Genetic Characterization of a Novel HIV-1 CRF07_BC/CRF55_01B Recombinant Form Identified in Jiangmen,China

Increasingly, recombination, with high mutation and replication rates, is posing problems in public health. Up to now, 98 circulating recombinant forms (CRFs) of HIV-1 have been defined (www.unaids.org/en/resources/fact-sheet). In China, the dominant genotypes of HIV include subtype B as well as CRF01_AE, CRF07_BC, and CRF08_BC. ^{1 –4} In addition, the prevalence of CRF55_01B ⁵ and the recombinant of CRF55_01B and CRF07_BC ⁶ is increasing. In the past decade, a sustained rise in unique recombinant forms (URFs) of HIV-1 have been observed in men who have sex with men (MSM) population due to multiple sexual partners and both homosexual and heterosexual transmissions. ⁷

In this study, we identified a new URF consisting of 07_BC and 55_01B. The specimen JM05 was collected in June 2018 from a man who claimed to be MSM and was detected positive for HIV-1 at the sexually transmitted diseases clinic in Jiangmen, Guandong province, China. He was married, but his spouse was negative for HIV-1. His CD4⁺ T cell count was 271 cells/μL. This study was approved by Medical Ethics Committee of Jiangmen Center for Disease Control and Prevention, and written informed consent was signed before sample collection.

RNA was extracted from plasma specimen and then reverse transcribed into cDNA, as described in the previous research. ⁶ In particular, the genomic sequences obtained by nested polymerase chain reaction (PCR) amplification were submitted to the HIV-1 BLAST Tool to detect whether it had sample contamination, and then were analyzed by online HIV-1 Sequence Quality Control Tool (https://www.hiv.lanl.gov/content/sequence/QC/index.Html) to exclude errors introduced in PCR.

The near full-length genome (NFLG) of JM05 was 8,510 nucleotides in length and aligned with reference sequences by using maximum likelihood methods in the MEGA v7.0.26 software to construct the phylogenetic trees (Fig. 1). One approach to analyze similarity between the NFLG of JM05 and HIV-1 reference strains was to use Simplot 3.5.1 software. Results revealed that JM05 was a recombinant form most likely composed of CRF07_BC and CRF055_01B (Fig. 2A). Bootscan analysis was subsequently carried out to position the recombination break points by using Recombination Detection Program v.4.95 (RDP4).

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FIG. 1.

Phylogenetic analysis of JM05_NFLG. The maximum-likelihood method based on the Kimura two-parameter model and 1,000 bootstrap replicates were applied. A black solid circle (•) marks JM05. NFLG, near full-length genome.

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FIG. 2.

Recombination breakpoint analyses of the novel identified JM05. (A) Similarity analysis between JM05 and reference sequences was performed using SimPlot 3.5.1. (B) Bootscan analysis was performed using RDP4 software configured with CRF07_BC (JX960601), CRF55_01B (KF927150), and subtype H (AF005496) as putative parental reference sequences. The x-axis shows nucleotide position of the new sequence. The y-axis shows the bootstrap value. (C) The results of recombination mapping analysis using the Recombinant HIV-1 Drawing Tool. The dotted line indicates an area similar to CRF55_01B. Color images are available online.

We identified eight unique recombinant points at nt 4954, 5230, 6037, 7045, 7942, 8369, 9002, and 9233, respectively (Fig. 2B). The breakpoints location confirmation and interlaced segments were also implemented in MEGA using the neighbor-joining trees. A mosaic pattern of the new strain JM05 was shown in Figure 2C. In addition to the sites identified by Simplot with window size 350 and RDP with window size 350, we observed a small region at positions 3,072–3,196, which seems to be like CRF55_01B, and the bootstrap value is <70%. Therefore, we show this as a dotted line area in Figure 2C.

Previous study has shown that CRF07_BC accounts for a high proportion of the HIV-infected MSMs in China. The first CRF07_BC strain was isolated from intravenous drug users in Yunnan, ⁸ and subsequently emerged in MSM population. HIV-1 CRF55_01B was first reported in 2012 and has been rapidly expanding in MSMs, ⁹ especially in the Pearl River Delta of China. ¹⁰ In 2014, an outbreak of CRF55_01B infection was reported in MSM population in the Southern city Shenzhen. ¹¹ A number of novel recombinant forms between CRF07_BC and CRF55_01B have been described. ^{12 –14} However, the mosaic structures of the five URFs consisting of CRF07_BC and CRF55_01B are different and may not come from the same ancestor (Fig. 3).

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FIG. 3.

Comparison of genomic structure of JM05 with other recombinant forms of CRF07_BC/CRF55_01B. HBX2 genomic pattern is used as the reference structure at the top of the plot. The red bar indicates the subtype B regions, whereas the green bar for CR55_01B, and the white bar for CRF07_BC. Color images are available online.

For exploring in drug mutations of the virus variant, the pol gene sequence was submitted to the HIV Drug Resistance Database (https://hivdb.stanford.edu/). Two mutations were found, M230I and V179E, which are uncommon and result in resistance to non-nucleoside reverse transcriptase inhibitors: M230I, which is responsible for an intermediate level of resistance to Nevirapine and Rilpivine, and V179E, which causes a low-level resistance to Efavirenz and Etravirine. Our findings indicate the potential drug resistance of the new URF and the need of continuous monitoring the spread of drug-resistant mutations.

In summary, we report a novel recombinant form of HIV-1 isolated from a male patient in Jiangmen, China. Identifying new recombinant viruses will contribute to the control of HIV/AIDS pandemic.

Sequence Data

The genome sequence has been deposited in GenBank with the accession number of MK697709.