Genetic Characteristics of Three Unique Recombinant Forms of HIV-1 in Yunnan,China

Sequence Note

Yunnan Province in China, which borders Myanmar, Laos, and Vietnam, is the first place that reported an epidemic of human immunodeficiency virus type 1 (HIV-1) in China. ¹ Because of the cocirculation of multiple HIV-1 subtype, such as subtypes B, C, and CRF01_AE, numerous intersubtype recombinant forms, including circulating recombinant forms (CRFs) and unique recombinant forms (URFs), have become common in Yunnan, ² as well as in its bordering region, Northern Myanmar. ³ One of the most important HIV-1 strains in China, CRF07_BC, originated in Yunnan in the early 1990s. ^4,5 Since then, HIV-1 recombinant forms have gradually become the major circulating strains in Yunnan, including the increasing number of second-generation recombinant forms. ^6,7 The monitoring of recent molecular epidemiological characteristics of HIV-1 is required for the prevention of complex recombinant forms in Yunnan, China.

From plasma samples of Burmese patients newly diagnosed with HIV-1 in Dehong Prefecture of Yunnan Province in 2017, three HIV-1 near full-length genomes were amplified and sequenced using previously described methods. ⁸ In brief, 5 mL of peripheral venous blood was collected with EDTA-2K plus vacuum tubes. Viral RNA was extracted from plasma samples using a High Pure Viral RNA Kit (11858882001; Roche Diagnostics Ltd., Mannheim) and was reverse transcribed using a PrimeScript II 1st Strand cDNA Synthesis Kit (6210A; TaKaRa Biomedical Technology Co. Ltd., Beijing). The HIV-1 near full-length genome was amplified using a TransTaq DNA Polymerase High Fidelity kit (AP131-13; Beijing TransGen Biotech Co., Ltd., Beijing) with a previously described strategy. ⁹ The five most similar sequences among the amplified sequences were blasted and downloaded from the Los Alamos National Laboratory HIV sequence database. The maximum likelihood tree and bootscanning plots were drawn by MEGA X and Simplot 3.5.1, as previously described. ^{8,10 –12}

The maximum likelihood tree of amplified sequences and their similar sequences showed that one of the amplified sequences (MM32M629) was in the outlier of the CRF07_BC and CRF08_BC clusters, indicating that it might have a close genetic relationship with CRF07_BC/CRF08_BC but might not be CRF07_BC/CRF08_BC (Fig. 1). One of the sequences (MM33M869) clustered with URFs of subtype B, C and CRF01_AE, implying that it was URF as well. To confirm whether it was the same strain as clustered sequences (KU820827 and KU820838), further bootscanning analysis was needed. The last amplified sequence (MM32F837) was in the cluster of recombinant forms of CRF01_AE and CRF07_BC, indicating that it might be a recombinant form of subtype B, C, and CRF01_AE as well.

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FIG. 1.

Maximum likelihood tree of HIV-1 near full-length genomes. The triangles indicate the sequences amplified from Burmese patients newly diagnosed with HIV-1 in Dehong Prefecture of Yunnan Province in China in 2017 by this study. HIV-1, human immunodeficiency virus type 1.

To explore the recombinant structure of amplified sequences and their most similar sequences, bootscanning plots were drawn by Simplot 3.5.1 (Fig. 2). In the pol region, the amplified sequence MM32M629 had a common subtype B fragment with CRF07_BC and CRF08_BC, which inserts into the subtype C skeleton, implying that it might be a second-generation recombinant form of CRF07_BC/CRF08_BC. These results were in accordance with the location of MM32M629 of the maximum likelihood tree, which was in the outlier of the CRF07_BC and CRF08_BC clusters. The amplified sequence MM33M869 had three identical breakpoints with CRF83_cpx and four identical breakpoints with URF KU820827, implying a possible recombinant relationship between these sequences. The amplified sequence MM32F837 had four identical breakpoints with URF FJ238521 and five identical breakpoints with URF MM044623, implying a possible recombinant relationship between these sequences as well. Notably, MM32F837 contained six CRF01_AE fragments, five subtype C fragments, and two subtype B fragments, which were separated by 12 breakpoints.

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FIG. 2.

Bootscanning plots of HIV-1 near full-length genomes of Burmese patients newly diagnosed with HIV-1 in Dehong Prefecture of Yunnan Province of China in 2017. The lines with different colors indicate the reference sequences with different HIV-1 subtypes.

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An increasing number of studies have reported that the second-generation recombination of HIV-1 took place in Yunnan in recent years. ^6,7 Sequences amplified and sequenced from Burmese patients newly diagnosed with HIV-1 in Yunnan by this study revealed that this process was still ongoing up to 2017. Furthermore, this recombination occurred not only between HIV-1 pure subtypes but also between CRFs and existing URFs, giving a partial explanation for the increasingly complex HIV-1 recombinant forms. One of the sequences amplified in this study (MM32F837) was as complex as having 12 breakpoints, almost catching up with the most complex HIV-1 strain in Southeast Asia, which has 14 breakpoints. ¹³

In 2013, we identified HIV-1 CRF83_cpx among drug users in Northern Myanmar. ⁸ In 2015, Chen et al. first reported the epidemic of CRF83_cpx among patients newly diagnosed with HIV-1 in Yunnan based on HIV-1 partial gag, pol, and env fragments. ¹⁴ In this study, we found that one HIV-1 near full-length sequence amplified from Burmese patients in Yunnan might be a second-generation recombinant form of CRF83_cpx. Owing to the limited HIV-1 molecular epidemiological information in Yunnan, we could not draw a conclusion as to whether CRF83_cpx originated in Yunnan or whether CRF83_cpx had transmitted from Myanmar to Yunnan; however, we could almost confirm that Burmese citizens in Yunnan play a critical role in HIV-1 cross-border transmission in the China–Myanmar border region. ^10,15 To monitor the spatiotemporal dynamics of HIV-1 strains in the China–Myanmar border region and to prevent the formation of complex HIV-1 recombinant forms, systematic surveys and immediate interventions are urgently needed.

In summary, we amplified and sequenced three HIV-1 near full-length genomes from Burmese patients newly diagnosed with HIV-1 in Yunnan Province in China in 2017. Phylogenetic and bootscanning analyses revealed that all of them were HIV-1 URFs, which might be second-generation recombinant forms of CRF07_BC/CRF08_BC, CRF83_cpx, and existing URFs. These results indicated that HIV-1 intersubtype recombination was still ongoing in Yunnan, China, calling for systematic molecular epidemiological studies and immediate targeted interventions.

Sequence Data

The sequences obtained in this study have been submitted to GenBank with accession numbers MT783422–MT783424. The protocol of this study was approved by the Ethics Committee of Kunming Institute of Zoology, Chinese Academy of Sciences (approval number: SWYX-2009021; approval date: January 7, 2009). All participants gave written informed consent.