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.
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.
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).
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.
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.
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 . 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.
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.
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