Identification of a Novel HIV-1 Recombinant Form (B/C) in Men Who Have Sex with Men in Shaanxi,China

Abstract

Many new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) of HIV-1 have been found in men who have sex with men (MSM) in recent years, in China. In this study, a unique HIV-1 recombinant genome (SN153) was characterized from an HIV-positive male infected through homosexual behavior in Shaanxi, China. The mosaic pattern had a complex intersubtype recombinant structure with six breakpoints, with three subtype C segments inserted into subtype B backbone. And three similar breakpoints with CRF07_BC were observed in the pol gene. The identification of the new URF suggested the genetic complexity of the HIV epidemic among MSM in Shaanxi province and the urgent need for epidemiological surveillance and their origin of the new recombination forms.

Because of the extensive genetic diversity, a substantial increase of HIV-1 subtypes, circulating recombinant forms (CRFs) and unique recombinant forms, has been found so far among different high-risk populations in China. Cocirculation and ongoing transmissions of different subtypes and CRFs in one area contribute to the continuing emergence of new intersubtype recombinants. Subtypes B and C circulating at the same time have already created new CRFs in China, including CRF07_BC,¹ CRF08_BC,² CRF57_BC,³ CRF61_BC,⁴ CRF62_BC,⁵ CRF64_BC,⁶ CRF85_BC,⁷ CRF86_BC,⁸ and CRF88_BC.⁹

Men who have sex with men (MSM) were marked as one of the most important risk populations of HIV-1 in China.¹⁰ From January to October in 2016, 46.4% cases of HIV-1 infection newly diagnosed in Shaanxi province were infected through MSM.¹¹ CRF01_AE, CRF07_BC, and subtype B were three predominant subtypes identified among MSM in a previous study.¹² In this study, the near full-length genome (NFLG) of a novel recombinant form (SN153) was harvested, deriving from subtypes B and C, which is different from any previously reported B/C recombinants.

Whole blood sample was collected from a 51-year-old man (SN153) on August 13, 2015, during a HIV-1 molecular epidemiology survey. The CD4+ T cell count was 57 cells/μL. Informed consent was signed before the sample collection. The patient (SN153) was married and diagnosed as HIV-1 positive in June 2015 through voluntary counseling and testing because of homosexual behavior. His CD4+ T cell count increased from 146 cells/μL in March, 2016, to 235 cells/μL in March, 2017, after receiving antiretroviral therapy in September, 2015. This study was reviewed by the Institutional Research Ethics Community of the Shaanxi Center for Disease Control and Prevention.

The viral RNA was extracted from plasma using QIAamp Viral RNA Mini Kit (QIAGEN) and then reversely transcribed into cDNA (SuperScript III Reverse Transcriptase; Invitrogen, Carlsbad, CA). The NFLG was amplified by nested polymerase chain reactions (nest-PCRs). The positive PCR products were sequenced and assembled by Sequencher v5.1. The NFLG sequence of SN153 was first codon aligned with subtyping reference sequences from the Los Alamos National Database (www.hiv.lanl.gov). Phylogenetic tree and subregion tree analyses were constructed by MEGA 6.05 using the neighbor-joining method with 1,000 bootstrap replications. Similarity plot and Bootscan analyses for SN153 were done using SimPlot software v3.5.1 to position the putative combination breakpoints. The parameters were a window size of 300 bp, a step size of 10 bp, and the neighbor-joining method using the Kimura two-parameter model with replicates of 500.

The NFLG sequence of SN153 was 8954 bp corresponding to the HXB2 nucleotide positions: 634–9,597. As shown in Figure 1, SN153 was much closer to subtype B clade beyond subtype C and other BC strains. The recombinant pattern analyses by Simplot v.3.5.1 revealed that the NFLG of SN153 composed of three subtype C segments inserted into the subtype B backbone, with the subtype B sequence (RL42), subtype C sequence (95IN21068), and subtype K sequence (96CM) as references (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid). There were six recombinant positions in the NFLG sequence of SN153 with five located in pol gene and one in vpr gene. The mosaic map of the new recombinant form SN153 is as follows: segment I (HXB2 nt 634–2632) from subtype B; segment II (HXB2 nt 2633–3046) from subtype C; segment III (HXB2 nt 3047–3333) from subtype B; segment IV (HXB2 nt 3334–4765) from subtype C; segment V (HXB2 nt 4766–4999) from subtype B; segment VI (HXB2 nt 5000–5827) from subtype C; and segment VII (HXB2 nt 5827–9597) from subtype B (Fig. 2). Subregion tree analyses confirmed the recombinant genetic origins (Supplementary Fig. S2). Segments I, III, V, and VII were clustered with subtype B. Segments I and VII might derive from subtype B-Thai, reference RL42 from China, with bootstrap values of 0.85 and 1.00, respectively. Segments II, IV, and VI were clustered with subtype C. Segment IV might derive from India C lineage with the bootstrap value 0.94.

FIG. 1.

A neighbor-joining phylogenetic tree of SN153 was constructed using MEGA 6.05 with reference sequences where the solid circle represents SN153. The stability of each node was assessed by bootstrap tests with 1,000 replicates and the bootstrap values >70% are shown at the corresponding nodes.

FIG. 2.

(A) Bootscanning analysis of the NFLG of SN153. Subtype B (RL42) and subtype C (95IN21068) are used as putative parental reference sequences, and subtype K (96CM) is used as an outgroup. The parameters of bootscanning analysis are as follows: window size of 300 bp; step size by 10 bp; tree algorithm, neighbor; distance model, Kimura; bootstrap replicate, 500. (B) The recombinant map of SN153 NFLG mosaic compared with CRF07_BC, created using the recombinant HIV-1 drawing tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). NFLG, near full-length genome.

Unlike other BC recombinants, CRF07_BC, CRF08_BC, CRF57_BC, CRF61_BC, CRF62_BC, CRF64_BC, CRF85_BC, CRF86_BC, and CRF88_BC were substituted by subtype B fragments into the subtype C parental backbone, whereas SN153 was the subtype B parental backbone with three subtype C fragments. Three similar breakpoints were observed between CRF07_BC and SN153 (Fig. 2), suggesting that those sites were hot recombinant regions. B/C recombinants sharing similar recombinant breakpoints with CRF07_BC or CRF08_BC were also observed in a previous report.¹³ It is still uncertain whether there has been a “parental” B/C recombinant creating the patterns of the shared recombinant positions. It needs to find more B/C recombinant forms for further investigation.

In this study, we reported a novel B/C recombinant form from the MSM population in Shaanxi province, with three segments from subtype C inserted into subtype B background. It suggested that the genetic lineages of HIV and newly recombinant strains found in MSM were comparatively complicated.³ The new emerging recombinant forms indicated that the local HIV-1 epidemic was active and molecular epidemiology should be reinforced.

Sequence Data

The NLFG sequence of SN153 has been deposited in GenBank under accession number MG742702.

Footnotes

Acknowledgements

This study was supported by Shaanxi province key S&T special projects of disease prevention and control (2014A5), and the authors thank the staff of Weinan CDC, Shaanxi Province, for their kind efforts in case identification, sample and epidemic data collection.

Author Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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