台灣新近分離新城病毒之研究

新城病（Newcastle disease ；ND）係由ND病毒(ND virus；NDV)所引起之家禽重要惡性呼吸、消化和神經系統性傳染病。近年來世界各地NDV抗原性的變異情形頗為常見，造成ND的爆發甚為普遍，台灣也於1999年跟隨全世界的ND瘟疫在某些地區爆發較多病例，並迅速擴及全台，其後仍偶見發生直到2008年。探索近年來台灣發生的ND病例大都發生於經ND疫苗免疫的雞群；而在診斷也出現與傳統血球凝集抑制（HI）診斷抗原(以Ishii株製成)無法產生交叉反應之新病毒株。本研究檢測新分離之NDV30株在腦內病性指數 ( ICPI:1.54-1.76)和平均死亡時間 ( MDT:36-48小時)檢測上均予歸為急性內臟型(VV) NDV (VVNDV) 。30株2002-2008年台灣地區所分離之NDVs中有29株(96.7%)屬於VIIe基因亞型，只有1株(3.3%)屬於VIIc基因亞型。VIIe基因亞型與其它VII基因亞型(VIIa、VIIb、VIIc及VIId)之核苷酸序列差異性分別為5.9-12.4%、9.3-12.8%、3.6-8.3%和3.9-6.8%。VIIe基因亞型與其它基因亞型(VIIa、VIIb、VIIc及VIId)之胺基酸序列差異度分別為5.9-15.2%、8.5-12.3%、3.3-12.3% 及5.0-9.5%。台灣地區分離NDV之核苷酸演化速率在VIIe亞型及VIIc亞型分別為9.98X104及3.21X104 bp/year。以F蛋白(aa 1-125)胺基酸序列之變異分析顯示，台灣分離VIIe基因亞型NDVs以其核苷酸序列之變異情形又可區分為VIIe1、VIIe2、VIIe3及VIIe4四小群。VIIe2之F蛋白胺基酸具有L23 F和T90  A之變異。VIIe3亞型則具有三個胺基酸序列的分別於特殊變異(E74  G 、A75 G及Q114  K)。VIIe4亞型具有三個胺基酸序列的特殊變異(I26  T， T29  A和A75  G)。病毒在這些胺基酸的變異或許是造成近年來臺灣地區分離NDVs之致病性和抗原性改變有關。因為發生在VIIe3和VIIe4小群之2 個重要胺基酸改變(E74  G、A75  G)恰好是 F蛋白質的中和抗體抗原決定位所在。台灣分離株HN蛋白大小為571個胺基酸。分析目前台灣分離NDV之HN蛋白之胺基酸序列發現有6個胺基酸 (R197  K、S200 L、H203 Y、E347 G、R352 T及S521 N) 發生變異，這些胺基酸的變異可能會影響HN蛋白的抗原性及其他功能。本實驗將原型及改變HN基因之二個病毒(TW/02-302HNM81I及TW84-1211HNI81M)之HN基因轉染至COS-7細胞中，在細胞表現I81M變異的HN蛋白若與M81I變異HN蛋白相較，會降低其對紅血球吸附的能力。以Sato (genotype III)及TW/02-301株(genotype VIIe) NDV製備鋁膠不活化疫苗，在經免疫21日齡蛋雞進行交叉保護性試驗，發現蛋雞免疫TW/02-301做成之自家疫苗對TW/02-301株攻擊的死亡率0%，對Sato株做成之死毒疫苗免疫組之死亡率為 42.6％，陰性對照組雞則全部死亡。台灣新近分離之VVNDV在F、HN及P/V基因已發生些許變異，此顯示台灣ND之疫情須以台灣新分離之VVNDV製備之不活化疫苗才能加以有效的控制。

