台灣禽類E型肝炎病毒血清流行病學調查及分離鑑定

Abstract

E型肝炎病毒(Hepatitis E virus; HEV)為人畜共通傳染病原，能夠在人類與哺乳類之間互相傳播。禽類HEV目前雖未被證實能夠人畜共通，但卻能造成禽類產業重大的經濟損失。為了了解禽類HEV在台灣的流行病學資訊，本論文從台灣12個地區61個雞場共收集了1,326隻禽類血清，並以酵素結合免疫吸附法進行禽類HEV抗體檢測。結果顯示雞場抗體陽性率(FPR)為95.1%，雞隻抗體陽性率(CPR)為40.57%，不同地區之CPR介於9%~58.48%之間。去除台灣東北地區最低的CPR數值9% (FPR=80%)後，總CPR為43.15% (FPR=96.4%)。以線性模式之變方分析法進行趨勢比較，種雞、蛋雞、以及地區之間的CPR並無顯著差異。本調查揭示了台灣雞場有嚴重的禽類E型肝炎病毒感染情形，而台灣西部區域又比東北區域嚴重。 此外收集4處雞場150隻雞隻之膽汁、肝臟、脾臟以及腸管等檢體進行禽類HEV之分離鑑定、定序，並進行親緣關係分析。首先，以3對預先準備的退化性引子，從150隻雞檢體中的一個膽汁檢體檢出禽類HEV。根據3對引子所獲得的禽類HEV片段，設計引子並進行病毒之全序列定序，依循由每次定序結果來修正設計引子的策略，總共設計了25支引子來進行全定序，結果顯示台灣禽類HEV RNA基因體全長為6,653個鹼基對。以基因庫中全序列或接近全序列的8株禽類HEV，加上1株豬型HEV作為外群，使用鄰位連接法、最大概似法以及貝氏推論法等親緣分析方式進行核酸以及蛋白質親緣關係鑑定。結果顯示台灣禽類E型肝炎病毒株與2012年發表之匈牙利病毒株(JN997392)同源性最高(核酸以及胺基酸之相似度各為86.5%與96.7%)，推測台灣禽類E型肝炎病毒株與其親緣關係高之匈牙利病毒株為禽類E型肝炎病毒之新型或第四型。 再者，開放閱讀框架2 (衣殼蛋白基因) 的後端185個鹼基對被用於檢測以及分析親緣關係。結果顯示，18株台灣禽類E型肝炎病毒分離株核酸相似度達100%，和其他國家病毒株相比，核酸相似度介於81.6% - 84.3%；親緣關係分析也顯示台灣禽類E型肝炎病毒株的特有性。

Hepatitis E virus (HEV) is a zoonotic pathogen transmitting between human and mammalian. Avian HEV may not be zoonotic, but is a causative pathogen of chickens to cause economic losses. To better understand the epidemiological profiles of avian HEV in Taiwan, this study used 1,326 chicken sera collected from 61 flocks in 12 administrative regions, and used ELISA to detect serum antibody against avian HEV. The flock seropositive rate (FPR) and chicken seropositive rate (CPR) were estimated. CPRs were estimated ranged between 9% and 58.48% depending on farm locations. Excluding the lowest 9% CPR (FPR=80%) samples from northeast Taiwan solely, comparisons made by ANOVA are significantly not different between breeders and layers and among geographical locations. Accordingly, CPR was estimated about 43.15% (FPR=96.4%) excluding samples from northeast Taiwan; and 40.57% (FPR=95.1%) from all detected sera, revealing that chicken farms in Taiwan have heavily been contaminated by avian HEV and which in the western Taiwan is even more seriously than in the northeastern region. Furthermore, specimens including bile, liver, spleen and intestine from 150 chickens of 4 poultry farms were used to isolate, identify, sequencing and analyze phylogenetic relationship of the avian HEV. In preliminary, an avian HEV strain was detected from bile specimen among 150 samples with 3 predesigned degenerative primers. Further, the sequence of detected avian HEV strain fragment was subsequently used to design primers and sequencing in one after another, accordingly, 25 primers have been designed and used to sequencing the avian HEV stain. Consequently, a full length of 6,653 bp was sequenced for Taiwan strain of avian HEV. And the phylogenetic relation was analyzed with using 8 complete or nearly complete sequences of avian HEV and 1 swine HEV as the outgroup from GenBank, with their encoded amino acids by neighbor joining, maximum likelihood and Bayesian inference methods. The concordant result indicates that Taiwan strain of avian HEV is closely related to one of Hungary strains reported in 2012 (GenBank No. 997392) with 86.5% homology of nucleotide and 96.7% of encoded amino acids, and might be classified with Hungarian strain as a new genic type or genic type 4. In addition, 185 bp locating in the 3’ end of ORF2 gene (capsid gene) of 18 isolates was sequenced and compared. The result indicates the nucleotide sequence identity of all 18 Taiwanese avian HEV strains was 100% in this region; and the identity between Taiwanese strains and foreign strains was 81.6% - 84.3%. The phylogenetic analysis also reveals the unique of the Taiwanese strain of avian HEV.