臺灣地區2002年第二型登革病毒基因變異及類種分析

Abstract

Dengue virus (DENV) is a member of Flavivirudae. There are 4 serotypes of dengue virus, DENV-1, DENV-2, DENV-3 and DENV-4. In Taiwan, the primary vectors of dengue virus are Aedes aegypti and Aedes albopictus. DENV can cause asymptomatic infection, mild dengue fever (DF), severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In recent years, there were indigenous cases every year in Taiwan. In 2002, there were 5336 cases including 241 DHF, and 21 fatal cases. Epidemiological study found that percentage of DHF cases increased in later period of the epidemic and more DHF cases in areas with high transmission intensity. Therefore, the aims of this study were: 1) to understand the association between the DENV-2 virus genetic variations and disease/epidemic severity, and 2) to explore the diversity of quasispeices in DENV-2 isolated from different temporal and spatial epidemiological characteristics. In the experimental design and methods, first, 22 DENV-2 (14 early, 8 middle/late stage) isolated in June to December, 2002 that cultivated for two generations in C6/36 cells. Theses viral genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) in fragments, and then analyzed differences in the full-length sequences of the nucleotides and amino acids. Besides, 12 (7 early, 5 middle/late stage) DENV-2 isolates were analyzed quasispecies of E protein region by clonal sequencing, calculated mean diversity and percentage of clones with nucleotide changes to quantitate the variations of quasispecies, and analyzed their tempo-spatial characteristics associated with diseases/epidemic severity. The results of full-length sequencing showed that the total identity of the DENV-2 nucleotides was 99.97 %. No specific sequence difference was found between DENV-2 from DF and DHF patients. Comparing the nucleotide/ amino acid sequences of DENV-2 to the consensus sequences of DENV-2 isolated in 2002, the numbers of nucleotide/amino acid differences between DF or DHF isolates were not significant (nucleotide: 2.92 vs 3.89, p= 0.41). Analyzing the temporal trend the DENV-2 from the early period of the epidemic were found to be with significantly more viral sequence identities to the consensus sequence than those DENV-2 from the middle and late periods (nucleotide difference: 2.00 vs 5.62, p= 0.0002). Moreover, the spatial trend demonstrate that DENV-2 obtained from areas with high transmission intensity in the early period of the epidemic had more nucleotide sequence identifies to the consensus sequences than those DENV-2 from areas with low transmission intensity in the same epidemic period. (mean: 1 vs 2.75, p= 0.10) The quasispecies of E protein of the 12 DENV-2s indicated that the mean diversities ranged from 0.003 % to 0.024 % and significantly increased with the increasing days of 4-6 than those of 0-3 after the onset of dengue illness. (d0-3: 0.010 %, d4-6: 0.018 %, p= 0.04). However, there were no significant differences in the diversities of DENV-2 quasispecies between mild DF and severe DHF cases, regardless 0-3 or 4-6 days after dengue onset. (0-3 days: 0.010 % vs 0.010 %, p= 0.64, 4-6days: 0.020 % vs 0.016 %, p= 0.4). Simultaneous analyses of tempo- and spatial factors revealed that DENV-2 quasispecies at days 0-3 after the onset of dengue illness were more homogeneous from areas with high transmission intensity and early period of the epidemic but this finding needs to increase sample size for better conclusion. In quantifying the titer of DENV-2 suspension grown from C6/36 mosquito cells), we discovered the plaque forming units (PFU)/copy number ratio at 5 days post-infection was lower in DENV-2 from later period of the epidemic than those DENV-2 in the early period, with overall range. 1/3000 to 1/16000. . In order to clarify the reasons of the difference, we selected one DENV-2 from early versus late periods of the epidemic grew them in mosquito C6/36 cells, harvested supernatants for sucrose gradient ultracentrifugation. The quantitative levels of DENV-2 were measured from each fraction of the 11 fractions as PFU/copy numbers for better comparison. The highest viral copy numbers all peaked in the same fraction (density 1.19g/ mL), regardless early or late DENV-2. Interestingly, late DENV-2’s certain low density fractions (1.07~1.13 g/ mL) involved high viral copy numbers but lower PFUs, implying the possible presence of defective virus. Whether these virus compositions might affect viral growth yields and/or host immunity, thereby leading to disease severity or higher DHF% or DHF/DF ratio, needs more studies to prove. In conclusion, DENV-2 obtained from the 4-6 days after the onset of dengue at an individual level and in the middle/late epidemic period of the 2002 epidemic at the population level contained more viral genetic variations and diversities of quasispecies than those DENV-2 from 0-3 days after dengue onset and early epidemic period, particularly in areas with high dengue clusters. Such a high homogeneity in the early epidemic period was also supported by the results from sucrose density gradients. More future experiments have to answer the mechanism involved in the association between increasing dengue virus diversity and epidemic severity through evolutionary selection of advantageous DENV-2 subvariant by analyzing the changing growth characteristics and other phenotypes of viruses in high/low sucrose density fractions and mixing experiments for verifying the possibility and the roles of defective interference particle in increasing DHF epidemic severity.