- 學位論文
比較南台灣2002年與2015年流行的第二型都會基因型登革病毒基因與表現型及流行病學特徵
Genetic, Phenotypic and Epidemiological Characteristics of the Two Cosmopolitan Genotype Dengue Serotype 2 Viruses Causing Epidemics in 2002 and 2015 in Southern Taiwan
摘要
台灣南部的高雄和台南自1987年幾乎每年有登革熱流行，在2001-2002年高雄和2015年台南均發生了第二型登革病毒所引起的登革熱大流行，分別造成5,041和22,770例登革熱確定病例；有趣的是在此兩波流行中病人的臨床症狀卻有差異。本研究目的有三︰(一)比較此兩波流行的臨床表徵與流行病學特徵，(二)比較造成此兩波登革熱流行的病毒株在基因型和表現型的差異，及(三)串聯臨床﹑流行病學與病毒學的結果，以綜觀流行全貌。 比對此兩波流行自病人血清中所分離得的第二型登革病毒株的全長核苷酸序列[高雄(Kaohsiung, KH) 40株、台南(Tainan, TN) 7株]，而高雄的病毒株的核苷酸彼此間之相同度高於99.872%，台南病毒株彼此間的相同性高於99.953%，因此從中挑選出分別代表兩波流行來自登革病患的病毒株，並利用Vero細胞(V)進行兩(V2)至三次(V3)病毒斑純化，再利用蚊子C6/36細胞進行病毒複製兩次(C2)，後續以純化後的各兩病毒代表株[2002-KH-A3L(V2C2)及2015-TN-6B1 (V3C2)]進行實驗。做法上，先分析臨床病人的流行病學與臨床病症數據，接着比對此兩病毒代表株在Vero細胞及蚊子C6/36細胞上的胞外及胞內的生長動力學與細胞毒性、在嬰兒倉鼠腎細胞(baby hamster kidney, BHK)的斑塊形態、細胞結合力、細胞穿透力、複製力、胞內與胞外非結構性蛋白1表現量(non-structural protein 1, NS1)及人類腸道細胞(Caco-2)的感染性等實驗，藉此比較造成這兩波流行的第二型登革病毒株在表徵上的差異及其與流行病學差異的關聯性；並以單變項 (卡方檢定、獨立學生t檢定)等統計法檢驗分析。 在流行病學特徵，發現2015年台南登革熱病例比2002年高雄女男比較低(1.01 vs 1.17, p<0.05)、平均年齡較低(45.66 ± 21.15 vs 47.52 ± 17.78歲， p<0.05)、腸胃道症狀率較高[12.23% (2785/22770) vs 4.30% (217/5041), p<0.0001]及病例的住院率較低[11.10% (2527/22770) vs 39.63% (1998/5041), p<0.001]；雖然兩波流行病例的致死率並無差異(0.56% vs 0.54%, p=0.29)，但2015年台南的登革住院死亡率顯著較高[(5.03% (127/2527) vs 1.25% (25/1998), p<0.05)。 演化分析引發此兩波流行的兩病毒代表株(2002-KH-A3L及2015-TN-6B1)，發現其均屬於都會基因型(Cosmopolitan genotype)的兩不同分群，但其在核苷酸的相同度為97.506%，胺基酸的相同度更高達99.322%。在病毒表現型上，生長動力學實驗顯示2015年台南代表株(2015-TN-6B1)在感染Vero細胞前期(感染後24與36小時)和感染C6/36細胞後24、72與120小時比2002-KH-A3L病毒株有較高的細胞外病毒量，且感染Vero細胞後24小時造成較高的細胞死亡數。兩株病毒在感染BHK細胞所產生的病毒斑塊形態和細胞結合能力均無差異，可是2015-TN-6B1病毒株在BHK細胞作用0、15與30分鐘後的穿透力(penetration)及的複製力均表現較佳。值得注意的的是2015-TN-6B1病毒株在感染Vero細胞後24﹑48及96小時所產生的胞內NS1也較多；而2002-KH-A3L病毒株在此同感染力而感染Vero細胞後48﹑72及96小時所產生的胞外NS1和NS1總量卻較高。更重要的是2015-TN-6B1病毒株感染人腸道Caco-2細胞後24、48及72時的胞外病毒量較高以及其在此細胞感染後96及144小時所呈現的病毒免疫螢光訊號也較高，再度證實2015台南病毒株在人腸道細胞的感染或複製力較高。 本研究的主要限制有四:(一)僅選出分別代表2001-2002年高雄和2015年台南兩波流行來自登革病患各一株的第二型登革病毒株；(二)除了病毒因素外，病人的登革感染史﹑群體免疫﹑共病、病人基因、病媒與環境﹑氣候及人為介入等因子均會導致此兩波流行的差異；(三)未測病人的免疫狀況，致無法更佳串連登革病毒與宿主的關係；及(四)尚未純化得不同的足量NS1蛋白以釐清其差異的機轉。 本研究發現引起2015年台南登革流行的第二型登革病毒株在生長動力學表現比導致2002年高雄登革流行的同血清型登革病毒株為佳，2015台南株在病毒的穿透力、複製力及胞內NS1量均表現較佳，可能因而造成2015年台南的流行幅度比2002年高雄為大；再加上2015台南株對於人腸道細胞的感染或複製力也較高，此可能解釋為何2015年台南登革病人有較高的腸胃症狀。未來應收集具時空流行病學特徵的的病人檢體，利用次世代定序分析在流行過程中病毒亞群的動態改變；再者，利用質譜議分析病毒NS1蛋白的轉譯後修飾等表徵，最後整合個人與族群層面的臨床、流行病學、病毒學與免疫學結果，定能明瞭為何某些流行較為嚴重的原因與機轉。
並列摘要
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.