登革病人體內病毒量、免疫複合體及細胞激素變化與疾病嚴重度之研究

登革病毒屬於黃病毒科中以節肢動物為媒介的病毒之一員，共有四種血清型，藉由埃及斑蚊及白線斑蚊叮咬人來傳播疾病；登革病毒感染後可能不會有任何症狀或是只有輕微的發燒，但是也可以引起登革熱（dengue fever, DF），出現的症狀包括出現發燒、頭痛、骨頭酸痛、肌肉痛、起疹子，或是引起登革出血熱（dengue hemorrhagic fever, DHF），會有皮下出血、腹水、肋膜積水等症狀，而且血小板會下降至10萬以下；嚴重的病例會因血壓下降引起休克，即是登革休克症候群。登革熱及登革出血熱是熱帶及亞熱帶地區公共衛生方面一個很重要的問題，估計每年有將近1億人感染登革病毒、25萬人出現登革出血熱。台灣在2002年爆發了大規模的第二型登革熱流行，病例尤其集中在高雄縣、市及屏東縣，有5388個登革熱確定病例，更出現了242個登革出血熱的病例。本研究第一部份在於採集登革熱及登革出血熱病人的連續檢體，利用即時定量反轉錄-多聚合酶鏈反應（real-time RT-PCR）以及酵素連結免疫吸附法（ELISA）來探討2002年台灣高雄地區登革病毒血清型第二型的流行中，病人血漿中病毒量的變化、含有病毒之免疫複合體（immune complex）的出現及各種細胞激素（cytokine）在病程中之變化和病人疾病嚴重度的關係。來自惠德醫院及阮綜合醫院總計125個病例，共有71位DF病人以及54位DHF病人，其中有19位為一次感染，106位為二次感染；125位病人中共有19位糖尿病患者，26位病人有高血壓，糖尿病和高血壓與DHF沒有相關。以106位二次感染的病人來分析其血漿中之登革病毒量之變化，在退燒前二天、退燒的前一天、退燒的第一天、第二天、第三天以及第四天，DHF的病人血漿中病毒量比DF的病人有顯著意義的高，這顯示退燒前二天至退燒的第二天血漿中的病毒量應可做為預測DHF的指標。更進一步分析其中13位病人可發現在發燒期，DF及DHF病人均可測到免疫複合體之存在，但是到退燒期DF病人大多測不到而在DHF病人中仍有大量免疫複合體存在，持續至退燒的第二、三天。以上顯示DHF病人清除登革病毒較DF病人慢。另一方面顯示DHF病人在退燒期仍有相當的登革病毒是以免疫複合體的形式存在血漿中，免疫複合體本身可活化補體且改變血管內皮細胞之通透性，這對DHF的免疫致病機轉上應有重要角色。進一步以二次感染的13個病人的連續檢體進行ELISA來測量病人血中cytokines（包括sIL-2R、IL-10、IFN-γ、sTNFR-II、IL-6、IL-8、TNF-ㄐ^的變化，比較DF和DHF病人，發現這些cytokines的量在DHF病人體內並沒有明顯的比DF高；將cytokine之peak或最高量出現的時間做整理，DF出現peak或最高量的時間點較早且較短，DHF則持續較久，可能表示其免疫系統活化較久，但仍待更多病例之研究以確認。第二部份則是利用病人血漿檢體，比較引起DF和DHF病人體內病毒E基因序列是否有差異，並探討在2002年流行期間登革病毒序列的變化。在2002年流行期間的DF和DHF的病人在E基因並沒有顯著的序列變化，顯示引起DF或DHF的病毒其基因序列差異不是在E基因。加入2001年所得到的序列來比較2001年至2002年的病毒E基因序列的變化，則發現在E基因第46個胺基酸的地方在2001年為T，在2002年時轉變為I。而在第291個及第1128個核苷酸（第97個及第376個胺基酸）分別有不改變胺基酸之變異。2002年前期（6月）及後期（11月）各1位DHF病人之登革病毒其全長基因體共8個地方有核苷酸改變，其中有2個改變胺基酸，其餘不改變胺基酸，大多在NS4B及NS5。本研究之整體目標是探討登革病人血漿中病毒量、免疫複合體及細胞激在病程中之變化及與疾病嚴重度之關係。本研究之重要發現為二次感染的DHF病人在退燒前二天至退燒第四天，其血漿中病毒量均較DF病人有顯著的高，顯示血漿中病毒量在這期間均可做為預測DHF的指標。此外，不同於DF病人，DHF病人在退燒期間血漿中仍持續有免疫複合體之存在，顯示其清除病毒較DF病人慢，而免疫複合體的持續存在，對DHF之免疫致病機轉應有重要角色。在比較DF及DHF病人體內病毒E基因序列方面，並沒有顯著的差異，顯示病毒株致病力差異的決定因子應是在E基因之外。

