Near Full-Length Genomic Characterization of a Novel HIV Type 1 CRF01_AE/CRF07_BC Recombinant Form Transmitted Between a Heterosexual Couple in Guangxi,China

HIV type 1 group M that contain nine genotypes (A, B, C, D, F, G, H, J, and K) and a great number of recombinant strains play a vital role in HIV-1 transmission. To date, at least 72 circulating recombinant forms (CRFs) and incalculable unique recombinant forms (URFs) have been identified. Therefore, the surveillance for recombinant strains is becoming a major part of HIV prevention and control. China is a country where multiple HIV-1 strains (subtypes/sub-subtypes, and CRFs) cocirculated. Four most prevalent strains include subtype B, CRF01_AE, CRF07_BC, and CRF08_BC, which together consist of more than 90% of all reported cases of infection ¹ . CRF07_BC and CRF08_BC, which can be called first generation CRFs in China, were believed to have generated from Yunnan province about 20 years ago.

In recent years, many different patterns of recombinant forms have been reported in China especially in regions like Yunnan and Jilin provinces where many different genotypes of HIV-1 strains cocirculated among the same risk population ^2,3 . Heterosexual transmission more than injecting drug users (IDUs) has become the major infection routes of HIV-1 epidemic since 2007 in China, and the CRF01_AE subtype is ascendant viral strain among heterosexual transmission population ^4,5 . Guangxi province, located in southwestern China near to Yunnan province and bordering Vietnam in the south, is one of the earliest areas of where cocirculation of multiple HIV-1 strains has been characterized. In previous researches, CRF01_AE and CRF08_BC were the major genotypes in Guangxi, followed by CRF07_BC ^{6 –8} . Cocirculation of multiple virus subtypes could create opportunities for the emergence of novel CRFs and URFs. But for many years, novel recombinant form of HIV-1 in Guangxi was rarely found ⁹ .

In this study, we identify new URFs composed of CRF01_AE and CRF07_BC. The sample was collected in November 2014 from a transmission event among the seroconversion couples in Wuzhou, Guangxi province. Epidemiological data reveal that the patient was female, married, and has no history of blood transfusion, injection drug use, or extramarital heterosexual activity. It's most likely that the husband became infected through intravenous drug use, then transmitted HIV virus to his wife, who has no risk factors for transmission beyond their sexual relationship. The sequence of the index (the husband) was not obtained because of antiviral therapy. The near full-length gene of the patient was amplified because of her different subtypes between pol and gag genes sequences.

The near full-length genome (NFLG) was amplified from the viral RNA, which was extracted from the blood plasma sample using a QIAamp Viral Mini Kit (QIAGEN, Hilden, Germany). The viral RNA was reverse transcribed into cDNA by the SuperScript III First-stand synthesis system Transcriptase (Invitrogen, Carlsbad, CA) following the instructions. The NFLGs were amplified with the template of near-endpoint diluted cDNA by the TaKaRa LA Taq (TaKaRa, Dalian, China) using the same nested polymerase chain reaction amplification conditions in both of the two rounds, as in the literature ¹⁰ . The positive products were purified using a QIAquick Gel Extraction Kit (QIAGEN) and sequenced by an ABI 3730XL sequencer using BigDye terminators (Applied Biosystems, Foster City, CA). All the sequences data were cleaned and assembled using Sequencher v5.1 (Gene Codes, Ann Arbor, MI). A BLAST search was conducted to exclude the potential laboratory cross-contamination. Finally, the sequence was aligned with HIV type 1 group M reference genotypes (A–D, F–H, J, K) and the CRFs (CRF01_AE, CRF08_BC, and CRF07_BC) reported in a previous study in China, which were downloaded from the Los Alamos national Laboratory HIV database (http://hiv-web.lanl.gov/). The codon-aligned nucleotide sequence was aligned with the reference viral strains (HXB2) by the BioEdit v7.1 using the Clustal W.

As shown in the Figure 1, the sequence of GXDA460B is closely related to CRF07_BC reference sequences, but clustered outside of the monophyletic branch. According to the results of that, the Recombinant Identification Program (RIP) (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html) was performed with all default settings except a windows size of 300 to analyze the recombinant structure of the NFLG sequence. The recombination form may contain CRF01_AE, B, C, CRF07_BC, and CRF08_BC (Fig. 2A), and the specific construct that was analyzed using Simplot v 3.5.1¹¹ showed that the NFLG genome was most likely composed of CRF01_AE and CRF07_BC (Fig. 2B). The break point analysis of the sequence (GXDY460B) was performed with the reference sequences (CRF01_AE and CRF07_BC as parents, genotype J as outgroup) according to the result of the last step by the same software using the Bootscan analysis function within the parameters were a window size of 300, a step size of 20. Phylogenetic analysis was performed to confirm the genotype of the mosaic fragments with MEGA 6.0 using the neighbor-joining method, under the Kimura two-parameter model with 1000 bootstrap replications ¹² .

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FIG. 1.

