Geographical Distribution of HIV-1 Genotypes Among MSM in 9 Chinese Cities
Figure 2 illustrates the HIV-1 genotype distribution among MSM in 9 Chinese cities (Table 1). The CRF01_AE genotype was most prevalent in 7 of the 9 cities (all except Chengdu and Dongguan): prevalence of CRF01_AE ranged from 25.0% in Dongguan to 81.0% in Shenyang. As shown in Figure 1A, we detected 2 distinct phylogenetic clusters of CRF01_AE (designated China's MSM clusters 1 and 2). Among the MSM subjects, 97.5% (353 of 362) of the circulating CRF01_AE strains belonged to either cluster 1 (157 of 362, 43.4%) or cluster 2 (196 of 362, 54.1%). A small fraction of the CRF01_AE strains (9 of 362, 2.5%) share a similar phylogenetic ancestry with strains found in typical Thailand CRF01_AE groups (Table 1; Fig. 1A). The prevalence of CRF01_AE cluster 1 is higher than that of cluster 2 in most cities, except Shenyang (capital of Liaoning province, northeastern China), where cluster 2 is the predominant lineage among the CRF01_AE strains (20.2% for cluster 1 vs. 77.5% for cluster 2) and among all HIV-1 strains circulating in Shenyang (165 of 263, 62.7%). CRF01_AE cluster 2 strains were detected in Beijing (24 of 163, 14.7%), Shanghai (3 of 20, 15.0%), Nanjing (2 of 18, 11.1%), and Changsha (2 of 23, 8.7%) but not in Jinan (0 of 10), Chengdu (0 of 63), Dongguan (0 of 16), or Kunming (0 of 7) (Table 1).
The HIV-1 CRF07_BC was detected among MSM in the 9 cities. The CRF07_BC cluster 3 accounted for 88.7% (94 of 106) of the CRF07_BC strains among Chinese MSM (Table 1; Fig. 1B). The prevalence of CRF07_BC cluster 3 ranged from 3.7% in Shenyang (10 of 263) to 47.6% in Chengdu (30 of 63). Chengdu and Dongguan were the only 2 cities where CRF07_BC cluster 3 prevalence (49.2% and 43.8%, respectively) was higher than CRF01_AE prevalence (31.7% and 12.5%, respectively) (Table 1).
In contrast, HIV-1 subtype B (United States–European), which is the predominant strain among MSM in Western countries,18–21 played a smaller role in China between 2009 and 2011. However, the presence of subtype B was significant in large Chinese cities: Beijing (30.7%, 50 of 163) and Shanghai (25.0%, 5 of 20) (Table 1). In contrast, subtype B was not detected in southern provincial cities: Changsha (0 of 23), Dongguan (0 of 16), and Kunming (0 of 7) (Table 1; Fig. 1C). Although sample sizes from these regions were small, the absence of subtype B may indicate that the HIV-1 epidemic among these MSM is largely influenced by locally common strains, either CRF01_AE or CRF07_BC. Different from the CRF01_AE and CRF07_BC clusters that are common across different cities, no trans-regional spread of subtype B strains was detected in this study, except for some local transmission clusters, such as those in Shenyang and Beijing (Fig. 1C). HIV-1 subtype B′, an important lineage of the viral strains responsible for the blood-borne HIV-1 epidemic in China, was rarely found (4 of 583, 0.7%) among Chinese MSM (Table 1; Fig. 1C). The geographical distribution of the HIV-1 strains and the distinct phylogenetic clusters (CRF01_AE clusters 1 and 2, and CRF07_BC cluster 3) responsible for the HIV-1 epidemic among Chinese MSM between 2009 and 2011 are schematically illustrated in Figure 2.
The genetic distances within CRF01_AE cluster 1, cluster 2, and the typical Thailand CRF01_AE groups were 0.030 ± 0.008, 0.028 ± 0.007, and 0.056 ± 0.011, respectively. This data agrees with the neighbor-joining phylogenetic tree, which shows that the sequences in CRF01_AE clusters 1 and 2 are more closely linked than those in the typical Thailand CRF01_AE groups. The genetic distance within CRF07_BC cluster 3 (0.012 ± 0.005) is the lowest among the 3 clusters and far lower than the genetic distance of the other CRF07_BC group (0.029 ± 0.006). The sequences in CRF07_BC cluster 3 were most homologous. The genetic distances within CRF01_AE clusters 1 and 2 and typical Thailand CRF01_AE groups are significantly different (P < 0.01), as are the genetic distances within CRF07_BC cluster 3 and the other CRF07_BC group (P < 0.01) (Fig. 3).