關鍵字

細胞激素；免疫複合體；登革病毒

並列摘要

Dengue virus is an arthropod-borne flavivirus that is transmitted to human by the mosquitoes, Aedes Aegypti and Aedes Albopitus. There are four serotypes（DEN-1, DEN-2, DEN-3 and DEN-4）of dengue virus. After dengue virus infection, the clinical presentations range from asymptomatic or a self-limited illness, dengue fever（DF）, to severe forms of disease, dengue hemorrhagic fever（DHF）and dengue shock syndrome（DSS）. DF is characterized by fever, headache, myalgia and abdominal pain. DHF/DSS is characterized by development of plasma leakage and low platelet count. The epidemics of dengue continue to be a major public health problem in the tropical and subtropical regions. It has been estimated that 100 millions of dengue infection and 250,000 cases of DHF occur annually. In the first part of this study, we investigated the dynamic change of plasma dengue viral load, virus-containing immune complex and cytokines in patients with different disease severity during a DEN-2 outbreak in Kaohsiung in 2002. One hundred twenty-five confirmed dengue patients from the Huider hospital and Yuan general hospital were included in this study. There were 71 DF patients and 54 DHF patients. There were 19 cases with primary infection and 106 cases with secondary infection. The ages of patients ranged from 8 to 82 years old. There is no statistical difference in gender or age between the DF and DHF groups. Among the 125 cases, there were 19 patients with diabetes mellitus（DM）and 26 patients with hypertension（HT）. DM and HT were not correlated with DHF. The levels of plasma dengue RNA of the 106 cases with secondary infection were determined during the course of infection. We found that plasma dengue viral load is significant higher in DHF than in DF patients during the period between 2 days before defervescence and 4 days after defervescence. We further analyzed the virus-containing immune complex in 13 cases of secondary infection, and we found that the level of immune complex is higher in DHF than in DF patients during the period between the last fever day and the first defervescence day. In these 13 cases, there is no significant difference in the plasma levels of cytokines, including sIL-2R, IL-10, IFN-γ, sTNFR-II, IL-6, IL-8, and TNF-?between the DF and DHF patients. Peak or maximum plasma of cytokines were found earlier during defervescence in DF patients than in DHF patients. In the second part of this study, we compare the E sequences of the viruses from DF and DHF patients, and the changes of the sequences during the epidemic. We found that there is no consistent change in the E sequences between DF and DHF patients. Comparing with 2001 viruses, there were three consistent nucleotides changes in the E sequences of the 2002 viruses, including one amino acid change from T to I at residue 46 and two silent mutations at nucleotide residues 291 and 1128 of the E gene. We also examined 2 full genome sequence of DEN-2 viruses of the early （June）and late（November）epidemic of 2002. There were 8 nucleotide changes, of which most were in the NS4B and NS5 genes. The overall objective of this study is to elucidate the relationship between plasma dengue viral load, virus-containing immune complex, cytokines and disease severity during the course of infection. The major findings of this study are that among the cases of secondary infection, DHF patients have significant higher levels of plasma viral load than DF patients during the period between 2 days before and 4 days after defervescence. This suggests that plasma viral load during this period may serve as a disease marker for DHF. In addition, while most DF patients have immune complex undetectable during defervescence, DHF patients continue to have immune complex in plasma up to 3 days after defervescence, suggesting a delay in the clearance of viremia in DHF patients. Persistent immune complex during defervescence may contribute to the immunopathogenesis of DHF. Finally, comparison of the E sequences from the DF and DHF patients revealed no consistent nucleotide changes, suggesting that the determinants of virulent strain associated with DHF may be outside of the E gene.

並列關鍵字

cytokines ； immune complex ； dnegue viral load

參考文獻

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國際替代計量

登革病人體內病毒量、免疫複合體及細胞激素變化與疾病嚴重度之研究

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