發(fā)布時間：2018-04-04 09:24

  本文選題：坦布蘇病毒　切入點(diǎn)：雛鴨　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：坦布蘇病毒(Tembusu virus,TMUV)感染是危害養(yǎng)鴨業(yè)的新發(fā)傳染病,對于我國養(yǎng)鴨業(yè)具有重要的經(jīng)濟(jì)意義。迄今為止,TMUV的分子致病機(jī)制尚不清楚。本研究以毒力存在差異的TMUV分離株為材料,對決定毒株間毒力差異的關(guān)鍵基因和位點(diǎn)進(jìn)行分析和鑒定,以期為闡明TMUV的分子致病機(jī)制提供數(shù)據(jù)。2014年10月,兩個3～4周齡麻鴨群發(fā)生了一種以癱瘓為特征的疾病。經(jīng)RT-PCR檢測、病毒分離、間接免疫熒光檢測和動物試驗(yàn),確定致病病原為TMUV。將第3代鴨胚分離株(GL株)經(jīng)腦內(nèi)途徑接種2日齡北京鴨,可復(fù)制出與自然病例相似的癥狀,在接種后3～6d雛鴨死亡率達(dá)100%。結(jié)果表明,該TMUV分離株對北京鴨雛鴨具有高致病性。為觀察TMUV不同毒株的毒力差異,以2日齡北京鴨為動物模型,用PS株、Y株和GL株經(jīng)腦內(nèi)途徑進(jìn)行感染試驗(yàn)。結(jié)果顯示,感染Y株和GL株后,雛鴨表現(xiàn)出嚴(yán)重的臨床癥狀和組織病理變化,且死亡率分別為80%和70%。而PS株未導(dǎo)致雛鴨死亡,僅影響雛鴨增重,所引起的組織病理學(xué)變化也較輕微。由此可見,Y株和GL株對雛鴨的毒力顯著高于PS株。基因組測序和序列比較結(jié)果顯示,在非編碼區(qū),PS株分別與Y株和GL株存在3個和4個堿基差異;在聚蛋白氨基酸水平,PS株分別與Y株和GL株存在16個和19個氨基酸差異,結(jié)果提示,這些差異位點(diǎn)可能是決定毒株間毒力差異的分子基礎(chǔ)。為鑒定TMUV毒力相關(guān)基因和位點(diǎn),構(gòu)建了PS株的反向遺傳操作技術(shù)平臺。用RT-PCR對PS株基因組進(jìn)行了分段擴(kuò)增,獲得5個(稱為A、B、C、D和E)基因片段,并由此構(gòu)建重組質(zhì)粒pBR322-AB和pBR322-BCDE。以這兩個質(zhì)粒為模板進(jìn)行PCR擴(kuò)增,并利用融合PCR方法獲得PS株的全長cDNA。經(jīng)體外轉(zhuǎn)錄,用純化后的RNA轉(zhuǎn)染BHK-21細(xì)胞,成功拯救出病毒。比較結(jié)果顯示,拯救病毒在BHK-21細(xì)胞上的生長特性以及對小鼠的致病性均與親本病毒相似,除遺傳標(biāo)記外,拯救病毒與親本病毒序列一致。在上述工作基礎(chǔ)上,以PS株基因組為骨架,分別構(gòu)建了含Y株不同基因區(qū)(5'UTR、3'UTR、E 和 NS1-3'UTR)的嵌合毒株(rPSY5'UTR、rPSY3UTR、rPSYE和 rPSYNS1-3'UTR)。致病性試驗(yàn)結(jié)果顯示,嵌合病毒rPSYE可使70%的雛鴨死亡,與Y株的毒力相近,由此可見,用Y株的E基因替換PS株的E基因,可顯著提高PS株對雛鴨的致病性,提示E基因是決定TMUV PS株和Y株毒力差異的關(guān)鍵基因。運(yùn)用定點(diǎn)突變技術(shù),將PS株E蛋白304位精氨酸(R)突變?yōu)閅株E蛋白對應(yīng)位置上的蛋氨酸(M),構(gòu)建了突變病毒 rPSYER304M。致病性試驗(yàn)結(jié)果顯示,用 rPSYER304M感染2日齡北京鴨,可引起60%的死亡率,與Y株所致死亡率相似,而親本拯救毒株rPS感染雛鴨的死亡率僅為10%。結(jié)果表明,E蛋白304位氨基酸(R/M)是決定TMUV PS株和Y株毒力差異的關(guān)鍵位點(diǎn)。
[Abstract]:Tembusu virus Tembusu virus (TMUV) infection is a new infectious disease that endangers the duck industry and has important economic significance for the duck industry in China.Up to now, the molecular pathogenesis of TMUV is still unclear.In this study, TMUV isolates with different virulence were used as materials to analyze and identify the key genes and loci that determine virulence differences among strains, in order to provide data for elucidating the molecular pathogenicity of TMUV.A disease characterized by paralysis was found in two 3-and 4-week-old ducks.By RT-PCR detection, virus isolation, indirect immunofluorescence detection and animal test, the pathogenic agent was identified as TMU V.The third generation of duck embryo isolate strain GL) was inoculated into the brain of 2 day old Beijing duck. The symptoms similar to those of natural cases could be replicated. The death rate of ducklings reached 100 at 3 ~ 6 days after inoculation.The results showed that the TMUV isolate had high pathogenicity to Peking duck ducklings.In order to observe the virulence difference of different strains of TMUV, two day old Beijing ducks were used as animal models. The infection test was carried out by using PS strain Y strain and GL strain through brain pathway.The results showed that after infection with Y strain and GL strain, ducklings showed severe clinical symptoms and histopathological changes, and the mortality was 80% and 70%, respectively.PS strain did not cause the death of ducklings, but only affected the weight gain of ducklings, and the histopathological changes were slight.Therefore, the virulence of Y strain and GL strain to ducklings was significantly higher than that of PS strain.The results of genomic sequencing and sequence comparison showed that there were 3 and 4 base differences between PS strain and Y strain and GL strain in non-coding region, 16 and 19 amino acid differences between PS strain and Y strain and GL strain at polyprotein amino acid level, respectively.The results suggest that these differential sites may be the molecular basis for the virulence difference among strains.In order to identify the virulence related genes and loci of TMUV, a platform for reverse genetic manipulation of PS strain was constructed.The genome of PS strain was amplified by RT-PCR, and five fragments were obtained, and the recombinant plasmids pBR322-AB and pBR322-BCDE were constructed.The two plasmids were used as templates for PCR amplification, and the full-length cDNA of PS strain was obtained by fusion PCR method.After transcription in vitro and transfection of BHK-21 cells with purified RNA, the virus was successfully saved.The results showed that the growth characteristics and pathogenicity of the rescue virus on BHK-21 cells were similar to those of the parent virus, except for genetic markers, the sequence of the rescue virus was consistent with that of the parent virus.On the basis of the above work, the chimeric strains rPSY5UTR3UTRE and NS1-3UTRE were constructed with the genome of PS strain as the skeleton, and the chimeric strains rPSY5UTR3UTRYE and rPSYNS1-3UTRYE were constructed respectively.The results of pathogenicity test showed that the chimeric virus rPSYE could kill 70% of ducklings and the virulence of Y strain was similar to that of Y strain. Thus, the pathogenicity of PS strain could be significantly improved by replacing the E gene of PS strain with Y strain.It is suggested that E gene is the key gene to determine the virulence difference between TMUV PS strain and Y strain.Using site-directed mutagenesis technique, the mutant virus rPSYER304M was constructed by mutating E protein 304 arginine R) into methionine MN on the corresponding position of Y strain E protein.The results of pathogenicity test showed that the death rate of Beijing duck infected with rPSYER304M for 2 days was 60%, similar to that caused by Y strain, while the death rate of parent saving strain rPS infected ducklings was only 10%.The results showed that the amino acid R / M at position 304 of E protein was the key site to determine the virulence difference between TMUV PS strain and Y strain.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王建昌;王金鳳;袁萬哲;劉立兵;;鴨坦布蘇病毒一步法RT-PCR與熒光定量RT-PCR檢測方法的建立[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2016年08期