關鍵字

F蛋白； HN蛋白；親源性分析

並列摘要

Newcastle disease (ND）is an important poultry disease with severely acute respiratory, digesive and neurological signs and syndromes caused by ND virus (NDV). Antigenic variation of the recently isolated NDVs has always been world widely reported. Which ends up with the ND outbreaks quite often been found reported elsewhere in the world. Some areas in Taiwan was following up with the world ND epidemic with more ND cases occurred, then wide spreading the whole island till 2008. Tracing the ND cases in Taiwan revealed that most of the outbreaks were happen in the ND vaccinated chicken. Serological detection of the serum of the new NDV infected chicken provided only very low hemagglutination-inhibition (HI) antibody titer with the ND antigen made of traditional strain (Ishii). Among the 30 local isolates of NDV molecularly studied in the experiment, 29 isolates (96.7%) were subgenotypely classified as VIIe except one isolate (3.3%) was VIIc. The nucleotide sequence difference of VIIe from VIIa、VIIb、VIIc及VIId is 5.9-12.4%, 9.3-12.8%, 3.6-8.3% and 3.9-6.8% respectively. The amino acid sequence difference of VIIe from VIIa、VIIb、VIIc及VIId is 5.9-15.2%, 8.5-12.3%, 3.3-12.3% and 5.0-9.5. The estimated evolution rate of the subgenotype VIIe and VIIc of the local isolated NDVs was 9.98X104及3.21X104 bp/year. The 29 local isolates were further sub-subgenotypely classified as VIIe1, VIIe2, VIIe3 and VIIe4 linages based on their nucleotide sequence. The amino acid residue difference at aa 1-125 of the VIIe2 linage provided 2 amino acid changes (L23 F and T90  A). The VIIe3 provided 3 amino acid changes (E74  G、A75 G and Q114  K). The VIIe4 also provided 3 amino acid changes (I26  T， T29  A and A75  G). Since the 2 aa residues (E74  G and A75  G) is just located at the neutralization antibody initiating site, the above aa changes in the recently local NDV isolates might be the reason in causing the change of the viral pathogeicity and antigenicity. The HN protein of NDV possessed 572 aa residues. The 6 aa residue changes (R197  K、S200 L、H203 Y、E347 G、R352 T及S521 N) of the HN protein in some of the local isolates might be provided some influence on its antigenicity and immunogenicity. The aa residue at 81 might provides some influence on the hemagglutination (HA) activity. A creaction of the 2 antigenic variant NDVs (TW/02-302HNM81I and TW84-1211HNI81M) for infecting COS-7 cell line revealed that the cell line covered with I81M HN protein was found with less HA activity than the cell line covered with M81I HN protein. A group of 21-day-old leghorn layers was vaccinated with ND oil emulsion inactivated vaccine prepared from Sato (genotype III) and/or TW/02-301 (genotype VIIe) strain, then challenged at 4 weeks post vaccination. A negative vaccinated group was also included. The negative vaccinated group was found with 100% mortality at one week post challenge. The TW/02-301 strain vaccine immunized group was all survive. Whereas the Sato strain vaccine immunized group was found with 42.9% mortality. The F, HN and P/V gene of the recently Taiwanese VVNDV isolates were found with some degree of genetic variation. End up with some change on its antigenicity and immunogenicity. A good control of the ND outbreaks in Taiwan has to be conducted by using the vaccine prepared from the recently isolated VVNDV.

並列關鍵字

Fusion protein ； HN protein ； phylogenetic analysis

參考文獻

Alexander DJ, Campbell G, Manvell RJ, Collins MS, Parsons G, McNulty MS. Characterisation of an antigenically unusual virus responsible for two outbreaks of Newcastle disease in the Republic of Ireland in 1990. Vet Rec 130: 65-68, 1992.

Alexander DJ, Manvell RJ, Lowings JP, Frost KM, Collins MS, Russell PH, Smith JE. Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathol 26: 399-418, 1997.

Alexander DJ, Reeve P, Poste G. Studies on the cytopathic effects of Newcastle disease virus: RNA synthesis in infected cells. J Gen Virol 18: 369-373, 1973.

Ballagi-Pordany A, Wehmann E, Herczeg J, Belak S, Lomniczi B. Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene. Arch Virol 141: 243-261, 1996.

Boursnell ME, Green PF, Campbell JI, Deuter A, Peters RW, Tomley FM, Samson AC, Chambers P, Emmerson PT, Binns MM. Insertion of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant. J Gen Virol 71: 621-628, 1990a.

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台灣新近分離新城病毒之研究

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