- 學位論文
評估抗登革熱血清對茲卡病毒感染的潛在抗體依賴性增強作用
The Evaluation of Potential Antibody Dependent Enhancement of Anti-Dengue Sera on Zika Infection
摘要
背景：登革熱病毒（DENV）和茲卡病毒（ZIKV）均為蚊媒傳播的黃病毒，在基因組結構上具有相似性。不同型别的DENVs的重複感染會引發登革熱重症的產生，目前推論是藉由抗體依賴性增強現象（ADE）具有相關性。台灣在2014年和2015年經歷了嚴重的登革熱疫情，其中大多數發生在高雄市等南部地區。雖然目前ZIKV在台灣尚未出現本土案例發生，但我們仍需確認與瞭解是否台灣登革熱患者一旦被ZIKV感染，會因為體內具有登革熱的抗體進而加速ZIKV的症狀。 目的：本研究欲瞭解先前存在的登革熱抗體是否會在爾後感染茲卡病毒後藉由ADE的 效應而促進茲卡病毒的感染。 材料與方法：ZIKV（PRVABC59）用於評估ADE特性。將ZIKV培育於C6 / 36和Vero細胞上，並使用噬斑測定法(Plaque assay)定量效價以及使用即時PCR（qRT-PCR）定量病毒。臨床登革熱患者的血清取自高雄醫科大學傳染病與癌症研究中心，並使用抗登革病毒IgG和IgM ELISA試劑盒進行酶聯免疫吸附測定（ELISA）以確定IgM / IgG濃度。急性期的IgM：IgG比率用於確定原發性和繼發性DENV感染。分析原發性和繼發性登革患者血清對於ZIKV感染的效應。抗體依賴性測定(ADE)試驗則用來評估登革熱患者血清對於ZIKV感染THP-1（表達FcrII的細胞）細胞的影響，並通過qRT-PCR測定細胞內ZIKV的效價。 結果：ZIKV分別使用C6 / 36和Vero細胞上培育與放大病毒。當細胞病變效應（CPE）（細胞形態和細胞分離的變化）達到~80％時，從細胞培養上清液中收集ZIKV。對ZIKV進行噬斑測定，確定ZIKV的滴度為7×103 PFU / ml。17個配對的血清皆使用抗登革病毒IgG和IgM ELISA測試，其中4對被鑑定為原發性感染，13對為繼發性感染。抗體依賴性增強試驗顯示，原發性感染的患者恢復期血清會促進ZIKV的感染。繼發性感染的血清，不論是急性期或恢復期皆會促進ZIKV的感染。之後統整比較登革熱急性期或恢復期的血清何者具有增強ZIKV的感染現象，結果顯示恢復期血清明顯表現出有較強的增強反應產生(P<0.05)。 結論：本研究證明抗登革熱病毒的抗體會藉由ADE來增強茲卡病毒的感染。
並列摘要
Background: Dengue virus (DENV) and Zika virus (ZIKV) are belonged to mosquito-borne flaviviruses and share similarities in genome structure. Heterotypic DENVs infection produces antibodies that are known to be associated with severe dengue, due to antibody-dependent enhancement (ADE), in which preexisting non-neutralizing antibodies leads to enhancement of infection. Several dengue epidemics have been reported in Taiwan, especially two large dengue outbreaks occurring in 2014 and 2015. The imported Zika cases have been detected in Taiwan. Recent studies indicate that antibodies against DENVs promote ZIKVs’ infection but the mechanism was still unclear. It is important to know that whether Taiwanese dengue patients are at risk of developing severe disease manifestation if they get infected with ZIKV. Aim: This study examined whether preexisting dengue antibodies from acute or convalescent dengue infection facilitates Zika infection via ADE infection. Materials & Methods: The ZIKV (PRVABC59) was used to evaluate the ADE properties. The ZIKV was propagated onto C6/36 and Vero cell lines and tittered using plaque assay and quantified using Real-Time PCR (qRT-PCR). Paired sera from previously infected dengue patients were obtained from Center for Infectious Disease and Cancer Research, Kaohsiung Medical University and were subjected to Enzyme-linked immunosorbent assay (ELISA) using Human Anti-Dengue Virus IgG and IgM ELISA Kit to determine the IgM/IgG concentration. The IgM:IgG ratio in the acute phase was used to determine primary and secondary DENV infection. The sera from primary and secondary DENV infection were analyzed for the potential enhancement of ZIKV. The antibody-dependent enhancement (ADE) assay was used to validate the enhancement properties of the ZIKV. The ADE was conducted by using ZIKV to infect THP-1 (a FcRII expressing cell) in presence of dengue patients’ sera and the intracellular virus titer was determined by qRT-PCR. Results: The ZIKV were propagated and amplified on C6/36 and Vero cells, respectively. The ZIKV was collected from the cell culture supernatants when the cytopathic effect (CPE) observed, which is the change of morphology and detachment of cells, reached ~80%. The ZIKV was subjected to plaque assay and the titer of the ZIKV was determined to be 7*103 PFU/ml. A total of 17 paired sera samples subjected to Human Anti-Dengue Virus IgG and IgM ELISA Kit with four samples identified as belonging to primary infection and thirteen as secondary infection. The ADE assay revealed that enhancement of ZIKV was only seen in the THP-1 cells pretreating with the antibodies from convalescent phase of primary DENV infection. In regards to secondary infection, most sera samples showed enhancement. All of the sample, with the exception of one sample, displayed enhancement (P<0.05). Unlike the primary DENV infection, some of the acute sera showed similar viral loads of enhancement to that of the convalescent phases. Each individual acute and convalescent phase sample was analyzed to determine which phase would have higher frequency of enhancement. The convalescent sera displayed greater frequency of enhancement increasing more than two folds compared to both control and acute phase. Conclusion: This study provides evidences that preexisting anti-dengue antibody induces ADE on Zika virus infection in the convalescent phase in primary infection and in both phases in secondary infection.