Evolutionary Characterization of the 3 Distinct HIV-1 Clusters Identified Among MSM in China
To determine the epidemic origins of the 3 major HIV strains among Chinese MSM, we estimated the tMRCA of the HIV-1 clusters (CRF01_AE clusters 1 and 2 and CRF07_BC cluster 3) by using the relaxed molecular clock model based on the nucleotide sequences of 1.0-kb pol gene. The analyses were carried out using nucleotide sequences of either the CRF01_AE or CRF07_BC strains determined in this study. Respective reference sequences sampled between 1990 and 2010 were downloaded from the Los Alamos HIV sequences database. The estimated evolutionary rates were 2.70 (2.05 – 3.42) ×10–3 and 1.51 (1.00 – 2.12) × 10–3 substitutions per site per year for CRF01_AE and CRF07_BC under the GTR + γ4 relaxed clock models with a constant-size model. The results were essentially similar to those previously estimated for HIV-1 CRF01_AE or CRF07_BC.21 As shown in the maximum clade credibility tree (Fig. 4), the median tMRCAs of clusters 1–3, calculated in a GTR + γ4 relaxed-clock model with a constant size model, were estimated to be 1999.1 (95% CI: 1996.2 to 2002.2), 2001.2 (95% CI: 1998.0 to 2004.3), and 2001.4 (95% CI: 1999.8 to 2004.8), respectively. Evolutionary and statistical assumptions do no significantly affect the estimated dates, and the effective sample size was >200.
This study represents the first large-scale (n = 583) HIV molecular epidemiological study of MSM that included multiple regions with varying degrees of HIV prevalence: Kunming and Chengdu >10%; Shenyang, Beijing and Nanjing 5%–10%; and Jinan, Changsha, Shanghai, and Dongguan 1%–5%.22 Our study identified 3 distinct lineages of HIV-1 strains (CRF01_AE clusters 1 and 2 and CRF07_BC cluster 3) (Table 1; Fig. 2) that accounted for more than three-quarters of the MSM HIV-1 infections in 9 Chinese cities (447 of 583, 76.7%) between 2009 and 2011 (Table 1). The CRF01_AE is the most prevalent HIV-1 genotype among Chinese MSM, accounting for 62.1% (362 of 583) of HIV-1 infections, followed by CRF07_BC (106 of 583, 18.2%). Notably, the pol sequence data sets from recently published MSM studies in Beijing,5,23,24 Shijiazhuang (capital of Hebei province, near Beijing),9 and Zhengzhou (capital of Henan province, near Beijing)10 also contain these 3 variants, and the overall trend of their reported prevalence agreed with our findings (see Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A417). Although the prevalence of subtype B (of United States–European origin) previously predominated among Chinese MSM,4 it accounted for only 15.9% (93 of 583) in this study. Subtype B′ (of Thai origin) played an even smaller role among Chinese MSM (4 of 583, 0.7%). The recent shift from subtype B to CRF01_AE prevalence among Chinese MSM agrees with the results from Beijing,5,23,24 Shijiazhuang,9 and Zhengzhou10 and further supports our finding that no widespread subtype B transmission cluster was detected although small local transmission clusters were found. Because of recruitment limitations, our sample sizes differed between cities and do not reflect regional HIV prevalence. Some cities yielded large samples, such as Shenyang and Beijing, whereas other heavily affected regions, such as Kunming, yielded relatively few subjects. Therefore, our estimate of HIV genotype distributions may be biased. However, we focused on the 3 distinct HIV-1 founding strains responsible for the growing HIV/AIDS epidemic among MSM in our 9 study cities, and at least 1 of the 3 clusters predominated in each of the cities.
The origins of these 3 lineages remain unknown. Database surveys found no sequences outside China that closely related to these 3. Surprisingly, we found that the vast majority of previously reported CRF01_AE sequences in heterosexual and IDU populations in Yunnan,25,26 Guangxi,27,28 and Fujian29 were on different phylogenetic branches from the 2 CRF01_AE clusters. However, many CRF01_AE sequences from the Liaoning heterosexual population7 have close phylogenetic links to MSM cluster 2. Additionally, a few CRF01_AE sequences from Yunnan30 or Guangxi28 infected cases with unknown transmission route and the heterosexual population in Liaoning7 and Beijing23 and Jiangsu IDUs31 were closely related to MSM cluster 1 (Fig. 1A). Similar to CRF01_AE cluster 1, none of the CRF07_BC sequences available in the database (n = 487) from non-MSM high-risk populations belonged to CRF07_BC cluster 3 except for a few CRF07_BC strains from Guangdong IDUs (6 of 35)32 (Fig. 1B). The close phylogenetic relationship between MSM and other high-risk population may imply the origin of 3 lineages, however, we cannot exclude the possibility that the above cases may also have homosexual contacts.
