Near Full-Length Genomic Characterization of a Novel Unique Recombinant (CRF01_AE/CRF07_BC) in Fuyang City of China

An important feature of HIV is its extreme genetic diversity attributed to its high-replicative capacity, error-prone nature, and the recombinogenic potential of the reverse transcriptase.¹ Up to now, 103 circulating recombinant forms (CRFs) and many unique recombinant forms (URFs) have been reported originated from the 9 subtypes (A, B, C, D, F, G, H, J, and K) of HIV-1 group M subtypes (www.hiv.lanl.gov). In China, the ratio of HIV infections conferred by CRFs is even >90%, almost all of them are recombinant forms of subtype B, B’, C, or CRF01_AE.^2,3 In recent years, the ratios of HIV infections conferred by CRFs and URFs have increased significantly in China. Up to now, a total of 24 CRFs have been identified in China, indicating that China has become a hot spot for HIV-1 recombination.

Fuyang city is locating in the western area of Anhui province of China, which is close to Henan province and also affected by serious HIV epidemic in former paid plasma donors in 1990s. In recent years, the number of new HIV infections has rapidly increased due to homosexual transmission among men who have sex with men (MSM) in the city.⁴ A recent phylogenetic study of HIV strains prevalent in MSM in Anhui showed that CRF01_AE accounted for 55.6% (74/133) of the infections, followed by CRF07_BC with 32.3% (43/133) and B with 5.3% (7/133).⁵ Obviously, CRF01_AE and CRF07_BC accounted for 87.9% of total HIV-1 infections in MSM in Anhui province. What is more, extensive and complex HIV-1 recombination between the B, C, and CRF01_AE genotypes have been identified in this region, including CRF01/B, CRF01/07, and CRF01_AE/CRF01_B/CRF07_BC.^5–7 Therefore, it is unsurprising to find the second-generation URF originated from CRF01_AE and CRF07_BC in this area.

In this study, we characterized a novel recombinant strain consisting of four segments from CRF01_AE and CRF07_BC, which was obtained from two epidemiologically unlinked HIV-1–positive individuals named FY184 and FY208. Patient FY184 was a 38-year-old unmarried HIV-positive male in Fuyang city, who was infected through homosexual contact and had the CD4+ T cell counts of 673 cells/μL. Peripheral blood was collected on March 14, 2018.

Patient FY208 denied to provide demographic information. The second phone interview was fulfilled to make sure that FY208 had no relationship with FY184. Peripheral blood sample was collected on March 27, 2018. Both participants signed informed consents before investigation and collection of peripheral blood.

For the near full-length genome (NFLG) amplification and sequencing, viral RNA was extracted from plasma samples using a high pure viral RNA kit (Roche, Basel, Switzerland) and reverse transcribed into cDNA by using Superscript IV first-strand synthesis system (Invitrogen). And then, the NFLG was amplified in two halves with High Fidelity Taq (Invitrogen) as described previously.⁸ Polymerase chain reaction positive products were purified and sequenced by Tianyi Huiyuan Life Science and Technology Company (Beijing, China) with several specific primers. All the sequence fragments were edited and assembled into contiguous sequences by using Contig Express software. Finally, the FY184's NFLG of 8,980 bp (from 633 to 9,613 according to HXB2 calibrator) and the FY208's NFLG of 8,930 bp (from 636 to 9,589 according to HXB2 calibrator) were obtained.

The NFLG sequences were submitted to a Basic Local Alignment Search Tool (Blast) to search for more similar sequences, but no sequence with high similarity (>95%) was found. The NFLG sequences collected from this study were further aligned with 29 reference sequences including two HIV-1 reference strains in each subtype and sub-subtype (A1, A2, B, C, D, F1, F2, G, H, J, and K), CRF01_AE, CRF07_BC, CRF08_BC, and one O group reference using HIV Align tool (https://www.hiv.lanl.gov/content/sequence/VIRALIGN/viralign.html).⁹ This alignment was then manually edited using BioEdit software. A phylogenetic tree was constructed by the neighbor-joining method based on Kimura 2-parameter model with 1,000 bootstrap replicates in MEGA6 software. For the purpose of recombination analysis, both sequences were submitted to the Recombinant Identification Program (RIP) (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) online software to obtain the recombinant breakpoints. Furthermore, phylogenetic analysis was further fulfilled based on each segment to explore the origin of each subtype region selected based on the RIP result. In addition to the 29 reference sequences previously used, 12 sequences of different clusters of CRF01_AE were added.¹⁰

The phylogenetic tree showed that the strain formed a distinct and well-supported (bootstrap value = 100%) monophyletic cluster, distantly related to subtype CRF01_AE (Fig. 1). As shown in the recombination analysis result (Fig. 2), the NFLG sequences were composed of CRF01_AE and subtypes CRF07_BC. The mosaic structure of the recombinant genome is composed of four segments as follows: segment I is CRF07_BC (790–1,854 nt), segment II is CRF01_AE (1,855–2,575 nt), segment III is CRF07_BC (2,576–4,748 nt), and segment IV is CRF01_AE (4,749–9,412 nt). Phylogenetic analyses of each subregion indicated that both CRF01_AE segments are originated from cluster 4 of CRF01_AE strains prevalent in China, which is mainly circulating among the MSM population in China.³ Therefore, the recombinant strains are more likely to be formed in MSM population rather than heterosexual or iv drug user population. Meanwhile, it is possible that there is a small region derived from CRF01_AE near the end of the second CRF07_BC region. This would explain why the two sequences fall just outside the CRF07_BC clade in the tree figure, built from this CRF07_BC region of the genome (Fig. 3).

In our study, two NFLG sequences with similar genomic mosaic schematics were identified. Both genomes contained CRF01_AE genomic backbones with two subtype CRF07_BC segments inserted separately. The emergence of the recombinant forms is complaint with the distribution of HIV subtypes circulating locally. Reporting of novel recombinants is helpful for understanding the evolution and genetic diversity of HIV in an area. The appearance of the novel CRF01/07 recombinant strains indicates that the genetic diversity of HIV-1 in this region is becoming higher and higher, which will interfere with the prevention and therapy of HIV infection. The surveillance of HIV epidemic in the area should be strengthened, especially in MSM population, which will facilitate the development of more effective preventive interventions.

Sequences Data

The gene sequences of FY184 and FY208 were deposited in the GenBank with accession nos. MN829458 and MN829459, respectively.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the NSFC (grant nos. 81773493 and 31800149), the State Key Laboratory of Pathogen and Biosecurity (AMMS), Anhui Provincial Program of Prevent Medicine & Public Health (grant no. 2017jk002), and National Grand Program on Key Infectious Disease Control (grant nos. 2017ZX10201101, 2018ZX10721102, and 2018ZX10732101-001-003).