Phylogenetic analysis of the NFLG. A neighbor-joining tree with 1,000 bootstrap replications was performed with the GXDY460B strain and standard reference sequences of HIV type 1 group M downloaded from HIV databases (http://hiv-web.lanl.gov/). The scale bar represents 2% genetic distance (0.02 substitutions per site). NFLG, near full-length genome.

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FIG. 2.

Recombinant Identification Program (RIP) and similarity plots analysis were performed for GXDY460B to identify parental subtypes. (A) Similarity distance analysis was performed using RIP from the Los Alamos National Laboratory HIV Database with default setting except for the window size of 300. (B) Similarity plots for GXDY460B using SimPlot software, with subtype reference strains CRF01_AE, B, C, CRF07_BC, CRF08_BC, and J. These reference strains were verified by RIP, which may contain the components of GXDY460B. CRF, circulating recombinant form.

The NFLG of GXDY460B contained about 12 interlaced segments coinciding with CRF07_BC (I, III, V, VII, IX, and XI) and CRF01_AE(II, IV, VI, VIII, X, and XII) (Fig. 3A).Five mosaic CRF07_BC (I, III, VII, IX, and XI)and six CRF01_AE (II, IV, VI, VIII, X, and XII;) were well identified by phylogenetic trees that the segments could cluster together with the reference strains of CRF07_BC or CRF01_AE with high bootstrap values (Supplementary Figure: Phylogenetic Analyses; Supplementary Data available online at www.liebertpub.com/aid). Based on the break points message from the phylogenetic tree, we can ensure that the recombinant form of GXDY460B was between CRF07_BC and CRF01_AE. The one CRF07_BC fragment (V) was too short (about 300 bp) to distinguish the specific subtype among the CRF07_BC, CRF08_BC and C. Region 2 and region 9 were used to identify the special subtypes with a reference sequence of CRF01_AE and CRF07_BC from China, because they were fairly large regions.The break points positions refer to the HXB2 coordinates, and they were located by the HIV Sequence Locator (www.hiv.lan.gov/content/sequence/LOCATE/locate.html). The location to the HXB2 of the recombinant were as follows: CRF07_BC (790–1184 nt), CRF01_AE (1185–2018 nt), CRF07_BC (2019–3537 nt), CRF01_AE (3538–4275 nt), CRF07_BC (4276–4589 nt), CRF01_AE (4590–5018 nt), CRF07_BC (5019–5897 nt), CRF01_AE (5898–6125 nt), CRF07_BC (6126–8322 nt), CRF01_AE (8323–8504 nt), CRF07_BC (8505–9041 nt), CRF01_AE (9042–9399 nt; Fig 3B).

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FIG. 3.

Bootscan plots and schematic representation of the GXDY460B NFLG's mosaic structure. (A) Bootscan plots of GXDY460B using CRF01_AE, CRF07_BC, and subtype J as subtype references. The parameters of SimPlot Bootscan analysis were set as default except for a window size of 300 and step size of 10. The break point positions refer to HXB2 coordinates located by the HIV Sequence Locator (www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). (B) Genomic map of GXDY460B. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

The predominant genotypes of HIV-1 including CRF01_AE, CRF08_BC, and CRF07_BC circulating in Guangxi mostly originated in IDU populations ¹³ . But in recent years, the main risk factor of HIV-1 infection has been transferred to the heterosexual population. CRF01_AE was first identified in China among the population of sexual transmission and IDUs in Yunnan and Guangxi province ^{14 –16} . It has been reported that CRF01_AE is a predominant genotype and is infected by heterosexuals in Guangxi province. CRF07_BC is one of the four major subtypes in Guangxi associated with heterosexual activity and IDUs. Although HIV-1 in Guangxi was found relatively late, its multiple transmission mode brings a big challenge to the prevention and control of HIV-1^17–20. According to previous research, the interaction of different infected population may produce a new recombination form ^9,21,22 . The recombinant strain has been reported among IDUs in southwest and northwest China ²³ . Furthermore, the recombination form between CRF01_AE and CRF07_BC has been found among men who have sex with men (MSM) population in Yunnan, Jilin, and Beijing ^22,24,25 . Cocirculation of multiple virus subtypes and multiple infection routes have existed for many years in Guangxi, but the recombinant strain was rarely reported among heterosexual transmission population because of its lower crowd confounding degree than MSM and IDUs. It is the first time that the URF between CRF01_AE and CRF07_BC was identified during heterosexual transmission in Guangxi.

In summary, we identified a new recombination form among heterosexual couple in Guangxi, which shows a new URF has emerged. Recombination between HIV-1 genotypes is the important mechanism to increase the viral strains complexity of HIV-1. URFs are associated with dual infections or super-infections in IV drug users and they are a symptom of an IVDU epidemic problem, but studying them will not end the problem or reduce new infections. The solutions involve drug treatment, needle exchange programs, and/or other ways to slow the spread of HIV within the IVDU and perhaps education methods or other interventions to help prevent the spread of the viruses out of the IVDU communities and into the heterosexual populations.