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研究生: 陳又瑄
Chen, Yu-Hsuan
論文名稱: 台灣2014至2019年豬生殖與呼吸綜合症病毒NSP2及ORF5基因序列分析
Sequence analysis of NSP2 and ORF5 genes of PRRSV in Taiwan from 2014 to 2019
指導教授: 邱明堂
Chiou, Ming-Tang
林昭男
Lin, Chao-Nan
學位類別: 碩士
Master
系所名稱: 獸醫學院 - 獸醫學系所
Department of Veterinary Medicine
畢業學年度: 108
語文別: 中文
論文頁數: 62
中文關鍵詞: 豬生殖與呼吸綜合症基因序列第5開放讀碼區第2非結構蛋白親源樹狀圖
外文關鍵詞: porcine reproductive and respiratory syndrome, genomic sequence, ORF5, NSP2, phylogenetic trees
DOI URL: http://doi.org/10.6346/NPUST202000010
相關次數: 點閱:42下載:8
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  • 豬生殖與呼吸綜合症 (porcine reproductive and respiratory syndrome, PRRS) 由PRRS病毒 (PRRS virus, PRRSV) 引起,此病自1980年晚期即嚴重危害全球豬隻產業。PRRSV之第2非結構蛋白 (non-structure protein 2, NSP2) 及第5開放讀碼區 (open reading frames 5, ORF5) 變異度大且與病毒之毒力相關,兩者常作為遺傳變異及流行病學之研究標的。目前,最普及用於控制PRRS方式為疫苗免疫,但疫苗針對異源性病毒株則僅具部分交叉保護力。儘管台灣於90年代中期開始開放使用PRRS減毒活毒疫苗 (modified live vaccines, MLV) 來控制此病,但本病仍持續活躍,而病毒之變異程度及其毒力改變應為疫情居高不下之原因。因此本研究目的為探討台灣2014至2019年PRRS病毒NSP2及ORF5基因序列變異程度。本研究挑選PRRSV陽性樣本增幅及選殖NSP2及ORF5基因片段,並參考26株國外PRRSV序列及32株台灣序列,以MEGA X及MegAlign軟體繪製親源樹狀圖與計算核苷酸及胺基酸相似度。依據親源樹狀圖、核苷酸及胺基酸相似度分析,本研究收集之98株PRRS病毒之ORF5基因序列屬於NA type,4株ORF5屬於EU type,22株NSP2則皆屬於NA type。台灣田間分離株主要由原型病毒株MD001演化而來,且與2014年以前自彰化、雲林及屏東三縣市分離之病毒株較為相似。於胺基酸序列分析,98株NA type ORF5變異程度最大之區域為第33個胺基酸,出現12種不同胺基酸 (amino acid, aa) 置換,比對VR-2332病毒株,其誘騙表位 (decoy epitope) (aa27-30)、主要中和表位 (aa37-45) 及N醣基化位點 (aa30、aa32/33/34) 皆出現不等程度之變異,另22株PRRSV NSP2胺基酸序列則皆屬於台灣特有之type D刪除模式,而這些胺基酸之變異或與疫苗效力不彰或豬場PRRS疫情難於控制有關。根據HS豬場ORF5基因序列及其保育豬隻死亡率分析顯示,核苷酸相似度與豬隻死亡率具負相關性。由於病毒之變異將影響診斷技術、疫苗研發及疫苗使用後之保護力,因此,定期監控於田間循環之PRRSV對PRRS之防控至關重要。

    Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) has led to significant economic losses in global swine industry since the late 1980s. Non-structure protein 2 (NSP2) and open reading frames 5 (ORF5) genes are the most variable regions of the PRRSV genome and are thus considered the important regions for studying the genetic variation and epidemiological evolution of PRRSV. The most effective method for controlling PRRS is vaccine immunization, but the vaccine is only partially cross-protective against heterologous virus strains. Although modified live vaccines (MLV) have been approved and used since the mid 90s, severe outbreaks continue to occur in Taiwan until now. Since viral variation and its virulence may be the cause of the high epidemic. The aim of the present study is to analysis the genetic diversity of NSP2 and ORF5 genes of PRRSV in Taiwan from 2014 to 2019. PRRSV-positive samples are selected for fragment amplification and gene cloning. Phylogenetic trees, nucleotide and amino acid (aa) sequence identities are constructed with 26 foreign and 32 Taiwanese sequences by MEGA X and MegAlign software. There are 98 strains of ORF5 gene sequences belonging to North American (NA) type and 4 strains belong to European (EU) type. Besides, 22 strains of PRRSV NSP2 are all NA type. The field isolates in Taiwan are mainly evolved from the prototype virus strain MD001, and are similar to those isolated in Changhua, Yunlin and Pingtung counties before 2014. Base on aa sequence analysis, the most variable site of 98 NA type PRRSV ORF5 is the 33th aa site with 12 different substitutions. The decoy epitope (aa27-30), main neutralizing epitope (aa37-45) and N glycosylation (aa30、aa32/33/34) site all showed different degrees of variation when comparing to VR2332. In addition, aa sequences of 22 PRRSV NSP2 strains belong to unique type D deletion mode in Taiwan. These aa variations may be the cause of poor vaccine efficacy or difficulty in controlling PRRSV outbreaks on pig farms. In addition, according to PRRSV ORF5 sequences and the mortality rates of nursery pigs on the HS farm, nucleotide identity has negative correlation with pig mortality. Since the mutation and recombination of virus will affect diagnostic technology, vaccine development and ability of protection, regular monitoring of PRRSV is critical to PRRS prevention and controll.

