發展偵測抗原之酵素連結免疫吸附分析以應用於病媒蚊中登革病毒篩檢

Abstract

登革病毒為目前最重要的蟲媒病毒(Abovirus)之一，造成熱帶及亞熱帶國家36億人遭受感染之威脅，是公共衛生的嚴重健康問題。由於目前尚無疫苗以及抗病毒藥物被成功發展，病患的治療多是針對症狀的緩解，而非真的消滅病毒。因此疾病的預防顯得更為重要，而疾病的預防與控制仰賴於病例、病毒、昆蟲學和生態學四個方面的監測。其中病媒蚊的病毒監測能了解流行期與非流行期間病毒傳播概況，有助於疫情預測以及病媒管控等公共衛生措施的及早介入，將損傷降到最低，並且沒有病例病毒監測的不顯感染問題，利於更確實監測病毒的流行情況，並且可以用來評估病媒管制措施是否有效。因此本論文致力於發展穩定的ELISA系統以應用於登革病媒蚊內病毒蛋白監測。我們藉由病毒、實驗室飼養蚊混入病毒以及胸腔注射感染蚊三個層面，系統性地評估此ELISA系統的偵測極限、敏感性、特異性、再現性、待測抗原穩定性，並實際應用於高雄市採集之野外病媒蚊檢體的篩檢。 本研究首先比較登革直接診斷兩個常用的標的，套膜蛋白(E protein)以及非結構性蛋白1(NS1 protein)在感染蚊中被ELISA測得的偵測極限，初步結果知不論為何種登革血清型兩種偵測極限皆無差異，NS1 protein的偵測在感染蚊中並未優於E protein的偵測，再加上E protein存在於病毒顆粒表面穩定性佳有利檢體保存。因此決定以E protein作為發展對象。本研究發展之E protein capture ELISA系統偵測極限為，含104 PFU/ml以上病毒量之感染蚊即可被偵測到，不受pool size大小的影響。在檢驗敏感性與特異性部分，本系統對於登革血清型四型感染蚊的檢驗敏感性分別為69.57%、66.67%、52.38% 、66.67%，但當蚊內所含病毒量達104 PFU/ml以上時，不論為何種血清型病毒之感染蚊敏感性都可達100%；檢驗特異性則分別為100%、93.75%、100% 、100%。此外，對於台灣現有的其他黃病毒屬病毒JEV及Culex flavivirus，本ELISA系統不具交叉反應，能有效鑑別蚊內登革病毒的感染。再現性的部分，由實驗結果知相同樣本的intra-assay CV介於2.13%~5.78%間；inter-assay CV則介於1.19%~8.03%間，具有好的再現性。接著是抗原穩定性的部分，本研究證實感染蚊中DENV1及DENV3套膜蛋白在室溫有乾燥劑的封閉式檢體箱至少可以保存14天，仍可被ELISA偵測到，穩定性佳。最後，我們將此ELISA系統應用於高雄市登革流行期採集的病媒蚊檢體篩檢上，檢驗陽性率為10.48%(13/124)、最小感染率(Minimum infection rate,MIR)則是31.33/1000。 綜合上述結果，本研究發展之DENV E protein capture ELISA在各項篩檢指標上都是不錯的，且確實能應用於野外病媒蚊檢體篩檢，未來極具潛力發展為快速篩檢試劑。

Dengue viruses, include 4 different serotypes (DENV-1 to DENV-4), are one of the most important arboviruses, which recently emerged as a severe public health problem in tropical and sub-tropical countries, including Taiwan. In the absence of effective vaccines and specific treatment for dengue, the prevention and control of disease rely on the surveillance of cases and mosquito vector. In this study, we developed an antigen-capture ELISA to detect the E protein of dengue virus in mosquito pools, and estimated its sensitivity, specificity, repeatability and stability in different aspects. The result can be summarized into the following: (1) The sensitivity of DENV-1 to DENV-4 infected mosquitoes were 69.57%、66.67%、52.38% and 66.67%, respectively. However, this assay was 100% sensitive in detecting pools with viral titer≧104 PFU/ml; (2) Mosquito pool size did not affect the detection limit; (3) The specificity of DENV-1 to DENV-4 infected mosquitoes were 100%、93.75%、100% and 100%; (4) This ELISA system did not cross-reactive with other flavivirus circulated in Taiwan, such as Japanese encephalitis virus(JEV) and Culex flavivirus; (5) According to the low intra-assay CV(2.13%~5.78%) and inter-assay CV(1.19%~8.03%), we demonstrated the adequate repeatability of this ELISA system; (6) Our data showed that DENV E protein remained stable in infected mosquitoes stored at room temperature with desiccants for at least 14 days and could still be detected by our ELISA system; (7) We applied this system to virological surveillance in field-caught mosquito pools.124 samples had been tested and we obtained the positive rate and the mini mum infection rate were 10.48%(13/124) and 31.33, respectively. In conclusion, this DENV E protein capture ELISA had the potential for being an inexpensive and convenient on-site assay that facilitate widely virological surveillance in mosquitoes.