Citation: Minghui An, Wei Song, Bin Zhao, Xue Dong, Lin Wang, Wen Tian, Xin Li, Lu Wang, Zhenxing Chu, Junjue Xu, Haibo Ding, Xiaoxu Han, Hong Shang. The Establishment and Spatiotemporal History of A Novel HIV-1 CRF01_AE Lineage in Shenyang City, Northeastern China in 2002–2019 .VIROLOGICA SINICA, 2021, 36(6) : 1668-1672.

The Establishment and Spatiotemporal History of A Novel HIV-1 CRF01_AE Lineage in Shenyang City, Northeastern China in 2002–2019

  • Corresponding author: Xiaoxu Han,, ORCID: 0000-0003-1427-8428
    Hong Shang,, ORCID: 0000-0001-5333-8943
  • Received Date: 24 May 2021
    Accepted Date: 28 June 2021
    Published Date: 23 August 2021
    Available online: 01 December 2021

  • 加载中
    1. An M, Han X, Zhao B, English S, Frost SDW, Zhang H, Shang H (2020) Cross-continental dispersal of major HIV-1 CRF01_AE clusters in China. Front Microbiol 11: 61
        doi: 10.3389/fmicb.2020.00061

    2. Chen J, Young NL, Subbarao S, Warachit P, Saguanwongse S, Wongsheree S, Jayavasu C, Luo CC, Mastro TD (1999) HIV type 1 subtypes in Guangxi Province, China, 1996. AIDS Res Hum Retrovir 15: 81–84
        doi: 10.1089/088922299311754

    3. Chen X, Ye M, Wang Y, Zhang C, Zheng YT (2020) Laos is affected by HIV CRF01_AE and the newly identified CRF97_01B. Virol Sin 35: 538–547
        doi: 10.1007/s12250-020-00215-4

    4. Cheng H, Zhang J, Capizzi J, Young NL, Mastro TD (1994) HIV-1 subtype E in Yunnan, China. Lancet 344: 953–954

    5. Dong MJ, Peng B, Liu ZF, Ye QN, Liu H, Lu XL, Zhang B, Chen JJ (2019) The prevalence of HIV among MSM in China: a largescale systematic analysis. BMC Infect Dis 19: 1000
        doi: 10.1186/s12879-019-4559-1

    6. Feng Y, He X, Hsi JH, Li F, Li X, Wang Q, Ruan Y, Xing H, Lam TT, Pybus OG, Takebe Y, Shao Y (2013) The rapidly expanding CRF01_AE epidemic in China is driven by multiple lineages of HIV-1 viruses introduced in the 1990s. AIDS 27: 1793–1802
        doi: 10.1097/QAD.0b013e328360db2d

    7. Han X, Dai D, Zhao B, Liu J, Ding H, Zhang M, Hu Q, Lu C, Goldin M, Takebe Y, Zhang L, Shang H (2010) Genetic and epidemiologic characterization of HIV-1 infection In Liaoning Province. China J Acquir Immune Defic Syndr 53(Suppl 1): S27-33

    8. Han X, An M, Zhang M, Zhao B, Wu H, Liang S, Chen X, Zhuang M, Yan H, Fu J, Lu L, Cai W, Takebe Y, Shang H (2013) Identification of 3 distinct HIV-1 founding strains responsible for expanding epidemic among men who have sex with men in 9 Chinese cities. J Acquir Immune Defic Syndr 64: 16–24
        doi: 10.1097/QAI.0b013e3182932210

    9. Hemelaar J, Elangovan R, Yun J, Dickson-Tetteh L, Kirtley S, Gouws-Williams E, Ghys PD, Isolation W-UNfH, Characterisation, (2020) Global and regional epidemiology of HIV-1 recombinants in 1990–2015: a systematic review and global survey. Lancet HIV 7: e772–e781
        doi: 10.1016/S2352-3018(20)30252-6

    10. Li X, Liu H, Liu L, Feng Y, Kalish ML, Ho SYW, Shao Y (2017) Tracing the epidemic history of HIV-1 CRF01_AE clusters using near-complete genome sequences. Sci Rep 7: 4024
        doi: 10.1038/s41598-017-03820-8

