Title & Institution: Professor, Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University

Email: jrwang@ncku.edu.tw

Topic in NCKU Research Week: Characterization of Quasispecies in Dengue Virus Infections

Abstract:

In 2015, there was a large dengue virus (DENV) outbreak in Southern Taiwan, resulting in more than 43,000 dengue cases, where 22,741 cases with 112 deaths were reported in Tainan City alone. Phylogenetic analysis on the capsid and envelope proteins revealed that all strains from the outbreak were classified as DENV serotype 2, cosmopolitan genotype. Due to the nature of RNA viruses, their high mutation rates produce a population of closely related but genetically diverse viruses, termed quasispecies. To determine the role of quasispecies in DENV disease severity, 22 samples (10 mild, 12 fatal) and 3 samples from one DENV infected patient from the 2015 outbreak were isolated and amplified with RT-PCR, sequenced with Next Generation Sequencing (NGS) using the Illumina MiSeq platform, and analyzed using QuasiRecomb and LoFreq programs. Haplotype analysis on samples from a single patient at different time-points showed changes in the number of haplotypes with time and the emergence of new haplotypes at day 5 post-infection.  According to the analysis of the mild and fatal case samples by QuasiRecomb, a total of 305 positions showed significant difference between mild and fatal cases. On the other hand, analysis by LoFreq showed 6 positions with p<0.05. Taking p<0.001 and p<0.05 from QuasiRecomb (4) and Lofreq (6) respectively, a total of 10 positions were selected, of which 1 position was located in the structural region, and the remaining majority were located in the non-structural protein (NS) regions, namely NS2A, NS3, and NS5. The NS region of DENV, like many other Flaviviruses, is responsible for the replication of new viruses. Therefore, with respect to the analyzed data, reverse genetics viruses of 10 selected positions were generated. Three of the variations found in the NS5 protein were shown to have better growth rates and stability than WT virus in both BHK-21 and Vero cells.  In conclusions, viral quasispecies found in isolates from the 2015 DENV epidemic, resulted in variations between mild and fatal cases, and these variations may play a role in DENV disease severity.

Keywords: Dengue virus, disease severity, next-generation sequencing, quasispecies

Personal Profile: Dr. Wang has the expertise, leadership and motivation in clinical and molecular virology. Her interests focus on the viral evolution of emerging viruses, especially on influenza viruses, enterovirus A71 (EV-A71) and dengue virus (DENV). Her researches are directed toward viral surveillance and to understand the genetic evolution of influenza virus, EV-A71, and DENV. In addition, her lab has defined various viral determinants for replication, viral fitness, antigenicity, and virulence by using reverse genetics viruses. Her laboratory has identified genotypic changes among clinical virus isolates in EV-A71 evolution. Furthermore, her lab. has compared and analyzed different dengue virus clinical tests for accurate, rapid and early detection of dengue virus infection. These findings increase the understanding of evolution, antigenicity, and pathogenesis, which will help for further vaccine and antiviral development.

Website link to personal research profile: http://mt.ncku.edu.tw/var/file/151/1151/img/3584/Jen-RenWang.pdf