Identification of a Novel HIV Type 1 Circulating Recombinant Form (CRF86_BC) Among Heterosexuals in Yunnan,China

The high efficiency replication, mutation, and recombination resulted in an extensive diversity and genetic complexity of human immunodeficiency virus type 1 (HIV-1). HIV-1 group M strains dominate the global AIDS pandemic and comprise 11 subtypes and sub-subtypes (A1, A2, B, C, D, F1, F2, G, H, J, and K), ¹ as well as 85 circulating recombinant forms (CRFs) reported in the Los Alamos National Laboratory HIV database and a great number of unique recombinant forms (URFs) (www.hiv.lanl.gov/content/sequence HIV/CRFs/CRFs.html).

Yunnan province, located in southwestern China, borders the opium-producing “Golden Triangle” regions composed of Myanmar, Laos, Thailand, and Vietnam and is regarded as the epicenter of the HIV-1 epidemic in China. Nearly all the major epidemic strains of HIV-1 in China, including B, B′, C, CRF07_BC, CRF01_AE, and CRF08_BC, and various URFs, have been detected here. ² Wide cocirculation and dual infection of subtypes B and C among high-risk individuals in Yunnan provide the opportunity for the generation of various novel CRFs. Originally, the HIV-1 epidemic in Yunnan was derived from both HIV-1 subtypes B and B′ (Thailand variant of subtype B) strains, subtype B′ had become the most widely epidemic subtype in western Yunnan among injecting drug users by the early 1990s. ³ Subsequently, HIV-1 subtype C strains were first identified among IDUs in Dehong in 1992. ³ Since then, numerous HIV-1 CRFs such as CRF07_BC, ⁴ CRF08_BC, ⁴ CRF57_BC, ⁵ CRF62_BC, ⁶ and CRF64_BC ⁷ have been reported in Yunnan in the past decade. Currently, sexual contact has become the dominant route of transmission of HIV-1, subtypes B and C have also spread out of former IDUs into the sexually transmitted population, and novel recombinant strains were arising frequently. ⁴ In this study, we identified a novel CRF designated CRF86_BC in heterosexual populations in western Yunnan and described their genomic characteristics.

Blood plasma was collected from three HIV-positive patients (13YNHS18, 13YNHS23, and 13YNHS26) who were infected through heterosexual transmission in Baoshan prefecture, Yunnan. Basic epidemiological information is shown in Table 1. There was no epidemiological link among these three individuals. The study was approved by the Yunnan Traditional Chinese Medicine Institute Ethics Committee. All participants supplied written informed consent for specimen collection and subsequent analyses.

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Table 1.

Demographic Information of Study Subjects Infected with CRF 86_BC

Strain name	Sampling year	Sampling region	Sex	Age	Risk factor	CD4 count	Accession no.
15YNHS18	2013	Baoshan, Yunnan	Male	32	Heterosexual	446	KX582249
15YNHS23	2013	Baoshan, Yunnan	Male	48	Heterosexual	107	KX582250
15YNHS26	2013	Baoshan, Yunnan	Female	30	Heterosexual	220	KX582251

The near full-length genome (NFLG) amplification and sequencing were conducted as previously described. ⁸ In brief, RNA was isolated from 280 μl plasma using the Virus RNA Mini Kit (Tiangen) according to the procedure described in the manual. Then the near full-length HIV-1 genome was amplified separately using reverse transcription-nested polymerase chain reaction as previously reported. ⁹ The generated products were analyzed by agarose gel electrophoresis, and the positive PCR samples were purified using the PCR Product Gel Extraction Kit (Tiangen) and were then sequenced by Invitrogen Co (Guangzhou, China).

All of the overlapped subgenomic DNA sequences were successfully obtained from three subjects. Then the sequenced fragments were edited and assembled into contiguous sequences using ContigExpress software. The sequencing data were aligned against the HIV-1 sequence database using NCBI BLAST search and manually edited with Bioedit 7.2.1 with reference to HXB2 to ensure accurate codon alignment. Phylogenetic and subregion tree analyses were carried out using the neighbor-joining method based on the Kimura 2-parameter model with 1,000 bootstrap replicates and a transition–transversion ratio of 2.0 applied in MEGA 6.02. The reference sequences relevant to HIV-1 epidemics in Asia, such as multiple subtype B strains (from Thailand, China, and Brazil), multiple subtype C strains (from India, China, and Europe), CRF57_BC, CRF61_BC, CRF62_BC sequences, and other CRFs that were detected in China were downloaded from the Los Alamos National Laboratory HIV sequence database. Recombination break points were determined using RIP and jpHMM included in the Los Alamos HIV database, and were further confirmed using SimPlot 3.5.1 software to perform bootscanning and informative-site analyses. Based on the information generated from jpHMM and Simplot, the structure of the new HIV-1 recombinant forms (B/C) was elucidated using the Recombinant HIV-1 Drawing Tool. The break point confirmation and origin of each segment were then analyzed using subtype phylogenetic analysis by the method already mentioned.