    11. Liu B, Sullivan SG, Wu Z (2007) An evaluation of needle exchange programmes in China. AIDS 21 (Suppl 8): S123–128
        doi: 10.1097/

    12. Vrancken B, Mehta SR, Avila-Rios S, Garcia-Morales C, Tapia-Trejo D, Reyes-Teran G, Navarro-Alvarez S, Little SJ, Hoenigl M, Pines HA, Patterson T, Strathdee SA, Smith DM, Dellicour S, Chaillon A (2020a) Dynamics and dispersal of local HIV Epidemics within San Diego and across the San Diego-Tijuana Border. Clin Infect Dis.
        doi: 10.1093/cid/ciaa1588

    13. Vrancken B, Zhao B, Li X, Han X, Liu H, Zhao J, Zhong P, Lin Y, Zai J, Liu M, Smith DM, Dellicour S, Chaillon A (2020b) Comparative circulation dynamics of the five main HIV types in China. J Virol 94(23): e00683–20.
        doi: 10.1128/JVI.00683-20

    14. Wang X, He X, Zhong P, Liu Y, Gui T, Jia D, Li H, Wu J, Yan J, Kang D, Han Y, Li T, Yang R, Han X, Chen L, Zhao J, Xing H, Liang S, He J, Yan Y, Xue Y, Zhang J, Zhuang X, Liang S, Bao Z, Li T, Zhuang D, Liu S, Han J, Jia L, Li J, Li L (2017) Phylodynamics of major CRF01_AE epidemic clusters circulating in mainland of China. Sci Rep 7: 6330
        doi: 10.1038/s41598-017-06573-6

    15. Xia Y, Pan XH, Zhang JF, He L, Luo MY, Jiang J, Yao JM, Wang H (2020) Analysis of new HIV-1 infection in MSM with seroconversion determined by limiting antigen avidity enzyme immunoassay. Zhonghua Yu Fang Yi Xue Za Zhi 54: 1232–1236 (In Chinese)

    16. Yu XF, Chen J, Shao Y, Beyrer C, Lai S (1998) Two subtypes of HIV-1 among injection-drug users in southern China. Lancet 351: 1250
        doi: 10.1016/S0140-6736(05)79316-8

    17. Zhang J, Xu JJ, Song W, Pan S, Chu ZX, Hu QH, Yu H, Mao X, Jiang YJ, Geng WQ, Shang H, Wang N (2018) HIV incidence and care linkage among MSM first-time-testers in Shenyang, China 2012–2014. AIDS Behav 22: 711–721
        doi: 10.1007/s10461-017-1840-4

    18. Zhao B, Song W, An M, Dong X, Li X, Wang L, Liu J, Tian W, Wang Z, Ding H, Han X, Shang H (2021) Priority intervention targets identified using an in-depth sampling HIV molecular network in a non-subtype B epidemics area. Front Cell Infect Microbiol 11: 642903
        doi: 10.3389/fcimb.2021.642903

  • 加载中

Figures(1) / Tables(1)

Article Metrics

Article views(5063) PDF downloads(7) Cited by()

Proportional views

    The Establishment and Spatiotemporal History of A Novel HIV-1 CRF01_AE Lineage in Shenyang City, Northeastern China in 2002–2019

      Corresponding author: Xiaoxu Han,
      Corresponding author: Hong Shang,
    • 1. NHC Key Laboratory of AIDS Immunology (China Medical University), National Clinical Research Center for Laboratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang 110001, China
    • 2. Key Laboratory of AIDS Immunology, Chinese Academy of Medical Sciences, Shenyang 110001, China
    • 3. Key Laboratory of AIDS Immunology of Liaoning Province, Shenyang 110001, China
    • 4. Department of Food Safety and Nutrition, Shenyang Center for Health Service and Administration Law Enforcement(Shenyang Center for Disease Control and Prevention), Shenyang 110001, China


