Near Full-Length Genomic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF107_01B) Identified Among Men Who Have Sex with Men in Heilongjiang Province of China

Human immunodeficiency virus type 1 (HIV-1) has high rates of mutation and replication generating various subtypes in the epidemic. ¹ Recombination between subtypes leading to a great deal of circulating recombinant forms (CRFs) and unique recombinant forms (URFs). ² As of July 2020, the Los Alamos National Laboratory HIV Database website (www.hiv.lanl.gov/conent/sequence/HIV/mainpage.html) recorded 105 CRFs and multiple URFs globally. Among them, China reported 22 kinds of CRFs, of which 17 kinds of CRFs are related to sexual transmission routes. Now, the main popular subtypes in China are CRF01_AE, CRF07_BC, and subtype B. ³ In this environment, a number of CRFs are forming by the random reorganization of CRF01_AE, CRF07_BC, and subtype B, such as CRF100_01C, CRF101_01B, CRF102_0107, CRF103_01B, and CRF104_0107. CRF100_01C was reported in 2018 among heterosexuals infected people in Yunnan, China. ⁴ CRF101_01B was reported in intravenous drug users in Yunnan in 2019. ⁵ CRF102_0107 was reported in the men having sex with men (MSM) population in Anhui in 2019, it is a recombinant strain formed by the second-generation recombinants CRF07_BC and CRF01_AE. ⁶ CRF103_01B was reported in Hebei in 2019, and may be exist in other provinces such as Beijing and Guizhou. ⁷ CRF104_0107 was identified form MSM at a sentinel surveillance site in Xi'an city, China, in 2020. ⁸

We used the pol gene region sequences collected in the laboratory in 2018. After adding reference strains, using Aliview software, a Neighbor-Joining phylogenetic tree was built. We found a cluster composed of six sequences (The cluster) in pol gene region, and The cluster had bootstrap values of 99%. Then sequences were analyzed by using the near full-length genome (NFLG) amplification and sequencing. Amplification and sequencing procedures of NFLG were performed as described previously. ⁹ Viral RNA experiments of subjects 151964 and 15010481 failed, whereas subjects HL18S17, HL18S37, HL18S214, and HL18S214 generated a new CRF (CRF107_01B). The reliability of tree branches was evaluated by 1,000 bootstrap replicates. The branches of HL18S17, HL18S37, HL18S214, and HL18S244 were marked with black diamond (Fig. 1). These patients were all men and unmarried. The other information such as specific age, Sample City, viral load, and CD4 count are shown in Table 1.

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FIG. 1.

Phylogenetic tree analysis of the NFLG sequences of CRF107_01B and the reference strain downloaded from the HIV database were used to construct the neighbor-joining phylogenetic tree including all subtype references of China. The reliability of tree branches was evaluated by 1,000 bootstrap replicates. The branches of HL18S17, HL18S37, HL18S214, and HL18S244 are marked with black diamond. NFLG, near full-length genome.

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Table 1.

Demographic Characteristics of Study Subjects Infected with CRF107_01B

Strain name	Gender	Risk factor	Age (years)	Ethnic group	Marital status	Education level	Sample date	Sampling region	Viral load	CD4 count
HL18S17	Male	MSM	26	Han	Unmarried	College	March 16, 2018	Xiang Fang, Heilongjiang	74,800	886
HL18S37	Male	MSM	22	Han	Unmarried	High school	March 29, 2018	Dao Li city, Heilongjiang	96,000	460
HL18S214	Male	MSM	26	Han	Unmarried	Senior	April 16, 2018	Ha Erbing, Heilongjiang	6.1 × 10^5	348
HL18S244	Male	MSM	39	Han	Unmarried	College	June 16, 2018	Ha Erbing, Heilongjiang	2.21 × 10^6	27

MSM, men having sex with men.

The recombination analysis was performed with Simplot 3.5.1, and analysis was set by a sliding window of 500 nucleotides (nt) advanced in 20 nt increments (Fig. 2). Recombination breakpoint analysis showed that the sequences consisted of CRF01_AE and subtype B. The map of the recombinant genome was as follows: I_B (790–2,199 nt), II_{CRF01_AE} (2,200–2,599 nt), III_B (2,600–4,999 nt), IV_{CRF01_AE} (5,000–5,699 nt), and V_B (5,700–9,500 nt) (Fig. 3). An NFLG phylogenetic tree showed that CRF107_01B was generated by three B (Western origin) segments and two CRF01_AE segments. CRF01_AE segments were described as cluster 5 lineage of CRF01_AE (CRF01-5).

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FIG. 2.

Recombination breakpoint analyses of CRF107_01B. (A) SimPlot was drawn by SimPlot3.5.1 software. The reference sequences (CRF01_AE and B) were similar to HL18S17, HL18S37, HL18S214, and HL18S244, and the subtype P was used as an out-group. (B) Bootscan plots showed that CRF107_01B consisted of CRF01_AE, and subtype B, and this graph has a window size of 500 bp and a step size of 20 bp. (C) The mosaic map of CRF107_01B was obtained using the Recombinant HIV-1 Drawing Tool of HIV database (https://www.hiv.lanl.gov/content/index). CRF, circulating recombinant form. Figures can be viewed in greater detail online.

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FIG. 3.

The phylogenetic trees of five mosaic segments (from I to V) defined by neighbor-joining in MEGA 6, and the bootstrap values of 80% and higher are shown. The sequences of CRF107_01B are marked with a solid black circle. CRF01_AE reference sequences is mainly divided into six unique lineages (CRF01-1–CRF01-6) in China. These CRF01_AE lineages were used as reference sequences in this article. Figures can be viewed in greater detail online.

CRF107_01B is the 18th CRF recorded by HIV database and formed by CRF01_AE and subtype B. Among the 18 CRFs, only CRF51_01B and CRF107_01B are formed with subtype B as the backbone, and the CRF01_AE fragments insert into the B backbone. CRF51_01B was reported in the sexually transmitted population in Singapore in 2011. One CRF01_AE fragment inserts into the pol gene region and the other inserts into the vif gene region of the B backbone. ¹⁰ Similar to CRF51_01B, CRF107_01B is also the insertion of two CRF01_AE fragments into the B backbone. However, the insertion positions of the two CRFs are different. The two insertion regions of CRF107_01B are pol and env, respectively. In addition to CRF55_01B, ¹¹ CRF59_01B, ¹² CRF67_01B, ¹³ CRF68_01B, ¹³ and CRF103_01B, ⁷ CRF107_01B is the sixth CRF formed by recombination of CRF01_AE and subtype B found in China.

In conclusion, we identified a novel HIV-1 CRF (CRF107_01B) among four patients without an obvious epidemiological linkage in Heilongjiang Province, China. The emergence of CRF107_01B makes the HIV epidemic more complicated.

Sequence Data

The sequences have been deposited in GenBank with accession numbers MT712388–MT712391.