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

陳又瑄(Chen Yu-Hsuan)

指導教授 ： 邱明堂 ； 林昭男

國立屏東科技大學/獸醫學院/獸醫學系所/碩士(2019年)

https://doi.org/10.6346/NPUST202000010

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

porcine reproductive and respiratory syndrome ； genomic sequence ； ORF5 ； NSP2 ； phylogenetic trees

參考文獻

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