    • Dear Editor,

      In China, HIV-1 CRF01_AE has always been one of the most prevalent subtypes, especially among sexually active population, since it was initially reported in Yunnan and Guangxi Provinces during the early 1990s (Cheng et al. 1994; Yu et al. 1998; Chen et al. 1999). Multiple independent introductions of CRF01_AE have led to the establishment of at least eight epidemic lineages in China, which have been circulating among different high-risk populations and have variable prevalence and geographic distribution (Li et al. 2017). Four main lineages were dominant among heterosexuals/injecting drug users (IDUs) and men who have sex with men (MSM); of these, two homosexual lineages accounted for about 60% cases (Wang et al. 2017), in line with that homosexuals are the most high-risk population in China nowadays (Dong et al. 2019). Shenyang is located in Northeastern China with > 8 million population and attract migrants from neighboring regions, which might directly or indirectly affect the HIV epidemic in neighboring cities, owing to the frequent population exchange. In Shenyang, multiple HIV subtypes have co-existed and heterosexuals, IDUs, and those who received blood transfusions were identified as the main risk groups historically (1997–2004) (Han et al. 2010). Presently, MSM became the most high-risk group with an HIV prevalence of 10.8% (Zhang et al. 2018), and the HIV epidemic was mainly driven by two lineages of CRF01_AE among this population, accounting for > 70% of all HIV-1 infected individuals in Shenyang. The transmission dynamics of viral lineages would reveal the epidemic characteristics of HIV through time and space and could provide key information for the performed interventions such as targeting the viral sources, sinks, and hubs throughout the viral transmission chains (Vrancken et al. 2020a, 2020b).

      We collected 3866 HIV-1 CRF01_AE pol sequences (HXB2: 2253-3314 nt) from a local HIV-1 drug-resistance database in Shenyang during 2002-2019. Of them, 1631 were from a follow-up cohort in the AIDS Clinic of the First Hospital of China Medical University, which accounted for over half of the HIV-1 infected individuals in Shenyang during 2002-2015; and 2235 were from all newly diagnosed HIV-1 cases in Shenyang during 2016-2019. In order to identify CRF01_AE epidemic of Shenyang, we reconstructed a phylogenetic maximum likelihood (ML) tree using the 3866 sequences collected in Shenyang and reference sequences (Li et al. 2017), and we found that except for three previously reported lineages (Li et al. 2017), a novel CRF01_AE lineage was circulating in Shenyang (Fig. 1A and Table 1). Moreover, the individuals of the novel lineage have significant characteristics for the variables of sex, age, risk group, location, occupation, education and marital status, compared with others (Table 1). This lineage was more prevalent among men born in the 1960s to 1970s who injected drug. Within this novel lineage, more individuals lived in Dadong and Shenhe District, most of whom were unemployed, divorced/widowed and graduated from high school. Next, we downloaded all available HIV-1 CRF01_AE sequences (HXB2: 2253-3314 nt; n = 10, 736) from 48 countries sampled between 1990 and 2019 in the Los Alamos HIV database ( The phylogenetic reconstruction revealed that although the novel lineage was unique to any reported lineage (support value = 100) and no one from the 10, 736 sequences was within the novel lineage, 40 Asian sequences (21 Thailand, 14 China, two Vietnam, two Singapore and one Myanmar); three North American sequences; five European sequences; and one Australian were observed within a branch outside this novel lineage (support value = 81) (Fig. 1B).

      Figure 1.  The identification and spatiotemporal history of a novel HIV-1 CRF01_AE lineage in Shenyang. A Phylogenetic analysis of all 3866 CRF01_AE sequences collected in Shenyang during 2002–2019. The blue shadows represent the previously reported HIV-1 CRF01_AE lineages in China and the red shadow represents the novel lineage in this study (branch support value = 0.7). B The phylogenetic analysis of 63 sequences in the novel lineage, and all available CRF01_AE pol sequences downloaded from the Los Alamos HIV database (LANL). The red shadow represents the novel lineage, and the similar sequences to the novel lineage (branch support value = 0.81), which are used in the time-scaled phylogenetic analysis. The both maximum likelihood trees (A and B) are reconstructed under GTR + I + G model with 1, 000 replicates in IQ-Tree v1.6.12. C The maximum clade credibility tree under the GTR + I + G substitution model, log normal relaxed molecular clock model, and non-parametric Bayesian SkyGrid mode performed in BEAST v1.10.4. The branch colors depict sampling locations. D Migration events of the novel lineage over Administrative Districts (a) and risk groups (b) in Shenyang City. The direction and number of all migration events (BF > 10) are presented here. Left and right sides are the source and destination of migration events, respectively. The fraction of two sides represents the proportion of migration events from sources towards destinations. MSM, men who have sex with men; HTS, heterosexuals; IDUs, injecting drug users

