Abstract
Recombinant forms contribute substantially to the genetic diversity of HIV-1. Recent studies have also revealed that three major viral strains (CRF07_BC, CRF01_AE, and subtype B) have been cocirculating among men who have sex with men (MSM) in Anhui, suggesting a high probability of generating new recombinants. In this study, we reported a novel CRF01_AE and CRF07_BC HIV-1 recombinant form in MSM in Fuyang city of China. Two near full-length genome (NFLG) named FY184 and FY208 were successfully obtained. The genomic composition analysis of the NFLG reveals that it was divided into four segments by three breakpoints, with two regions of CRF07_BC inserted into a CRF01_AE backbone's gag and pol regions. The CRF01_AE regions were originated from a subcluster lineage of CRF01_AE, which is mainly circulating among MSM in China. The emergence of a novel recombinant of CRF01_AE/CRF07_BC is indicative of the increasing genetic diversity of the HIV epidemic in MSM in Anhui.
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 (
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 (
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).
Phylogenetic tree analysis. A neighbor-joining tree was created with full-length genomes of our FY184 and FY208 strain (labeled with two black dots) sequences and the reference sequences of subtype A–D, F–H, J, K, CRF01_AE, CRF07_BC, CRF08_BC, and group O (
RIP was performed for MN829458 and MN829459 to identify parental subtypes. 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. RIP, recombinant identification program.
Subregion tree analyses of FY184 and FY208 genomes.
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.
Footnotes
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).