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

錢智恆

doi:10.6342/NTU201702283

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比较南台湾2002年与2015年流行的第二型都会基因型登革病毒基因与表现型及流行病学特徵

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

钱智恒 , 硕士　　导师：金传春　　

英文 DOI： 10.6342/NTU201702283

登革热；台湾；台南；高雄；肠胃道症状； 2002年； 2015年； Dengue fever ； Taiwan ； Tainan ； Kaohsiung ； Gastrointestinal symptoms ； 2002 ； 2015

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摘要 │ 参考文献 (81) │ 国际计量

摘要〈TOP〉

台湾南部的高雄和台南自1987年几乎每年有登革热流行，在2001-2002年高雄和2015年台南均发生了第二型登革病毒所引起的登革热大流行，分别造成5,041和22,770例登革热确定病例；有趣的是在此两波流行中病人的临床症状却有差异。本研究目的有三︰(一)比较此两波流行的临床表徵与流行病学特徵，(二)比较造成此两波登革热流行的病毒株在基因型和表现型的差异，及(三)串联临床﹑流行病学与病毒学的结果，以综观流行全貌。

比对此两波流行自病人血清中所分离得的第二型登革病毒株的全长核苷酸序列[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间之相同度高於99.872%，台南病毒株彼此间的相同性高於99.953%，因此从中挑选出分别代表两波流行来自登革病患的病毒株，并利用Vero细胞(V)进行两(V2)至三次(V3)病毒斑纯化，再利用蚊子C6/36细胞进行病毒复制两次(C2)，後续以纯化後的各两病毒代表株[2002-KH-A3L(V2C2)及2015-TN-6B1 (V3C2)]进行实验。做法上，先分析临床病人的流行病学与临床病症数据，接着比对此两病毒代表株在Vero细胞及蚊子C6/36细胞上的胞外及胞内的生长动力学与细胞毒性、在婴儿仓鼠肾细胞(baby hamster kidney, BHK)的斑块形态、细胞结合力、细胞穿透力、复制力、胞内与胞外非结构性蛋白1表现量(non-structural protein 1, NS1)及人类肠道细胞(Caco-2)的感染性等实验，藉此比较造成这两波流行的第二型登革病毒株在表徵上的差异及其与流行病学差异的关联性；并以单变项 (卡方检定、独立学生t检定)等统计法检验分析。

在流行病学特徵，发现2015年台南登革热病例比2002年高雄女男比较低(1.01 vs 1.17, p<0.05)、平均年龄较低(45.66 ± 21.15 vs 47.52 ± 17.78岁， p<0.05)、肠胃道症状率较高[12.23% (2785/22770) vs 4.30% (217/5041), p<0.0001]及病例的住院率较低[11.10% (2527/22770) vs 39.63% (1998/5041), p<0.001]；虽然两波流行病例的致死率并无差异(0.56% vs 0.54%, p=0.29)，但2015年台南的登革住院死亡率显着较高[(5.03% (127/2527) vs 1.25% (25/1998), p<0.05)。

演化分析引发此两波流行的两病毒代表株(2002-KH-A3L及2015-TN-6B1)，发现其均属於都会基因型(Cosmopolitan genotype)的两不同分群，但其在核苷酸的相同度为97.506%，胺基酸的相同度更高达99.322%。在病毒表现型上，生长动力学实验显示2015年台南代表株(2015-TN-6B1)在感染Vero细胞前期(感染後24与36小时)和感染C6/36细胞後24、72与120小时比2002-KH-A3L病毒株有较高的细胞外病毒量，且感染Vero细胞後24小时造成较高的细胞死亡数。两株病毒在感染BHK细胞所产生的病毒斑块形态和细胞结合能力均无差异，可是2015-TN-6B1病毒株在BHK细胞作用0、15与30分钟後的穿透力(penetration)及的复制力均表现较佳。值得注意的的是2015-TN-6B1病毒株在感染Vero细胞後24﹑48及96小时所产生的胞内NS1也较多；而2002-KH-A3L病毒株在此同感染力而感染Vero细胞後48﹑72及96小时所产生的胞外NS1和NS1总量却较高。更重要的是2015-TN-6B1病毒株感染人肠道Caco-2细胞後24、48及72时的胞外病毒量较高以及其在此细胞感染後96及144小时所呈现的病毒免疫萤光讯号也较高，再度证实2015台南病毒株在人肠道细胞的感染或复制力较高。

本研究的主要限制有四:(一)仅选出分别代表2001-2002年高雄和2015年台南两波流行来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﹑群体免疫﹑共病、病人基因、病媒与环境﹑气候及人为介入等因子均会导致此两波流行的差异；(三)未测病人的免疫状况，致无法更佳串连登革病毒与宿主的关系；及(四)尚未纯化得不同的足量NS1蛋白以厘清其差异的机转。

本研究发现引起2015年台南登革流行的第二型登革病毒株在生长动力学表现比导致2002年高雄登革流行的同血清型登革病毒株为佳，2015台南株在病毒的穿透力、复制力及胞内NS1量均表现较佳，可能因而造成2015年台南的流行幅度比2002年高雄为大；再加上2015台南株对於人肠道细胞的感染或复制力也较高，此可能解释为何2015年台南登革病人有较高的肠胃症状。未来应收集具时空流行病学特徵的的病人检体，利用次世代定序分析在流行过程中病毒亚群的动态改变；再者，利用质谱议分析病毒NS1蛋白的转译後修饰等表徵，最後整合个人与族群层面的临床、流行病学、病毒学与免疫学结果，定能明了为何某些流行较为严重的原因与机转。

并列摘要〈TOP〉

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.

参考文献 ( 81 ) 〈TOP〉

Akey, D. L., et al. (2014). "Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system." Science 343(6173): 881-885.
连结：
Anderson, J. R. and R. Rico-Hesse (2006). "Aedes aegypti vectorial capacity is determined by the infecting genotype of dengue virus." The American journal of tropical medicine and hygiene 75(5): 886-892.
连结：
Avirutnan, P., et al. (2010). "Antagonism of the complement component C4 by flavivirus nonstructural protein NS1." Journal of Experimental Medicine: jem. 20092545.
连结：
Avirutnan, P., et al. (2006). "Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement." Journal of Infectious Diseases 193(8): 1078-1088.
连结：
Avirutnan, P., et al. (2007). "Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E." PLoS Pathog 3(11): e183.
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