      Total(n=2235) Novel lineage(n=48) P-value
      Male 2101 38(79.17%) < 0.001
      Female 132 9(18.75%)
      Unknown 2 1(2.08%)
      Birth period
      < 1960 167 1(2.08%) < 0.001
      1960s 323 20(41.67%)
      1970s 333 21(43.75%)
      ≥1980 1410 5(10.42%)
      Unknown 2 1(2.08%)
      Risk groupa
      MSM 1839 11(22.92%) < 0.001
      HTS 320 2(4.17%)
      IDUs 41 34(70.83%)
      Unknown/other 35 1(2.08%)
      Dadong 298 16(33.33%) < 0.001
      Shenhe 187 14(29.17%)
      Heping 285 4(8.33%)
      Tiexi 295 4(8.33%)
      Huanggu 320 4(8.33%)
      Yuhong 231 2(4.17%)
      Hunnan 188 1(2.08%)
      Unknown/other 431 3(6.25%)
      Officer 204 1(2.08%) < 0.001
      Unemployed 1395 43(89.58%)
      Famer 57 1(2.08%)
      Unknown/other 579 3(6.25%)
      Primary school 100 1(2.08%) < 0.001
      Junior high school 572 26(54.17%)
      Senior high school 475 17(35.42%)
      College 1056 3(6.25%)
      Unknown/other 32 1(2.08%)
      Marital status
      Single 1360 14(29.17%) < 0.001
      Married 465 11(22.92%)
      Divorced/widowed 404 22(45.83%)
      Unknown/other 6 1(2.08%)
      a: MSM, men who have sex with men; HTS, heterosexuals; IDUs, injecting drug users.

      Table 1.  The demographic characteristics of CRF01_AE-infected individuals among all newly diagnosed cases during 2016–2019

      We also used Bayesian molecular clock and phylogeographic approaches to reconstruct its epidemic history over time and space and identify transmission hotspots to guide targeting interventions. After verification of temporal signals in TempEst, the time-scale phylogenetic tree, reconstructed using 63 sequences from Shenyang, 56 sequences close to the novel lineage, and 42 reference sequences (An et al. 2020), revealed that the median time to most recent common ancestor (tMRCA) of the novel lineage was 2006.44, with a narrow time range corresponding to the 95% highest posterior density (HPD) interval of 2003.56–2008.07. The long branch away from the node of the novel lineage was also observed in the maximum clade credibility (MCC) tree (Fig. 1C), similar to the ML tree (Fig. 1A and 1 B). Additionally, the novel lineage had been further divided into two sub-lineages. The tMRCAs of HIV-1 CRF01_AE from Africa and Thailand were estimated to be 1970.68 and 1981.07, respectively, of which the 95% HPD were 1960.67–1982.97 and 1979.58–1985.8, respectively (Fig. 1C), consistent with previous studies (Feng et al. 2013; Han et al. 2013). The estimated evolutionary rate based on pol sequences (HXB2: 2253–3314 nt) was 1.36 (1.121.59) 9 × 10–3 substitutions/site/year. Finally, we estimated the well-supported direction and number of migration events based on the BF > 10 calculated under Bayesian stochastic search variable selection (BSSVS) using phylogeographic inference (Fig. 1D). Dadong District was the initial and dominant source (77.27%), from which the virus had spread to Heping and Shenhe District. Shenhe and Heping were the secondary centers, of which Shenhe was also the main destination (59.09%), suggesting it may be a potential hub of the viral spread. Between risk groups, the virus had been transmitted by IDUs to MSM, who were the main risk population of HIV-1 infection in Shenyang, accounting for 84.62% of all migration event.

