Isolation and Genetic Evolution of Dengue Virus from the 2019 Outbreak in Xishuangbanna, Yunnan Province, China

Background: Dengue virus (DENV) can be divided into four serotypes—DENV-1, DENV-2, DENV-3, and DENV-4. In humans, infection leads to dengue fever (DF), dengue hemorrhagic fever, and dengue shock syndrome, both widely prevalent in tropical and subtropical regions. In 2019, a severe outbreak of DF occurred in Xishuangbanna, Yunnan province. Objective: To investigate the etiology and genotype of the causative agents of this severe dengue outbreak in Xishuangbanna. Methods: Between October and November 2019, the sera of patients clinically diagnosed with DF were collected in the first People's Hospital of Xishuangbanna. RNA was extracted from the sera and amplified by RT-PCR with flavivirus primers. Flavivirus-positive sera were then used to inoculate Aedes albopictus cells (C6/36); viral RNA was extracted from these cells, amplified, and sequenced with DENV E gene-specific primers. Sequence splicing and nucleotide homology genetic evolution analysis were carried out by biological software (DNAStar). Unique mutations in the E genes of isolated DENV were analyzed by SWISS-MODEL and PyMOL. Results: Of the 60 samples collected from DF patients, 39 tested positively with flavivirus primers. The DENV was isolated from 25 of the 39 positive seras, of which 20 showed cytopathic effects (CPE) and 5 were no CPE. In these 25 isolated nucleic acids, 21 strains of DENV-1, 3 strains of DENV-2, and 1 strain of DENV-3 were identified according to the sequence of E protein. In the four unique mutations (D52, Y149, L312, T386), D52 and Y149 in the E protein of DENV-1 were predicted to be exposed on the surface of the prefusion conformation. Conclusion: The 2019 outbreak of DF in Xishuangbanna area of Yunnan Province consists of at least three serotypes of DENV-1, DENV-2, and DENV-3, and the sources of these virus strains are of mixed and complicated origin.