The NFLG sequences from the three subjects were 8,656, 8,705, and 8,742 bp in size for strains 15YNHS18, 15YNHS23, and 15YNHS26, respectively, spanning from the most of gag gene to part of three long terminal repeat corresponding to the location 841-9554 of HXB2 strain. Phylogenetic analysis revealed that these three sequences formed a distinct monophyletic branch with a bootstrap value of 100%, distantly related to all known HIV-1 subtypes/CRFs (Fig. 1). Furthermore, the recombination structures were determined on the basis of RIP and boot scanning analyses. The three NFLGs indeed share the same but novel recombination forms to be composed of B and subtype C, with four subtype B fragments inserted into the pol, vpr, vpu, env, and nef regions of the subtype C backbone (Fig. 2A). A total of eight recombinant break points were found at HXB2 positions according to informative sites analysis. To characterize the recombinant structure, genomic map was obtained by the Map-Draw Tool available at the Los Alamos HIV sequence database (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) (Fig. 2B).

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FIG. 1.

Phylogenetic analyses of the NFLG nucleotide sequence of CRF86_BC. The representing different HIV-1 group M reference sequences were used to construct the neighbor-joining phylogenetic tree. The sequences of CRF86_BC (15YNHS18, 15YNHS23, and 15YNHS26) are marked in •. The stability of the nodes was assessed by bootstrap analysis with 1,000 replications, and only bootstrap values 100 are shown at the corresponding node. The scale bar represents 5% genetic distance. HIV-1, human immunodeficiency virus type 1; NFLG, near full-length genome.

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FIG. 2.

Recombination break point analyses of CRF86_BC. (A) Bootscan analysis was conducted using a window size of 300 bp and a step size of 40 bp along with reference strains of B, C, and representative HIV-1 M subtypes. (B) Genomic structure of CRF86_BC. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). LTR, long terminal repeat.

Subregion tree analyses further confirmed the break points of the three NFLG sequences as follows: I (841–2,638 nt) C, II (2,639–3,192 nt) B, III (3,193–3,508 nt) C, IV (3,509–4,533 nt) B, V (4,534–5,717 nt) C, VI (5,718–6,193 nt) B, VII (6,194–8,498 nt) C, VIII (8,499–8,980 nt) B, and IX (8,981–9,554 nt) C, using HXB2 as a reference. Recombinant structures of these strains were distinct from the any known CRFs reported to date, and the strains were isolated from three HIV-1-infected patients without obvious epidemiological linkage in Yunnan. Therefore, these new recombinants are now designated CRF86_BC. Moreover, subregion tree analyses also revealed that the backbone fragment of subtype C (regions I, III, V, VII, and IX) was most likely from the Indian and Chinese C lineage (IN/CN), and the inserted segments of subtype B (regions II, IV, VI, and VIII) originated from the Thai and Chinese B lineage (TH/CN) (Fig. 3). This finding agreed with the previous many reports, such as CRF07_BC, CRF08_BC, CRF57_BC, CRF64_BC, and the newly identified CRF85_BC.

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FIG. 3.

Subregion tree analyses of the CRF86_BC genome. The phylogenetic trees of the nine mosaic fragments identified by bootscan analysis were constructed using the neighbor-joining method based on the K-2 model in MEGA. The reliability of tree branches was evaluated by 1,000 bootstrap replicates, and bootstrap values more than 0.7 were considered stable. The sequences of CRF64_BC isolates are marked in •. Regions I, III, V, VII, and IX represent subtype C and regions II, IV, VI, and VIII represent subtype B.

The areas on the China–Myanmar border were regarded as the “hotspots” for HIV-1 recombination occurrence. Extensive and complex HIV-1 recombination between the B′, C, and CRF01_AE genotypes have been identified in this region. Various newly identified CRFs of HIV-1, such as CRF57_BC, CRF62_BC, CRF64_BC, and CRF65_cpx, have been identified in Dehong. ⁹ Similarly, Myanmar also has one of the highest rates of HIV-1 infection in Southeast Asia. ¹⁰ Some URFs between two of the three subtypes B, C, and CRF01_AE have also been found in high-risk populations in central Myanmar. ¹¹ A higher prevalence (86.1%) of URFs that cover all four recombinant forms between the subtypes B, C, and CRF01_AE was found in northern Myanmar. ¹² Recently, a novel HIV-1 CRF01_AE/B/C recombinant was described in northern Myanmar. ¹³ In this study, we identified a novel recombinant form (CRF86_BC) in Baoshan prefecture, which is located in the western region of the Yunnan province and shares its border with the Dehong prefecture and Myanmar. Our previously reported CRF78_cpx consisting of CRF01_AE, B′, and C was also detected in this region. ² Taken together, these findings indicated that the high frequency of multiple recombinant events between B′, C, and CRF01_AE were constantly occurring in Baoshan. Furthermore, to verify whether the novel CRF strains circulating in Baoshan have been affected by the HIV-1 variants circulating in the two adjacent regions (Dehong prefecture and Myanmar), further molecular epidemiological survey is needed to monitor the recombination history and phylogenetic linkage of the strains on the HIV epidemic in the China–Myanmar border.

The emergence of CRF86_BC may pose a great challenge for developing an effective HIV-1 vaccine. A better understanding of the evolving complexity of the HIV-1 epidemic will provide important information for the development of HIV-1 vaccine and novel drug for HIV-1. Therefore, it is necessary to strengthen the continuous surveillance of the genetic diversity of HIV-1 in Yunnan, China.

Sequences Data

The NLFG sequences of isolated 15YNHS18, 15YNHS23, and 15YNHS26 have been deposited in GenBank under accession numbers KX582249, KX582250, and KX582251, Supplementary Data; Supplementary Data available online at www.liebertpub.com/aid).