應用反轉錄聚合鏈反應診斷家禽流行性感冒病毒及區分血清亞型

Abstract

中 文 摘 要 此實驗應用聚合連鎖反應作為快速檢測之技術，以應用在家禽流行性感冒臨床病例之快速診斷；首先我們於具有高保留性之核蛋白基因上設計出用來檢測A型流行性感冒病毒之通用性引子，經以此技術應用於家禽流行性感冒之種毒株及由野鳥分離之病毒株，結果均可以準確的檢出；而因家禽流行性感冒病毒與新城雞病病毒所造成之病害十分相似，故參考先前已設計用來偵測NDV之引子配合偵測家禽流行性感冒病毒之引子，發展單管多引子之RT-PCR使之能同時偵測家禽流行性感冒病毒與新城雞病病毒。進一步為了區別新城雞病病毒疫苗株與野外病毒株之混淆，我們亦參考先前已發表之台灣地區新城雞病野外病毒株序列，設計出用來檢測野外病毒株之引子，並應用在進行單管多引子RT-PCR中，經以動物試驗進行檢出效果評估，具有可應用於臨床檢測之適用性，作為快速區別家禽流行性感冒病毒或新城雞病病毒之感染。 為了能快速的檢出AIV感染及區分出其血清亞型，我們針對每一種HA與NA亞型之基因設計出一對具特異性之引子，以供作為以RT-PCR區分血清亞型之用，在HA亞型方面，由目前所有各亞型已公開之基因序列設計出之引子，應用於已知亞型之病毒株及由野鳥所分離之病毒株之亞型區分上，並將產物以定序後確認之，結果均可區分出病毒之血清亞型；而在NA亞型方面，由於目前對於其基因序列的研究只偏重在N1、N2亞型，能供作我們進行分析設計引子之基因序列的資訊相當少，所以設計引子之通用性也相對的受到限制，在應用上效果並不佳。 對於台灣地區所分離之AIV，雖有分離到H7亞型之病毒株，但目前並未發現有具高病原性之病毒株，而從家禽中只分離到2株H6N1亞型病毒株，其均屬於低病原性毒株，在臨床上均無引起明顯之症狀；而就基因族譜之分析上，在核蛋白基因分析上，確認為禽源之毒株，在宿主性及地域性上與雁鴨分離株和香港97年H5N1分離株有相近之關係。

Abstract This study uses RT-PCR for the rapid identification of avian influenza virus (AIV) in clinical cases. We designed a pair of universal primers according to the conserved sequences of the viral nucleoprotein gene. This pair of primers was able to amplify specific PCR products from standard AIV strains and viruses isolated from free-living birds. Because the clinical symptoms of AIV are very similar to those of Newcastle virus (NDV), we developed a multiplex RT-PCR procedure to identify and differentiate AIV and NDV in a single PCR reaction. This multiplex RT-PCR procedure could also differentiate vaccine strains of NDV from field isolates. Experiments using chickens inoculated with AIV and NDV showed that the multiplex RT-PCR could serve as a rapid diagnostic procedure for AIV and NDV. For rapid typing and subtyping of AIV, we designed a pair of specific primers for each HA subtype and NA subtype. For the HA subtyping, the primers we designed were based on the published nucleotide sequences of the HA genes. We found that RT-PCR using these primers could correctly subtype all the standard AIV strains and viruses tested. For the NA subtyping, because only very limited numbers of NA gene sequences are published, the primers we designed gave unsatisfied results in NA subtyping of AIV strains tested. Though the H7 subtype viruses were isolated in Taiwan, they are not highly virulent strains. The two H6N1 viruses we isolated from chickens are also low pathogenic strains, and produced no apparent symptom. Phylogenetic analysis based on the nucleoprotein sequences revealed that viruses of avian origins are closely related to viruses isolated from anatidae in term of host specificity, and are closely related to H5N1 viruses isolated in Hong Kong in term of geographic distribution.