Near Full-Length Genome Sequence of a Novel HIV-1 Recombinant Form (CRF01_AE/07_BC) Detected Among Men Who Have Sex with Men in Zhejiang,China

HIV/AIDS remains to be one of the most important threats to public health in China. From 2006 to 2014, men who have sex with men (MSM) have accounted for a sharply increasing proportion from 2.5% to 25.8% among newly reported HIV/AIDS cases nationwide. ¹ A national cross-sectional study showed a 4.9% of overall prevalence of HIV infection among the MSM population. ² Zhejiang, standing on the southeastern coast, is one of the economic developed provinces in China, attracting large number of migrants from all parts of country annually. Since Zhejiang identified the first HIV-1 case in 1985, ³ the cumulative number of reported HIV/AIDS cases has reached 14,703 by the end of 2013, becoming the highest number of reported cases in southeastern China. CRF01_AE and CRF07_BC are two predominant HIV-1 circulating strains in Zhejiang. Among MSM, the proportion of CRF01_AE and CRF07_BC is 64.2% and 31.7%, respectively. ⁴ It is certain that cocirculation of various subtypes among MSM can accelerate the generation of a novel HIV-1 recombinant form.

Here, we detected a novel HIV-1 second-generation recombinant form (CRF01_AE/CRF07_BC), designated as 16ZJ305, by near full-length genome (NFLG) analyses, isolated from an HIV-positive consenting male patient among the MSM population in Zhejiang Province, which is different from the CRF01_AE/CRF07_BC recombinant viruses previously reported in China.

In this study, an HIV-1-positive plasma sample was collected from a 27-year-old male merchant (Patient 16ZJ305) in the provincial capital city of Zhejiang Province, Hangzhou. 16ZJ305 is a Chinese citizen of Han ethnicity, residing in Xiaoshan District, which is the biggest district of Hangzhou. He is an individual businessman after graduation from college. He was diagnosed as HIV positive on April 30, 2016 after he had suffered a long-term oral ulcer and was recruited from the Zhejiang Provincial Center for Disease Control and Prevention (ZJCDC). A self-report by 16ZJ305 shows that he was infected through homosexual behavior. 16ZJ305 was highly active antiretroviral therapy treatment naive before his blood sample was collected. His CD4⁺ T cell count was 6 cells/μL and viral load was 70,800 copies/mL on May 17, 2016 when the plasma was collected. Written informed consent was obtained from the subject before sample collection, and was approved by the institutional review board of the First Affiliated Hospital, School of Medicine, Zhejiang University.

The amplification and sequencing of the new generated NFLG were performed as previously described. ⁵ Recombination breakpoints were determined using RIP and jpHMM (www.hiv.lanl.gov) and SimPlot. Breakpoint confirmation and origin of each segment were then analyzed by phylogenetic trees. The standard subtype reference alignment file, including all HIV-1 group M, group P, CRF01_AE, CRF07_BC, and CRF08_BC, was downloaded from the Los Alamos National Laboratory HIV Database (www.hiv.lanl.gov). Nucleotide sequences were first aligned using HIV Align (www.hiv.lanl.gov) and then adjusted by using BioEdit. Phylogenetic trees were constructed in FastTree + FigTree using maximum likelihood method with GTR model and 1,000 bootstrap replications to confirm the subtype of mosaic fragments. ⁶ Branches with bootstrap values >0.7 were considered as phylogenetic clusters.

The NFLG sequence amplified from Patient 16ZJ305 was 9,001 nucleotides (nt) in size (relative to the HXB2 nucleotide numbering system: positions 638 to 9642) and the blast search data show that no evidence of sample contamination was detected. As shown in Figure 1, 16ZJ305 clustered with CRF07_BC, CRF08_BC, and C subtype reference sequences (bootstrap value 100%), but formed a distinct monophyletic branch different from CRF07_BC, CRF08_BC, and C subtype reference sequences.

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FIG. 1.

Phylogenetic tree analysis of the near full-length genome nucleotide sequence of the subject, 16ZJ305. The tree was constructed through the maximum likelihood method with 1,000 bootstrap replicates. The solid black circle denotes 16ZJ305. All reference sequences of the HIV-1 group M subtypes and CRFs were downloaded from the HIV database (www.hiv.lanl.gov/). The scale bar represents a 0.04 genetic distance. CRF, circulating recombinant form.