2 田浪;馮曉聲;郭吉余;錢雪橋;;禽坦布蘇病毒研究進(jìn)展[J];中國畜牧獸醫(yī);2016年02期

3 王玉倩;薛秀花;;實(shí)時熒光定量PCR技術(shù)研究進(jìn)展及其應(yīng)用[J];生物學(xué)通報(bào);2016年02期

4 黃瑜;蘇榮茂;傅光華;萬春和;陳紅梅;傅秋玲;陳珍;施少華;程龍飛;林建生;;禽坦布蘇病毒感染的宿主及臨床表現(xiàn)[J];中國獸醫(yī)雜志;2014年11期

5 萬春和;施少華;黃瑜;;禽坦布蘇病毒包膜蛋白E的基因特征[J];福建農(nóng)業(yè)學(xué)報(bào);2014年08期

6 寇甜甜;丁天兵;;黃病毒屬病毒E蛋白結(jié)構(gòu)和功能研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2014年03期

7 傅光華;陳紅梅;黃瑜;萬春和;傅秋玲;程龍飛;施少華;林建生;;種番鴨源禽坦布蘇病毒分離鑒定及基因組序列分析[J];福建農(nóng)業(yè)學(xué)報(bào);2014年04期

8 唐雷;葛俊偉;韓雪;劉志鵬;;反向遺傳操作技術(shù)的發(fā)展及趨勢[J];畜牧獸醫(yī)科技信息;2014年03期

9 韓凱凱;李銀;黃欣梅;趙冬敏;劉宇卓;謝星星;游園;;鵝源坦布蘇病毒RT-LAMP快速檢測方法的建立[J];浙江農(nóng)業(yè)學(xué)報(bào);2014年01期

10 葛平萍;刁有祥;唐熠;劉鑫;高緒慧;于春梅;曲俊姿;;鴨坦布蘇病毒山東株的分離鑒定及其特性[J];中國獸醫(yī)學(xué)報(bào);2014年01期

相關(guān)博士學(xué)位論文 前1條

1 王笑言;鴨產(chǎn)蛋下降相關(guān)新病毒的分子鑒定與生物學(xué)特性研究[D];中國農(nóng)業(yè)大學(xué);2015年

，

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