and southeast provinces. According to their description, most of them frequently visited China for drug injection or heroin smuggling because of the cheaper price and higher purity of drugs. Some escaped to China to evade imprisonment. All of them shared needles or apparatuses in China and Taiwan, and some of them had sexual contact with commercial sexual workers in China. It is reasonable to speculate that CRF07_BC might have been introduced into Taiwan from China by the heroin trafficking route to cause an outbreak among IDUs in Taiwan. Eight IDUs visited other countries in Southeast Asia, but because their viral strains clustered with those of the other IDUs, it is more likely that they were infected during this epidemic in Taiwan and not during their past travels in Southeast Asia.
Two isolates of subtypes B and 1 of CRF01_AE were identified among our population. They might have contracted HIV from another route of transmission, distinct from the outbreak in our study. Although no outbreak of subtype B or CRF01_AE was reported among IDUs in Taiwan, continued monitoring is important because needle sharing is common. Recently, McCutchan et al reported a new CRF, CRF15_01B, derived primarily from CRF01_AE, with only the env region (roughly bases 5700 to 7800 in AF516184) being derived from subtype B among IDUs and heterosexual exposures in Thailand.29 This finding suggests that we will face an even more complex genetic diversity of HIV-1 in the future.
Phylogenetic analysis of the env gene or heteroduplex mobility is the most popular approach to the study of HIV-1 subtype, based on the great variability of the env sequence. However, other nucleotide sequences that vary significantly may also be useful for this purpose. The PR and RT genes, for example, are studied to identify mutations conferring antiretroviral drug resistance and may subsequently be used in managing patients appropriately. Aside from providing antiretroviral drug resistance data, the PR and RT sequences have been used to distinguish HIV-1 subtypes or CRFs by the phylogenetic approach.30-34 A recent study demonstrated the emergence of unique recombinant forms of HIV-1 CRF01_AE/B in Kuala Lumpur, Malaysia, by using phylogenetic analysis of the PR and RT genes.35 The isolates of CRF07 and CRF08 were mostly subtype C, which accounted for its initial classification as subtype C, but there were several regions that were far from subtype C and closer to subtype B. These regions were located in the p24 domain of gag, the PR and RT domain of pol, first half of Rev and Tat protein, Vpu, and the Nef (http://hiv-web.lanl.gov/content/hiv-db/CRFs/CRFs.html). RIP analysis of the PR and RT of pol sequences identified in our study population revealed similar breakpoints in the pol coding region with CRF07_BC, distinct from CRF08_BC. The yield rate of sequencing the PR and RT using the TRUGENE HIV-1 Genotyping Kit was superior to methods using primers based on env. Viral sequences amplification failed in 18 patients using the latter method, but successfully identified the PR and RT sequences using the TRUGENE HIV-1 Genotyping Kit. Our study revealed that the PR and RT of pol gene can be used for concurrent surveillance of circulating HIV-1 subtypes and antiretroviral drug resistance genotyping (data not shown).
Injection drug use is the initial mode of HIV transmission in many Asian countries with concentrated HIV epidemics, including China, Malaysia, Nepal, and Vietnam.36-38 Countries with extensive heterosexual spread of HIV, including Thailand, Myanmar, and India, also have high rates of HIV prevalence in injecting drug users. HIV epidemics can occur very rapidly among IDUs when needle sharing is widespread. Based on our study, all of the patients shared needles or other apparatuses used for injection, especially the solvent (water), and frequently reused syringes. Sharing needles and other injection equipment is a very efficient way of transmitting HIV because HIV is inoculated directly into the bloodstream and HIV may remain viable for up to 4 weeks in syringes.39 Spread of HIV from IDUs to their sexual partners and the general population may have a great impact on public health. In many countries, IDUs often trade sex for drugs, have multiple sexual partners, and do not use condoms regularly. Epidemiological and molecular studies indicate that the recent heterosexual spread of HIV is often related to a prior IDU epidemic.11-13
The global spread of HIV is dynamic and unpredictable, and the regional balance of strains can shift quickly. Our report indicates that HIV-1 CRF07_BC has been introduced into Taiwan, causing an outbreak among IDUs. This finding provides information relevant to antiviral therapy and vaccine development in Taiwan and may assist public health workers in the prevention of HIV-1 spread.
The authors are greatly indebted to the study patients for their participation. This work was supported by the Center for Disease Control of the Department of the Health, Taiwan, Republic of China (DOH94-DC-1112).
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Keywords:Copyright © 2006 Wolters Kluwer Health, Inc. All rights reserved.
CRF07_BC; HIV-1; injection drug user; Taiwan