Genetic, Phenotypic and Epidemiological Characteristics of the Two Cosmopolitan Genotype Dengue Serotype 2 Viruses Causing Epidemics in 2002 and 2015 in Southern Taiwan

• Main Authors: Chi-Hang Chin, 錢智恆
• Other Authors: Chwan-Chuen King
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/51335302875945379052

碩士 === 國立臺灣大學 === 流行病學與預防醫學研究所 === 105 === Kaohsiung and Tainan (located in southern Taiwan) have experienced annual epidemics of dengue since 1987. There were two large-scale outbreaks caused by serotype-2 dengue virus (DENV-2), in Kaohsiung (KH) in 2001-2002 and in Tainan (TN) in 2015, resulting in 5041 and 22,770 total cases, respectively. However，the clinical manifestations of these two epidemics were different. This study had 3 specific aims: (1) to compare epidemiological and clinical manifestations of these two epidemics, (2) to evaluate virological characteristics of the two DENV-2 viruses genotypically and phenotypically, and (3) to integrate clinical, epidemiological and virological characteristics for better understanding the differences in overall picture of these two epidemics. Full-length viral nucleotides sequences, obtained by using Sanger sequencing, of the isolated viruses from 2001-02 KH and 2015 TN patients’ serum samples were analyzed in order to acquire the representative virus strains of the corresponding epidemics. The full-length nucleotides sequences of 47 virus strains (40 of KH, 7 of TN) within the same epidemic shared extremely high identities (>99.872% for Kaohsiung strains and >99.953% for Tainan strains). Representative virus strains of these two epidemics were plaque-purified two (V2) to three(V3) times from Vero cells (V) and then amplified in Aedes albopictus C6/36 cells (C). Epidemiological data and clinical manifestation of laboratory-confirmed dengue patients of these two epidemics were analyzed. Growth kinetics and cell toxicity of these two representative DENV-2 strains [2002-KH-A3L (V2C2) and 2015-TN-6B1 (V3C2)] were evaluated by collecting infected supernatant and Vero cells at different time points [0, 24, 36, 48, 72, 96 hour post-infection, (h.p.i.)]. Focus-forming assay (FFA) and real-time polymerase chain reaction (qPCR) were used to quantify viral RNA copies in both infected supernatant and cells. Trypan blue was used to quantify survived cells to assess cell toxicity. Viral plaque morphologies, and other phenotypic abilities in cell binding, penetration, replication and expression levels of intracellular and extracellular non-structural protein 1 (NS1), using in-house ELISA to quantify, in baby hamster kidney (BHK) or Vero cells were also examined. Virus infectivity in human colon epithelial cells (Caco-2) were evaluated through growth kinetics and immunofluorescent assay (IFA). Univariate analyses (Chi-square and unpaired t-test) were used to compare the differences between these two epidemics. Comparing to those results of the 2001-2002 Kaohsiung DENV-2 epidemic, epidemiological analyses showed that the 2015 Tainan epidemic had lower female to male ratio (1.01 vs 1.17, p<0.05), lower mean age (45.66 ± 21.15 vs 47.52 ± 17.78 years old, p<0.05), higher frequencies of gastrointestinal symptoms [12.23% (2785/22770) vs 4.30% (217/5041), p<0.0001], lower hospitalization rate [11.10% (2527/22770) vs 39.63% (1998/5041), p<0.001]. Although the case-fatality rate had no difference (0.56% vs 0.54%, p=0.29), the hospitalization fatality rate of the 2015 Tainan epidemic was significantly higher [(5.03% (127/2527) vs 1.25% (25/1998), p<0.05). Evolutionary analyses found that these two DENV-2 strains (2002-KH-A3L and 2015-TN-6B1) all belong to the different clades of the Cosmopolitan genotype. However, they had high viral sequence identity percentages in amino acids (99.322%) and nucleotides (97.506%). Growth kinetics of these two purified viruses revealed that the 2015 Tainan DENV-2 (2015-TN-6B1) strain replicated more efficiently (24 and 36 h.p.i.), and caused more cell death at 24 h.p.i. than those of the 2002 Kaouhsiung DENV-2 (2002-KH-A3L) strain. There was no significant difference in the plaque morphologies or cell binding abilities in BHK cells. However, the penetration ability at 0, 15, 30 minutes post-incubation and replication ability of the 2015-TN-6B1 strain in BHK cells were superior to those from the DENV-2 2002-KH-A3L strain. The intracellular levels of NS1 in these cells infected with the 2015-TN-6B1 strain (MOI=1) at 24, 48, 96 h.p.i were higher. By contrast, those infected with the 2002-KH-A3L strain at 48, 72, 96 h.p.i. showed higher extracellular and total levels of NS1. Most importantly, the human intestine Caco-2 cells infected with the 2015-TN-6B1 strain at 24, 48 and 72 h.p.i demonstrated higher extracellular viral loads shown by FFA than those of the Caco-2 infection with the 2002-KH-A3L. In addition, these Caco-2 cells infected with the 2015-TN-6B1 at 96 and 144 h.p.i. also revealed higher intensities of fluorescent signal in IFA staining of viral antigen either E or NS1. These results showed that the 2015-TN-6B1 strain had higher infectivity or replication efficiency in human intestinal cells than those of the 2002-KH-A3L strain. There are four major limitations of this study. First, we only chose one DENV-2 viral strain from the dengue patient to represent each of the corresponding epidemics. Second, other factors besides viral attribute, such as herd immunity, comorbidity, past history of DENV infection with different serotypes, vector factors, climate, environmental factors, intervention strategies and human behavior may also contribute the differences in these DENV-2 caused two major epidemics. Third, the individual immunity was not measured for better conclusion on the interactions between DENV-2 and human host. Fourth, we failed to purify NS1 of these two representative strains up to suffiecient amount to examine the difference in the mechanism of NS1 in viral pathogenesis. In conclusion, the 2015 TN DENV-2 strain showed better performance in growth kinetics with better penetration, replication abilities and higher levels of intracellular NS1 than those of the 2002 KH strain. This might explain why the 2015 Tainan epidemic was more severe. Moreover, the 2015 TN strain showed better infectivity and higher replication efficiency in human intestinal Caco-2 cells which is well-correlated with the higher percentages of gastrointestinal symptoms in the 2015 epidemic. Future effort needs to collect specimens from different time points of the various epidemic waves with tempo-spatial epidemiological characteristics for analyzing dynamics of DENV subpopulations through epidemic process, using next generation sequencing. In addition, the combination of further viral phenotypic studies can be done to examine the post-translation modification of NS1, using mass spectrometry and proteomics analyses. Based on this study, the integrated results from clinical, epidemiological to virolgical and immunological results at both individual and population levels can give us the best clues to fully understand the reasons why certain epidemic is more severe than others.