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Emergence of HIV-1 CRF01_AE/B unique recombinant forms in Kuala Lumpur, Malaysia

Tee, Kok Keng; Pon, Chee Keong; Kamarulzaman, Adeebaa; Ng, Kee Peng

Basic Science

Objectives: To investigate the molecular epidemiology of HIV-1 and to screen for the emergence of intersubtype recombinants in Kuala Lumpur, Malaysia.

Design: A molecular epidemiology study was conducted among HIV-1 seropositive patients attending the University Malaya Medical Center (UMMC) from July 2003 to June 2004.

Methods: Protease (PR) and reverse transcriptase (RT) gene sequences were derived from drug resistance genotyping assay of 100 newly diagnosed or antiretroviral-naive patients. These were phylogenetically analysed to determine the subtypes and recombination breakpoint analyses were performed on intersubtype recombinants to estimate the recombination breakpoint(s).

Results: CRF01_AE predominated in Kuala Lumpur with 65% in both PR and RT genes. B subtype was detected at 14% and 12% in PR and RT genes, respectively. C subtype was present at 1% in both genes. Overall, the concordance of PR and RT genes in discriminating subtypes/circulating recombinant forms (CRF) was high at 96%. In this study, novel CRF01_AE/B intersubtype recombinants were detected at high prevalence (22%), including those isolates with subtype discordance. Thai variants of CRF01_AE and B subtype were involved in the genesis of these unique recombinant forms (URF). Interestingly, 19 CRF01_AE/B intersubtype recombinant isolates shared similar recombination breakpoints in both PR and RT genes. Several distinct URF were also identified.

Conclusion: PR and RT genes can be utilized for subtype/CRF assessment with high degree of agreement, allowing concurrent surveillance of circulating HIV-1 subtypes with antiretroviral drug resistance genotyping tests. The emergence of highly identical CRF01_AE/B intersubtype recombinants suggests the possibility of the appearance of a new circulating recombinant form in Kuala Lumpur.

From the Department of Medical Microbiology and aMedicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Received 6 September, 2004

Revised 21 October, 2004

Accepted 2 November, 2004

Correspondence to K.P. Ng and K.K. Tee, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. E-mails:;

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HIV-1 subtypes are clustered together to form clades; group M (‘main’), group O (‘outlier’) and group N (non-M/non-O) [1–3], with group M circulating worldwide. To date, there are nine established HIV-1 subtypes (A, B, C, D, F, G, H, J and K) and 16 circulating recombinant forms (CRF) ( ). HIV-1 is characterized by its extremely high genetic diversity. Rapid turnover of virions in CD4 lymphocytes, error-prone reverse transcriptase (RT) activity and the presence of RNA dimer are some of the factors contributing to this variation [4,5]. The extensive variability of HIV-1 has a huge impact on the epidemiology, diagnosis and disease pathogenesis and on the development of effective vaccine and long lasting antiretroviral therapy.

Heteroduplex mobility assay or phylogenetic analysis of the env gene is the most popular approach to study HIV-1 subtypes, based on the great variability of the env sequence. However, other nucleotide sequences that vary significantly may also be useful for this purpose. The protease (PR) and RT genes, for example, are studied to identify mutations conferring antiretroviral drug resistance and subsequently used in managing patients appropriately [6]. Besides providing antiretroviral drug resistance data, the PR and RT sequences have been utilized to distinguish HIV-1 subtypes or CRF by phylogenetic approach [7–14].

In Malaysia, with an estimated total of 58 000 HIV infections and 8300 AIDS cases reported until December 2003, CRF01_AE and B subtype have been the predominant subtypes among heterosexuals and injecting drug users (IDU), respectively [15,16]. Cocirculation of two or more subtypes/CRF in any population is believed to play an important role in generating intersubtype recombinant strains such as a CRF01_AE/B recombinant if the circulating subtypes are CRF01_AE and B [17–23]. Evidence showed that recombination between subtypes/CRF involving the pol gene is also common [17,19,20]. Thus the study of drug resistance mutations provides an alternative to screen for intersubtype recombinants of epidemiological significance.

The genetic characterization of HIV-1 strains in Malaysia has not been extensively investigated despite the importance of HIV infection in neighbouring Thailand. In this study, we evaluated the feasibility of using PR and RT genes to distinguish HIV-1 subtypes in Kuala Lumpur, and found evidence of the emergence of novel intersubtype recombinants.