In addition, we observed indications suggesting the geographical origins of the viral strains. Although our sample sizes differed and several were insufficient for drawing definitive conclusions, we saw some patterns in the geographical distribution of these 3 lineages (Fig. 2; Table 1): (1) CRF01_AE cluster 1 was found throughout China but more frequently in the southern and eastern coastal regions [Kunming/Yunnan (4 of 7, 57.1%); Nanjing/Jiangsu (8 of 13, 61.5%); Shanghai (9 of 20, 45.0%); and Jinan/Shangdong (5 of 10, 50%)] than in other regions (χ2 = 17, P < 0.001). In contrast, (2) CRF01_AE cluster 2 was more highly concentrated in Shenyang/Liaoning in northeastern China (198 of 311, 63.7%) than in other regions (<20%) (χ2 = 198, P < 0.001). Finally, (3) CRF07_BC cluster 3 was most common in south-central provinces [Chengdu/Sichuan (30 of 63, 47.6%); Changsha/Hunan (9 of 23, 43.5%); Dongguan/Chuangdong (7 of 16, 43.8%)] (χ2 = 92, P < 0.001). These observations suggest that clusters 1, 2, and 3 may originate from southern-eastern coastal, northern, and south-central regions in China, respectively. Alternatively, the apparent distribution differences may simply reflect differences in the founding effects of the variants among the MSM populations of different regions.
In summary, the results of phylogenetic analysis based on pol sequences and the geographical distribution analysis matched the estimates in our recent MSM phylodynamic study on primary HIV infection based on viral near-full-length sequences.16 This agreement suggests that (1) CRF01_AE cluster 1 may have originated in southern/southwestern provinces and spread north along the southeastern coast, and that (2) CRF01_AE cluster 2 strains may have originated around Yunnan province, but the strains could have spread directly to the northeast. However, maximum clade credibility tree analyses (Fig. 4) revealed that the initial appearances of cluster 1 and cluster 3 strains could not be separated into a clear time gradient and that the number of some cluster 2 strains was insufficient to draw definitive time-related conclusions. These limitations do not negate the aforementioned suggestions. Rather, the lack of a clear time gradient implies that the spread of cluster 1 strains among MSM probably occurred in a short time, and that MSM from different regions across China likely had close contact. More historical samples and longer HIV sequences may be helpful to investigate the definitive origins of the 3 lineages responsible for China's MSM epidemic.
As shown in Figure 3, the nucleotide diversity of CRF07_BC cluster 3 (0.012 ± 0.005) is lower than that of CRF01_AE cluster 1 (0.030 ± 0.008) and cluster 2 (0.028 ± 0.007) (P < 0.01). The high level of genetic homogeneity of CRF07_BC cluster 3 suggests that it emerged later than clusters 1 and 2 among Chinese MSM. However, the estimated evolutionary rate was lower for CRF07_BC than CRF01_AE under the GTR + γ4 relaxed-clock models with a constant-size model. Consistent with this finding, Bayesian molecular clock analyses estimated that tMRCAs for clusters 1, 2, and 3 are 1999, 2001, and 2001, respectively (Fig. 4). To reduce the impact of uneven sample sizes in the Bayesian MCMC analysis, we selected as many sequences as possible from regions with smaller sample sizes and only a few phylogenetically representative sequences from large-sample regions, such as Shenyang, Beijing, and Chengdu. Taken together, these data indicate that the CRF07_BC cluster 3–driven epidemic among MSM occurred later than that driven by CRF01_AE clusters 1 and 2.
In this study, we detected a total of 18 other recombinant strains (18 of 583, 3.1%) that harbored recombination break points in the 1.0-kb pol gene. We noticed that B/CRF01_AE recombinants were remarkably common in Dongguan (5 of 16, 31.2%), Nanjing (3 of 18, 16.7%), and Changsha (2 of 23, 8.7%) (Table 1). The genotyping method used in this study was based on partial genome sequences, covering only 10% of the HIV-1 genome. The prevalence of recombinants may be underestimated if the break points located outside the genome region were analyzed. So the true prevalence of recombinants in Dongguan, Nanjing, and Changsha could be considerably higher than we noted. The epidemic trend of the new recombinants merits further study.
In conclusion, using a large study population with recent samples, we demonstrated that more than three-quarters of recent HIV-1 infections among Chinese MSM were caused by 3 distinct lineages of HIV-1 strains (CRF01_AE clusters 1 and 2; CRF07_BC cluster 3). These lineages are responsible for the recent upsurge in the HIV/AIDS epidemic among Chinese MSM, demonstrating a remarkable founding effect. This could, in turn, suggest that Chinese MSM engage in extremely high-risk behaviors and warrant urgent implementation of effective preventive measures to limit the epidemic.
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HIV-1; CRF01AE; CRF07BC; men who have sex with men; phylogenetic tree analysis; Bayesian molecular clock analysis
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