Identification and characterization of a novel HIV-1 circulating recombinant form (CRF59_01B) identified among men-who-have-sex-with-men in China

Abstract

The HIV-1 epidemic among men-who-have-sex-with-men (MSM) continues to expand in China. A large-scale national survey we conducted on HIV-1 strains among MSM in 11 provinces in China from 2008 to 2013 (n = 920) identified a novel transmission cluster consisting of six strains (0.7%) that belonged to a new circulating recombinant form (designated CRF59_01B). CRF59_01B contains two subtype B segments of U.S.-European origin (in the pol and vpu-env regions) in a CRF01_AE backbone. CRF59_01B is the second CRF (after CRF55_01B) circulating primarily among MSM in China. CRF59_01B occurs at a low frequency (less than 1%), but it was detected in four different provinces/regions in China: Liaoning (northeast China) (n = 3); Hunan (central China) (n = 1); Guangdong (south China) (n = 1); Yunnan (southwest China) (n = 1). One additional recombinant strain was detected in a heterosexual individual in Liaoning province but is not the focus of this paper. Bayesian molecular clock analyses indicate that CRF59_01B emerged as a result of recombination between CRF01_AE and subtype B around the year 2001. The emergence of multiple forms of recombinants and CRFs reflects the ever-increasing contribution of homosexual transmission in China's HIV epidemic and indicates an active HIV transmission network among MSM in China.

Figure 1. HIV-1 genotype distribution among MSM in China and map of China depicting the occurrence of CRF59_01B.

(A) The overall genotype distribution of HIV-1 strains among MSM newly diagnosed during 2008–2013 in 11 provinces in China (n = 920). (B) Map of China. Study sites (11 provinces) and the proportion of CRF59_01B among HIV-1 strains in their respective provinces.

Figure 2. Neighbor-joining tree analysis of the near full-length nucleotide sequences of CRF59_01B.

The neighbor-joining tree was constructed using the near full-length nucleotide sequences (8.8 kb) (HXB2: 790–9600 nt) of CRF59_01B strains identified in six epidemiologically-unlinked individuals [five MSM (Bi) and one heterosexual] from China (Table 1). These strains are compared with the reference sequences of all known subtypes/sub-subtypes as well as CRFs relevant to this study, including CRF15_01B, CRF34_01B and CRF52_01B from Thailand; CRF51_01B from Singapore; CRF33_01B, CRF48_01B, CRF53_01B and CRF54_01B from Malaysia; and CRF55_01B from China (http://www.hiv.lanl.gov/content/index). Bootstrap values (>70) of the respective nodes are indicated. 01th = Thailand CRF01_AE; 01af = African CRF01_AE; Chinese MSM clusters 1 and 2 = CRF01_AE variants associated with transmission among MSM in China.

Figure 3. Recombination breakpoint analyses of CRF59_01B.

(A) Bootscanning plot analysis. Analyses were performed using CRF01_AE (90TH.CM240) and subtype B (83FR.HXB2) as parental subtypes, and subtype C (95IN21068) as an out-group with a moving window of 350 nt with a step of 50 nt. Numbers represent nucleotide positions relative to the HXB2 genome. (B) The deduced subtype structure. Black = subtype B (of U.S.-European origin); gray = CRF01_AE; blank = no sequence data available. (C) Subgenomic phylogenies estimated using the neighbor-joining method from alignments representing Regions I, III, V (CRF01_AE) and the concatenated II+IV (subtype B) region. Bootstrap scores greater than 70% are indicated at corresponding nodes. 01th = Thailand CRF01_AE; 01af = African CRF01_AE; clusters 1 and 2 = CRF01_AE variants associated with transmission among MSM in China.

Figure 4. Maximum clade credibility (MCC) trees of CRF59_01B.

The MCC tree was obtained by Bayesian Markov Chain Monte Carlo (MCMC) analysis of the concatenated CRF01_AE [(Regions I+III+V) (HXB2: 790–2569; 2719–6418; 8244–9600)] regions and subtype B [(Regions II+IVa) (HXB2: 2570–2718; 7626–8243)] regions, using a relaxed clock model in GTR+G4 with a constant coalescent model. Analyses were implemented in BEAST v.1.6.0. HIV-1 subtype C sequences are used as an out-group. The medians of the estimated tMRCAs with 95% highest probability density (HPD) (in parentheses) and the posterior probability (>0.95) of the nodes relevant to this study are indicated. (C) The distribution of the posterior probability of the estimated tMRCAs for CRF59_01B as well as the related CRF01_AE lineages (top) and subtype B lineages (bottom).