Background
HIV-1 CRF65_cpx strain carries drug-resistant mutations, which raises concerns about its potential for causing virologic failure. The CRF65_cpx ranks as the fourth most prevalent on Hainan Island, China. However, the origin and molecular epidemiology of CRF65_cpx strains in this area remain unclear. This study aims to estimate the spatial origins and dissemination patterns of HIV-1 CRF65_cpx in this specific region.

Methods
Between 2018 and 2021, a total of 58 pol sequences of the CRF65_cpx were collected from HIV-positive patients on Hainan Island. The available CRF65_cpx pol sequences from public databases were compiled. The HIV-TRACE tool was used to construct transmission networks. The evolutionary history of the introduction and dissemination of HIV-1 CRF65_cpx on Hainan Island were analyzed using phylogenetic analysis and the Bayesian coalescent-based approach.

Results
Among the 58 participants, 89.66% were men who have sex with men (MSM). The median age was 25 years, and 43.10% of the individuals had a college degree or above. The results indicated that 39 (67.24%) sequences were interconnected within a single transmission network. A consistent expansion was evident from 2019 to 2021, with an incremental annual addition of four sequences into the networks. Phylodynamic analyses showed that the CRF65_cpx on Hainan Island originated from Beijing (Bayes factor, BF = 17.4), with transmission among MSM on Hainan Island in 2013.2 (95%HPD: 2012.4, 2019.5), subsequently leading to an outbreak. Haikou was the local center of the CRF65_cpx epidemic. This strain propagated from Haikou to other locations, including Sanya (BF > 1000), Danzhou (BF = 299.3), Chengmai (BF = 27.0) and Tunchang (BF = 16.3). The analyses of the viral migration patterns between age subgroups and risk subgroups revealed that the viral migration directions were from "25–40 years old" to "17–24 years old" (BF = 14.6) and to "over 40 years old" (BF = 17.6), and from MSM to heterosexuals (BF > 1000) on Hainan Island.

Conclusion
Our analyses elucidate the transmission dynamics of CRF65_cpx strain on Hainan Island. Haikou is identified as the potential hotspot for CRF65_cpx transmission, with middle-aged MSM identified as the key population. These findings suggest that targeted interventions in hotspots and key populations may be more effective in controlling the HIV epidemic.