由台灣鵝分離家禽網狀內皮增生症病毒與序列分析

Abstract

目前有關家禽網狀內皮增生症病毒 (Avian Reticuloendotheliosis Virus, REV) 的研究以雞與火雞為主，與鵝相關的研究很少，本研究的目的是了解台灣現場鵝隻REV疾病現況，利用聚合酶鏈反應 (PCR) 以及血清中和試驗來檢測REV感染情形。於一家白羅曼鵝場病例中將buffy coat接種到DF-1細胞，分離到九株REV分離株，完成其中一株全段前病毒基因定序，將此株REV與台灣雞REV分離株共四株與國外REV進行親源性分析，比較國內外REV的基因序列差異。結果顯示此分離株與其他株的gag跟pol基因相似度分別是96.9到99.9%以及96.7到99.9%之間，env基因相似度為95.9到100%之間，LTR是在REV基因序列中差異最大的區域，此分離株與其他株LTR基因相似度為74.9到99.8%之間，因為在LTR區域中含有較大片段的插入和缺失。根據以上序列比對以及親源性分析之結果，顯示此分離株與其他REV毒株相當接近，表示REV在不同品種的禽類宿主基因的變異性很小。塗鍍濃縮純化的REV全病毒搭配標示過氧化氫酶之單株抗體以架構阻斷型酵素連結免疫吸附反應 (ELISA)，以用來檢測水禽REV抗體，實驗結果顯示每孔需要塗鍍600 ngREV全病毒搭配50倍稀釋標有過氧化氫酶之抗體才能達到OD值接近於1之效果，但此條件不符合經濟效應。以PCR檢測四場鵝隻血液，結果顯示血液REV核酸陽性率為28.8%，以血清中和試驗檢測抗體，其陽性率為60.3%，顯示台灣鵝隻REV感染情況為相當普遍。

Many reticuloendotheliosis virus (REV) associated studies have been focused on chicken or turkey, but rarely on goose. The purpose of this study was to investigate the REV occurrence in geese in Taiwan. PCR and serum neutralization test were used to detect REV infection. Nine REV isolates from a white Roman goose farm were isolated by inoculating buffy coat onto the DF-1 cell line. The complete proviral sequence of one REV was determined. The relationship of gene sequences of four Taiwanese REVs were compared with the reference REVs by the phylogenetic analysis. The percent of nucleotide identity of gag and pol ranged from 96.9 to 99.9% and 96.7 to 99.9%, respectively. The percent of nucleotide identity of env ranged from 95.9 to 100%. The LTR was the most divergent region of genome with 74.9 to 99.8% nucleotide identity containing deletion and insertion. It indicates that this isolate is closely related to other REVs and the gene diversity among REV isolates in different avian hosts is minimal. For developing a blocking enzyme-linked immunosorbent assay (ELISA) to detect antibody in waterfowls, the concentrated and purified REV was used as coating antigen and monoclonal antibody was labeled with horseradish peroxidase as tracer. The optical density value was close to 1 on the condition that 600 ng of REV per well was coated with 50-fold diluted tracer. The REV detection by PCR showed 28.8% positive rate and antibody detection by serum neutralization test showed 60.3% positive rate in four goose farms. Thus REV infection is common in geese in Taiwan.