    中文摘要 I
    Abstract III
    誌  謝 V
    目  錄 VI
    圖表目錄 IX
    第 1 章 前 言 1
    第 2 章 文獻回顧 3
    2.1 豬生殖與呼吸綜合症病毒 3
    2.1.1 歷史背景與經濟衝擊 3
    2.1.2 病毒介紹 4
    2.1.2.1 病毒學 4
    2.1.2.2病毒基因之多樣性 4
    2.1.2.3 Glycoprotein 5 5
    2.1.2.4 Non-structural protein 2 6
    2.1.3 傳播及感染途徑 7
    2.1.4 致病機制 7
    2.1.5 臨床症狀 8
    2.1.6 病理病變 9
    2.1.7 診斷檢測 10
    2.1.7.1免疫組織化學染色 (immunohistochemistry, IHC) 10
    2.1.7.2聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 11
    2.1.7.3血清學 11
    2.1.7.4病毒分離 (virus isolation, VI) 12
    2.1.8 預防及控制 12
    第 3 章 材料與方法 14
    3.1 實驗設計 14
    3.2 病例收集 15
    3.3 樣本前處理及核酸萃取 15
    3.4 反轉錄即時聚合酶連鎖反應 (reverse transcription real-time polymerase chain reaction, RT-qPCR) 16
    3.5 聚合酶連鎖反應 (polymerase chain reaction, PCR) 16
    3.6 膠體電泳分析 17
    3.6.1 瓊脂膠體製備 17
    3.6.2 電泳分析 17
    3.7 PCR產物之純化及含氮鹼基A之接合 18
    3.8 PCR產物之選殖 19
    3.8.1 接合作用 (ligation) 19
    3.8.2 轉形 (transformation) 19
    3.8.3 菌株篩選 (blue and white screening) 19
    3.9 PRRSV ORF5 基因序列分析 20
    3.10 PRRSV NSP2 基因序列分析 21
    3.11 圖表繪製與統計分析 21
    第 4 章 結 果 22
    4.1 樣本收集 22
    4.2 PRRSV ORF5親源樹狀圖分析 22
    4.3 PRRSV ORF5核苷酸序列分析 23
    4.4 PRRSV ORF5胺基酸序列分析 23
    4.5 PRRSV NSP2胺基酸序列分析 24
    4.6 屏東縣HS豬場之PRRSV ORF5基因序列 24
    4.6.1 PRRSV ORF5親源樹狀圖分析 24
    4.6.2 PRRSV ORF5核苷酸序列分析 25
    4.6.3 PRRSV ORF5胺基酸序列分析 25
    4.6.4 PRRSV ORF5變異程度與保育豬隻死亡率分析 25
    第 5 章 討 論 44
    參考文獻 48
    作者簡介 62

    Figure 1. Phylogenetic trees based on the ORF5 of 102 Taiwanese PRRSV strains and 26 reference PRRSV strains.. 27
    Figure 2. Phylogenetic trees based on the ORF5 of 98 Taiwanese PRRSV strains and 32 reference PRRSV strains. 29
    Figure 3. Schematic amino acid of PRRSV GP5. 31
    Figure 4. Alignment of amino acid sequences of NSP2 from 41 PRRSV strains.. 32
    Figure 5. Phylogenetic trees based on the ORF5 of 20 Taiwanese PRRSV strains from the HS pig farm and 5 reference PRRSV strains. 34
    Figure 6. The nucleotide sequence identity and divergence of MD001, MLV and 20 Taiwanese PRRSV strains from the HS pig farm in this study. 35
    Figure 7. The amino acid sequence identity and divergence of MD001, MLV and 20 Taiwanese PRRSV strains from the HS pig farm in this study. 36
    Figure 8. Comparison of relationship between mortality of nursery pigs and the ORF5 nucleotide identity on the HS pig farm from January 2016 to March 2019. 37
    Figure 9. Linear regression between mortality of nursery pigs and the ORF5 nucleotide identity on the HS pig farm from January 2016 to March 2019. 38

    Table 1. Primers and ZNA probe used in the multiplex real-time PCR assay for the M gene 39
    Table 2. Primers used for RT-PCR assay for the NSP2 and ORF5 gene 40
    Table 3. PRRSV isolates reported previously worldwide used for comparison and constructing the phylogenetic tree in this study 41
    Table 4. PRRSV strains isolated from field outbreaks in Taiwan between 1991 to 2013 used for comparison and constructing the phylogenetic tree in this study 42
    Table 5. PRRSV isolates reported previously worldwide used for NSP2 amino acid sequence comparison in this study 43

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