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Materials and methods

Clinical samples

Blood samples in EDTA from 100 randomly selected newly diagnosed or therapy-naive HIV-1 seropositive patients (Abbott AXSYM System, Abbott Laboratories, Illinois, USA) were collected from the HIV clinic of the University Malaya Medical Center (UMMC) and plasma samples were stored at −80 °C. Ethical approval was obtained from the institution's ethical committee.

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PR and RT nucleotide sequence determination

HIV-1 RNA was extracted by column purification method (QIAamp Viral RNA Mini Kit, Qiagen, Hilden, Germany) and reverse transcribed to cDNA before nested PCR was performed. For PR gene, primers PI-1685 (5′-GGAATTTTCCTCAGAGCAGACCAG-3′) and PI-2209 (5′-TCTTCTGTCAATGGCCACTGTTTAAC-3′) were used in the first PCR and primers PI-1685 and PI-2172 (5′-CCATTCCTGGCTTTAATGTTACTGGTAC-3′) were applied in the second PCR. For RT gene, primers RT-2955 (5′-GCTTTACCTTAATCCCTGCATAAT-3′) and A-35 (5′-GGTTGTACTTTAAATTTCCCAATTAGTCC-3′) were used in the first PCR followed by primers B887-2 (5′-CTGTACCAGTAACATTAAAGCCAGG-3′) and RT-2923 (5′-GCCCAATTTAGTTTTCCCACTAAT-3′) in the second PCR. The nucleotide sequences of PR and RT were determined by cycle-sequencing dideoxy chain termination method on an automated DNA sequencer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems, Foster City, California, USA). Codons 1–96 for the PR and 20–255 for the RT were sequenced and studied.

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Phylogenetic analysis

The electropherograms were analysed and adjusted manually. All PR and RT sequences were analysed by the BLAST 2.0 program at NCBI ( to search for sequence similarities to previously reported sequences. Sequences were aligned with HIV-1 strains of various reference subtypes and CRF obtained from the Los Alamos HIV Database ( and the NCBI using profile alignment option of CLUSTAL X version 1.83. Regions that could not be aligned ambiguously were omitted from the analysis. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 [24]. Evolutionary distances were calculated using the Kimura 2-parameter model with a transition: transversion ratio of 2.0 [25]. Phylogenetic trees were constructed using the neighbour-joining method. The reliability of the branching orders was confirmed by bootstrap analysis with 1000 replicates. Bootscan analysis was performed using SimPlot version 2.5 to identify recombination breakpoints in samples with undetermined subtypes or CRF. Putative parental strains of different subtype and CRF were selected from Los Alamos HIV Database for bootscan analysis. Subregion confirmatory tree analyses were carried out to confirm the subtype in each fragment.

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HIV-1 genetic subtypes and recombinant strains circulating in Kuala Lumpur

From July 2003 to June 2004, 100 plasma samples were collected from newly diagnosed or antiretroviral-naive patients consisting of 81% male and 19% female. The major ethnic group in this study was Chinese (65%) followed by Malay (22%), Indian (9%) and others (4%). The reported transmission routes were heterosexual (57%), men who have sex with men (MSM) (12%), IDU (6%), heterosexual and IDU (4%), bisexual (2%), mother-to-child (MTC) (1%) and unknown transmission route (18%). Fig. 1a and b display the phylogenetic relationship of the 100 samples in both PR and RT genes and the subtype assignment is summarized in Table 1. Based on PR gene analysis (Fig. 1a), CRF01_AE was the predominant strain circulating among HIV-1 patients in Kuala Lumpur, with a prevalence of 65% and B subtype was present at 14%. C and G subtypes were each detected from two male patients from India and Democratic Republic of Congo, respectively. This is the first reported detection of G subtype in Malaysia. Interestingly, 19 of these samples, thought to be unique recombinant forms (URF), appeared to be closely related to CRF01_AE but not sufficient to cluster into any known subtypes or CRF.

Fig. 1

Fig. 1

Table 1

Table 1

In the RT gene (Fig. 1b), the predominant circulating strain was CRF01_AE consisting of 65% of the specimens. B subtype was present at 12% and C and H subtypes contributed only 1% each. Sample 04MYKL1721, which was a G subtype in the PR gene, however was designated as H subtype in the RT gene. Similar to the findings noted in the PR gene, 21% of the patients remained unclassified to any known subtypes or CRF by RT gene, but clustered closely to CRF01_AE. In these 21 samples, 20 of them were clustered in a monophyletic group with the remaining sample, 03MYKL1590, branching out from this group.

