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By using molecular network technology to analyze the transmission characteristics of the CRF55_01B strain in Yunnan Province, we aimed to identify high-risk transmitters, trace the origin time, location, transmission routes and transmitted drug resistance(TDR) of the virus, and provide a data basis for subsequent surveillance. Demographic data and laboratory test results were collected from all newly diagnosed and treatment-naïve individuals infected with the CRF55_01B strain in Yunnan Province, from January 2020 to December 2024. The molecular network of the CRF55_01B strain was constructed via the paired gene distance method, with Cytoscape 3.10.1 software employed to visualize and analyze the network. BEAST software was used to construct the MCC tree and estimate posterior probabilities, while SpreaD3 was employed to visualize spatiotemporal diffusion and calculate Bayes factors for transmission path analysis. Drug resistance analysis was conducted via the Stanford HIV Drug Resistance Database (HIVdb). A total of 68 newly diagnosed and treatment-naïve patients infected with the CRF55_01B strain in Yunnan Province were enrolled. With 0.018 as the optimal genetic distance threshold, a molecular network was constructed, yielding a network access rate of 57.35% (39/68). A total of 14 molecular clusters were formed, most of which consisted of two sequences. Among 68 participants, 8.82% (6/68) had drug-resistant infections, while the drug resistance mutation rate of the tested strains was as high as 98.53% (67/68). Half of the drug-resistant patients (3/6) were incorporated into the molecular network, leading to the formation of a single drug-resistant transmission cluster. Analysis of transmission network signatures (TNSs) identified 7 high-risk transmitters. Bayesian phylogenetic analysis revealed that the most recent common ancestor (tMRCA) of the CRF55_01B strain dated back to 2002.03; the strain entered Qujing City, Yunnan Province via heterosexual transmission in 2005.20, and was simultaneously detected in Pu’er City and Wenshan Prefecture in the same year. In approximately 2006.16, it was first found among the men who have sex with men (MSM) population in Yunnan Province. Since then, it has spread bidirectionally between MSM and heterosexual populations. Multiple transmission patterns were observed, including intra-familial, local, cross-regional, and multi-partner transmission, accompanied by transmitted drug resistance (TDR). Imported cases were mainly from Hubei and Guangdong Provinces, with Honghe Prefecture serving as the primary entry point in Yunnan. The CRF55_01B strain was introduced into Qujing City, Yunnan Province, via heterosexual contact around 2005.20. Thereafter, the virus exhibited a bidirectional spread between MSM and heterosexual populations, demonstrating diverse transmission patterns including intra-familial, local, cross-regional, and multi-partner transmission. Meanwhile, evidence of drug-resistant strain transmission was detected. Molecular transmission network analysis further revealed that over 40% of potential concealed transmission sources remained unincorporated into the network. Based on this, it is recommended to adopt differentiated monitoring strategies, with a focus on strengthening the monitoring of drug-resistant strains in core hub areas and precise interventions for mobile populations. Not applicable.