      Preliminary phylogenetic analyses (Fig. 1A and 1B) revealed that the novel lineage was independent of other previously reported CRF01_AE lineages (Li et al. 2017), and no sequence was clustered within this novel lineage. Furthermore, the tMRCA of the novel lineages was 2006 (Fig. 1C), far later than other known lineages that typically appeared during the 1980s and 1990s in China (Feng et al. 2013). Notably, there were more Southeast Asian sequences located outside the lineage (Fig. 1C), suggesting that the ancestral strain of the lineage was introduced to Shenyang directly or indirectly from other Southeast Asian countries(Chen et al. 2020; Hemelaar et al. 2020) and the novel lineage has most likely sustained only by local transmission since its entrance to Shenyang, and yet not spread elsewhere. However, among newly diagnosed cases in Shenyang between 2016 and 2018, recent infections could be found in this lineage (Zhao et al. 2021), according to the results of HIV-1 limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) (Xia et al. 2020); this predicted that the novel lineage was spreading and required urgent interventions to prevent its expansion.

      The viral migration history (Fig. 1D) revealed that IDUs living in Dadong District were viral sources and the strains have been circulating in some main districts of Shenyang (Dadong, Shenhe, Heping, Huanggu, Tiexi and Hunnan). As a large part of the transmissions from Dadong and toward Shenhe District, interventions focusing on the two areas would likely reduce viral spreading on a large scale. Moreover, Shenyang, as the capital of Liaoning Province and the political, economic, and cultural center of Northeast China, attracts more labor migration, rural–urban migration, and circular population migration; this could directly or indirectly affect the HIV epidemic of neighboring areas.

      Additionally, identifying the individuals who are more likely to be at the highest risk of being infected or infecting others will be directly actionable in HIV prevention. From the demographic information of the patients of the novel lineage (Table 1), more elders (> 50 years) and IDUs were observed, most of who were living in Dadong and Shenhe District, coinciding with the Bayesian inferences (Fig. 1D). Owing to the needle exchange programs, from 2000, that freely provided needles to IDUs, both the HIV incidence and prevalence among IDUs have rapidly decreased (Liu et al. 2007). Now MSM have become the dominant risk group for HIV infection. Obviously, the number of MSM was followed by IDUs among all newly diagnosed cases in Shenyang during 2016–2019 (Table 1). Importantly, in this novel lineage, the strains have been transmitted from IDUs to MSM (Fig. 1D), indicating that this lineage has the potential to further expand via homosexual contact. Other demographic characteristics related to occupation, marital status and education would also provide more information to identify the intervention targets (Table 1). Further, more individuals who have marriage history were observed within lineage, inferring that they as the higher risk transmitter would have more chances to transmit virus to their partners via heterosexual contact.

      Taken together, the interventions targeting geographical hotspots and high-risk groups would have direct benefits on reducing risks of HIV transmission and infection. Here, a focus on IDUs and MSM in Dadong and Shenhe District, such as enhancing health education and increasing testing frequency, will likely have the maximum effect in controlling the epidemic of this novel lineage locally and elsewhere. Such continued molecular monitoring can uncover more active viral transmission chains to ensure rapid response to new situations of the HIV epidemic.

    • We wish to thank the staff of the First Affiliated Hospital of China Medical University and Shenyang Center for Disease Control and Prevention, and all participants of this study. This work was funded by the Mega-projects of National Science Research for the 13th Five-Year Plan (2018ZX10721102-006-003); the National Natural Science Foundation of China (81871637); CAMS Innovation Fund for Medical Sciences (2019-I2M-027); and Scientific Research Funding Project of Liaoning Province Education Department (QN2019005).

    • The authors declare no competing interest.

    • This study was approved by the Medical Ethics Committee of the First Hospital of China Medical University and informed consent was obtained (ethics number 2019–153-2 and 2019–150-2).

    Figure (1)  Table (1) Reference (18) Relative (20)



    DownLoad:  Full-Size Img  PowerPoint