Overall, the concordance between PR and RT genes in subtype, CRF and URF discrimination was high (96%). Four samples, 03MYKL1554 (BPRCRF01_AERT), 03MYKL1590 (CRF01_AEPRURFRT), 04MYKL1721 (GPRHRT) and 04MYKL1903 (BPRURFRT) showed subtype discordance in PR and RT genes. The presence of URF in both PR and RT genes was 19% and 21% respectively but considering 03MYKL1554, 03MYKL1590 and 04MYKL1903 as a URF, the overall prevalence of URF in this study was 22%, indicating a high frequency of URF circulating among HIV-positive population in Kuala Lumpur. To summarize, Table 2 shows the distribution of subtypes/CRF/URF among different groups with risk practices. CRF01_AE and B subtype were the predominant strain among heterosexuals and MSM patients, respectively. Interestingly, most of the IDU and heterosexual/IDU patients were harbouring URF but none of them with B subtype.

Table 2

Table 2

It was observed that 19 of the 22 URF samples were concordant in both PR and RT genes. Details of all the URF samples are summarized in Table 3. Of note was the high proportion of Malay ethnic at 50%. Various mode of transmission were associated with this group and most of the patients were newly diagnosed.

Table 3

Table 3

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HIV-1 CRF01_AE/B URF circulating in Kuala Lumpur

All unclassified samples from both PR and/or RT genes, which were thought to be a URF, were subjected to bootscan analysis by SimPlot. HIV-1 B′ subtype isolate RL42 and CRF01_AE isolate 93TH253 were selected as the putative parental strain, with C subtype isolate 92BR025 as a background strain. The bootstrap values were plotted for a window of 120 base pair (bp) (for PR gene) and 140 bp (for RT gene) moving in increments of 20 bp along the alignment.

All 19 concordant URF samples in both PR and RT genes showed similar bootscanning plots in both genes. A representative sample (03MYKL1317) is shown in Fig. 2a and b. In the PR gene encoded by gag–pol, one breakpoint was detected approximately at nucleotide (nt) 2348 (HXB2 numbering) where the 5′ subregion prior to the breakpoint was B′ subtype and the 3′ subregion was CRF01_AE (Fig. 2a). In the RT gene, one breakpoint at 2855 nt was estimated, with B′ subtype at 5′ subregion and CRF01_AE at the downstream segment after the breakpoint (Fig. 2b). These were then confirmed by subregion confirmatory tree analysis on both PR and RT genes (data not shown). In general, the longer RT genome (708 bp) provided a better bootscan image resolution than PR gene (291 bp).

Fig. 2

Fig. 2

Sample 03MYKL1554 was not subjected for bootscan analysis because both PR and RT genes were clustered as B subtype and CRF01_AE respectively (Fig. 1a and b), showing no evidence of recombination within these genes. Sample 03MYKL1590 when analysed demonstrated three recombination breakpoints in the RT gene (Fig. 2c) but the PR gene was not analysed as it was a CRF01_AE. The three breakpoints in the RT gene were estimated at position 2675 nt, 2895 nt and 3215 nt, the highest number of breakpoints found among all URF samples in this study. The small 5′ subgenome of the RT gene was estimated as B′ subtype until 2675 nt, followed by CRF01_AE (2676–2895 nt) and B′ subtype (2896–3215 nt). The 3′ segment was predicted as CRF01_AE from 3216 nt to 3313 nt. Finally, the PR gene for 04MYKL1903 was B subtype but the RT gene (Fig. 2d) had one recombination breakpoint at 3096 nt.

From all the URF samples analysed, B′ subtype and CRF01_AE were involved in the genesis of intersubtype recombinants with most of them (n = 19) sharing a similar recombination profile. This allowed the identification of these URF as CRF01_AE/B intersubtype recombinants, the first of its kind to be reported in Malaysia.

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In addition to testing for antiretroviral resistance, PR and RT gene sequences can be utilized for subtype and CRF discrimination. In the present study, CRF01_AE was found to be the dominant subtype by both PR and RT genes. B subtype was present to a much lower level and a single C subtype was isolated from a patient from India. The presence of CRF01_AE/B intersubtype recombinant in both genes is high, circulating as the second highest strain. The overall frequency of CRF01_AE/B intersubtype recombinant screened in this study, including those samples with discordant subtype assignment in both PR and RT genes, is 22%. In brief, the concordance between PR and RT genes in subtype (and CRF01_AE/B intersubtype recombinant) discrimination is high.