SimPlot analysis shows that the NFLG sequence of 16ZJ305 was composed of CRF01_AE and CRF07_BC, with one region of CRF01_AE inserted into a CRF07_BC backbone (Fig. 2A). And bootscanning analysis of the NFLG sequence reveals that the two unique recombination breakpoints corresponded to HXB2 nucleotide positions 6398 and 8742, located in tat/rev and env gene regions, respectively, and divided the NFLG into three regions: region I (HXB2, 638-6397), CRF07_BC; region II (HXB2, 6398-8741); region II I (HXB2, 8742-9642) (Fig. 2B). Similar results were obtained using online software RIP (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) and jpHMM-HIV (http://jphmm.gobics.de/submission_hiv.html). The final map results of the schematic structure are shown in Figure 2C. The profile of the recombinant structure of 16ZJ305 is distinct from that of the CRF01_AE/CRF07_BC recombinants previously reported in China.

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FIG. 2.

Recombinant analyses of the near full-length genome nucleotide sequence of 16ZJ305. (A) The simplot analysis between 16ZJ016 and the reference sequences. (B) Bootscanning analysis of 16ZJ305 using CRF01_AE, CRF07_BC, and subtype J as reference sequences. The bootscan window was 300 bp with a step size of 20 bp using SimPlot version 3.5.1. (C) The results of the Recombinant HIV-1 Drawing Tool with the two breakpoints of 16ZJ305 according to HXB2 nucleotide sequence numbering.

Subregion tree analysis of 16ZJ305 futher confirmed the two recombinant breakpoints identified by bootscanning analysis (Fig. 3). It also demonstrated that the CRF01_AE region (II) in 16ZJ305 belonged to CRF01_AE subcluster 4 lineage, but formed a distinct monophyletic branch from CRF01_AE - Cluster4. A potential reason for this could be that there was a very short region of CRF 07_BC inserting into CRF01_AE region (HXB2 8339-8401). The CRF07_BC regions (I and III) in 16ZJ305 clustered with CRF07_BC reference sequences (bootstrap value 97.7% and 100%), and belonged to CRF07_BC Cluster 1. In summary, the parental origins of the CRF01_AE and CRF07_BC regions of novel second-generation recombinant (16ZJ305) could be from CRF01-4 and CRF07-1.

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FIG. 3.

The subregion trees of one CRF01_AE and two CRF07_BC segments of 16ZJ305 were established by the methods shown in Figure 1 and based on the recombinant breakpoints shown in Figure 2C.

Recombination between different HIV-1 subtypes is a primary mechanism that contributes to the genome complexity of HIV-1, and new recombinant strains are frequently found in regions where various subtypes are prevalent. CRF01_AE and CRF07_BC are two predominant HIV-1 circulating strains among MSM in Zhejiang. ⁷ To date, two NFLG sequences of HIV-1 CRF01_AE/CRF07_BC second-generation recombinants have been identified in Zhejiang Province. 15zj032 comprised three CRF01_AE and four CRF07_BC fragments, ⁸ whereas 15zj016 with three fragments of CRF07_BC was inserted into the CRF01_AE backbone. ⁹ In the study, a novel circulating recombinant forms (CRF) recombinant strain 16ZJ305 was characterized with a CRF01_AE fragment inserted into a CRF07_ BC parental backbone. Although the three novel second-generation recombinants are unrelated variants, they may indicate that diverse recombination forms exist between CRF01_AE and CRF07_BC prevailing in the Zhejiang Province. Since CRF_01AE and CRF_07BC have become the two major strains of HIV-1 in China especially among young MSM populations, ¹⁰ increasing recombinant strains of two strains have been reported in Beijing, Guangxi, Jilin, Sichuan, and Shanxi. ^{5,9,11 –13} The emergence of new second-generation recombinants indicated the presence of complex transmission networks of different HIV subtypes/CRF infections among sexual transmissions in this region. Therefore, an effective and continuous HIV-1 molecular epidemiologic monitoring among high-risk populations is indispensable, which is of great importance in understanding the dynamics of the HIV-1 epidemic in China.