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標題: A Survey of Avian Polyomavirus (APV) Infection and Viral Genetic Characteristics in Taiwan during 2012-2013
作者: Yi-Hsin Lai 賴奕欣
關鍵字: 鳥多瘤病毒;病毒核酸序列;臺灣;avian polyomavirus;viral genetic characteristic;Taiwan
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鳥多瘤病毒可造成多種類鸚鵡之急性、致死性及高傳染性的疾病,近年來在世界各國針對APV所做的流行病學研究均以PCR作為診斷工具,而本研究亦以PCR做檢測。收集的材料來自於臨床病例、鸚鵡繁殖場的糞便拭子及死亡鳥隻檢體,總計有443個樣本並可分類22個屬別以及41個種別。除了以PCR方法檢測病毒核酸,在剖檢檢查可見感染APV的鳥隻有肝臟腫大出血、脾臟腫大且有多發局部性壞死灶等特徵性的肉眼病變。另外,於組織病理學檢查,可見感染細胞呈現細胞核腫大並有特徵性的嗜鹼性核內包涵體,其中以脾臟及腎小管上皮細胞較容易觀察之。本實驗所設計用以診斷APV感染之PCR 引子對,其目標增幅產物為VP1基因片段,大小約為272 bp,此檢測方法亦具有良好的敏感性與特異性。以此PCR試驗檢測結果APV總陽性率為37.47% (166/443),顯示APV持續存在於台灣的事實。另外雙重基因定序設計針對病毒之VP 2/3及t/T antigen之基因片段作分析,得知本研究中所分離出20株病毒株針對此兩段部分基因的定序比對相似度高達98.3%以上,表示此20株台灣株針對此部分基因片段具有高度保留特性。進一步針對此兩段基因片段與來自於世界各國已發表之病毒株做比對,其結果亦不具有顯著性差異。然而,傳統型PCR有可能形成非特異性的產物而且其敏感性仍須再提高,這些傳統型PCR所包含的缺點可藉由發展新式定量型即時PCR來彌補。本研究所設計的即時PCR試驗是利用SYBR Green螢光系統來做標定,並使用與傳統型PCR相同之診斷引子對。此即時PCR可偵測到的病毒最低濃度為101 病毒之複製單位,此外藉由帶有其他種鸚鵡病原核酸材料包括鸚鵡喙羽病病毒、鸚鵡披衣菌、大腸桿菌以及沙門氏菌,並未有任何產物被增幅出來,顯示此試驗具有良好的特異性。即時PCR試驗除了有良好的特異性與敏感性,其所具備的再現性以及降低檢測過程中檢體之間汙染的風險,因此適合發展為快速檢測APV感染之臨床診斷試驗。

The avian polomavirus (APV) is an acute, lethal, and highly contagious viral disease in most parrot species. Recently, the worldwide epidemiological studies of APV infection in psittacine birds were performed by PCR. In this study, we collected fecal and tissue samples from clinical cases and a breeding aviary. For APV prevalence, 443 samples from 22 genera and 41 species of psittaciform birds were checked by PCR assay. Confirmed by necropsy exam which indicated hepatomegaly with hemorrhage and swollen spleen in the sick birds. In further histopathologic exam, the characteristic basophilic intranuclear inclusion bodies could be found in karyomegalic cells in spleen, renal tubular epithelium, and liver. Based on the sequence of the VP1 gene of APV from Genbank, we designed a PCR assay with high sensitivity for APV detection. The total positive rate was 37.47% (166/443) indicating APV persistently existing in Taiwan. Multiple gene sequencing assays were performed at combined region of VP2 and VP3 and the gene encoding region of t/T antigens. The similarity of 20 isolates of VP2/3 and t/T antigen coding regions from Taiwan ranged from 98.3–100.0%. This investigation revealed that APV isolated from Taiwan was highly conserved. The sequencing results of 20 isolates in this study also indicated that there was no significant difference by comparing with worldwide sequences in the tested gene regions. However, the public conventional PCR assays for APV detection might amplify non–specific products and the sensitivity of the assays should be improved. These drawbacks could be avoided by developing the quantitative real–time PCR (qPCR) assay, which showed improved rapidity, sensitivity, reproducibility, and reduced the risks of contamination. In the current study, the qPCR assay based on SYBR Green Ⅰ fluorescence and the primer set designed at the conserved region of VP1 gene was developed. The detection limit of the qPCR assay reached 1×101 copies of viral DNA, and the assay was highly specific without amplifying any product from other avian viral and bacterial genomes (including PBFD, Chlamydia, E. coli and Salmonella Typhimurium). This newly developed qPCR is a suitable tool for rapid clinical investigations
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