Bootscanning plots revealed that these 19 CRF01_AE/B intersubtype recombinant samples had a unique recombination profile in both PR and RT genes which have never been reported previously in any of the other known CRF01_AE/B recombinants or with CRF15_01B from Thailand [17–23]. The PR and RT genome structures of these URF were the results of recombination of B subtype and CRF01_AE with similar breakpoints, reflecting common ancestry from the same recombination event(s). Fig. 3 shows the deduced gag–pol gene structures of CRF01_AE/B intersubtype recombinants detected in this study. Besides the 19 homologous samples, another three samples (03MYKL1554, 03MYKL1590 and 04MYKL1903) also showed unique recombination profiles. A segment of 66 bp between the PR and RT gene was not amplified and examined in this study because the in-house drug resistance genotyping assay is designed to study the PR and RT gene individually. In Fig. 3, a ‘virtual’ recombination breakpoint is expected within this segment of 66 bp in samples 03MYKL1554, 03MYKL1590 and the group of 19 samples because the subtype assignment in the genomes before and after this segment is discordant.

Fig. 3

Fig. 3

In this study, PR and RT genes derived from drug resistance genotyping assay were successfully assigned to a known subtype, CRF or an intersubtype recombinant, suggesting the feasibility of using PR and RT gene sequences for subtype/CRF/URF assessment, particularly CRF01_AE, B, C subtypes and CRF01_AE/B intersubtype recombinant. However, a practical method that sequences the PR and RT genes including the 66 bp will allow more reliable phylogenetic and especially bootscan analysis. For better subtype assignment, it would be necessary to sequence another region, for instance, the env gene.

One discordant sample between G and H subtype in PR and RT gene, respectively, was unlikely to be classified as a GPRHRT URF due to small sample size. Further assessment of the subtype for this sample by env subtyping might be needed. This indicates that phylogenetic analysis on PR and RT genes may not be adequate in differentiating G and H subtype efficiently. It is however the first detection of this subtype in Malaysia in a patient from Congo.

B subtype and CRF01_AE, previously known as E subtype, have been circulating in Malaysia since the 1990s where B subtype was the predominant subtype among IDU and CRF01_AE among heterosexuals [15,16]. However the increasing frequency of CRF01_AE among antiretroviral-naive IDU diagnosed between 1998 and 2002 has been observed in Kuala Lumpur, and has overtaken B subtype as the predominant strain (unpublished data) [26–31]. In this study, B subtype was not present among IDU (and hetero/IDU) patients but CRF01_AE/B intersubtype recombinant was the dominant strain, reflecting a major shift in the proportions of pure B subtype and CRF01_AE to CRF01_AE/B intersubtype recombinant [17]. The presence of CRF01_AE/B intersubtype recombinant among different groups with risk practices, especially among IDU, has diluted the dominance of B subtype and CRF01_AE, implying that the breakpoints shared among the recombinants may be beneficial to the viral fitness or transmissibility. Besides being capable of generating drug resistant recombinant [32,33], the increasingly complex genetic recombination of HIV-1 may complicate future clade matched vaccine development.

In conclusion, the presence of a novel recombination structure among 19 CRF01_AE/B intersubtype recombinants in Kuala Lumpur has provided evidence of the emergence of a new candidate of circulating recombinant form that would have a recombination pattern different from the already described CRF15_01B. Full genome characterization of these CRF01_AE/B intersubtype recombinants is needed for further confirmation and elucidation.

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The authors thank the staff in the HIV/Viral Hepatitis Laboratory and Infectious Diseases Unit at University Malaya Medical Center, Kuala Lumpur, Chatchawal Sa-Nguansilp, Dr. Sunee Sirivichayakul and Prof. Kiat Ruxrungtham, Chulalongkorn University, Bangkok, Thailand, Dr. Robert E. Oelrichs, Burnet Institute, Melbourne, Australia, Dr. Sodsai Tovanabutra, Henry M. Jackson Foundation, Rockville, Maryland, USA and Dr. Yutaka Takebe from National Institute of Infectious Diseases, Tokyo, Japan for their assistance in this study. We also like to thank Prof. Lam Sai Kit for his critical reading of the manuscript.

Sponsorship: Supported by the IRPA grant (Grant number: 06-02-03-1015) from the Ministry of Science, Technology and Innovation, Malaysia.

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Malaysia; phylogenetic analysis; circulating recombinant forms; HIV-1 intersubtype recombinants; unique recombinant forms; molecular epidemiology

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