Laboratory Diagnosis and Genetic Analysis of Dengue Virus Epidemic in Taiwan
Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, 129 DENV were isolated from 628 patient sera. Sequencing of C, prM, and E genes from 45 DENV strains was performed and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Compared with other genotypes, several variations were found. The major substitution was C-A314G (K73R) found in the capsid region, however, reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells when compared with the wildtype virus. Next generation sequencing (NGS) of three sera collected from one DENV-infected patient at different time-points showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged whereby leucine mutated to isoleucine and was identified in NS4A-115. Quasispecies analysis of DENV-1, -2, and -3 revealed that the variations were all located in the nonstructural protein region of the genome, namely NS2B, NS3, and NS4A. In conclusion, a newly emerged substitution in the capsid region was found in the 2015 isolates. This C-A314G (K73R) substitution showed lower replication rate and stability than wildtype virus, thus indicating that the other variants in the viral population may contribute to the 2015 outbreak. Additionally, haplotype analysis showed that the DENV viral population continually changes overtime to adapt to its environment.