一株蛋鸡新城疫病毒的分离鉴定及全基因组序列分析

为研究云南新城疫病毒（NDV）分子流行特性和致病性,对云南省1个蛋鸡场的组织样品进行病原分离,通过血凝、血凝抑制试验和RT-PCR检测分离毒株,证明为NDV。通过病毒最小致死量（MLD）、鸡胚半数感染量（EID₅₀）、鸡胚平均死亡时间（MDT）、脑内接种致病指数（ICPI）和静脉接种致病指数（IVPI）测定病毒毒力,结果EID₅₀为10^-4.4,MLD为10^-4,MDT为97.5 h,ICPI为0,IVPI为0,显示分离株为弱毒株。高通量测序及遗传进化分析发现,分离毒株基因组全长为15 198 bp,从3'端到5'端有6个结构基因依次为NP-P-M-F-HN-L,各基因之间有1~48个核苷酸间隔。全基因组、F和HN基因遗传进化分析证明分离毒株属于Class Ⅰ分支,F基因ORF全长为1 662 bp,编码553个氨基酸,F蛋白裂解位点氨基酸序列为112-ERQERL-117,符合NDV弱毒裂解位点特征,HN基因ORF全长为1 851 bp,编码616个氨基酸,在HN基因保守区域234-NRKSCS-239位氨基酸序列没有发生变异。F蛋白有6个糖基化位点比较保守,HN蛋白有6个潜在的糖基化位点,但与LaSota相比存在缺失的和多出的糖基化位点。F蛋白半胱氨酸残基分布在76、199、338、347、362、370、394、399、401、424、514、523位氨基酸,F蛋白中性抗原位点在72、75、79和170位氨基酸发生了变异,HN蛋白受体结合部位和NA活性位点氨基酸为174R、401E、416R、526Y,未发生变异。本研究首次报道了云南省蛋鸡新城疫ClassⅠ毒株的全基因序列特征,为新城疫病毒ClassⅠ毒株研究奠定了基础。     

一株鸭源新城疫病毒毒力基因分析及其对鸭的致病性研究

为了解中国鸭源新城疫病毒(Newcastle disease virus,NDV)的毒力特点,从2009年广东地区发病鸭群中分离和鉴定出1株新城疫病毒(简称NDV-104),对其生物学特性、致病性和融合蛋白(fusion,F)、血凝素神经氨酸酶(hemagglutinin-neuraminidase,HN)基因进行了研究。结果显示,分离株的MDT、ICPI和IVPI分别为56.4 h、1.95和1.64,结合F蛋白裂解位点(112~117位)的氨基酸序列分析,确定了分离株为新城疫强毒。致病性结果表明,分离病毒对雏鸭具有感染性和致病性。F基因遗传进化结果显示,分离株属于基因Ⅶd亚型。F、HN蛋白的氨基酸同源性结果表明,分离株与2000年以来国内外分离到的基因Ⅶ型NDV同源性较高,分别为96.6%~99.3%和97.0%~99.7%,而其与常用疫苗株B1、V4、Clone30、Mukteswar和LaSota的F、HN蛋白氨基酸序列的同源性较低,且与LaSota株和V4株的同源性最低,分别仅为88.1%和87.6%,说明分离株与经典新城疫病毒毒株存在一定的差异。     

NDV长春株生物学特性及与国外毒株HN蛋白基因的同源性比较

以新城疫病毒（NDV）长春株接种9日龄SPF鸡胚增殖后,进行了蚀斑纯化（三代）鉴定,差数、蔗糖密度梯度离心提纯和生物学特性鉴定。结果表明,该病毒的鸡胚平均致死时间（MDT）大于168h,雏鸡脑内致死性（ICPI）为0.25,雏鸡静脉致死性（IVPI）为0,血凝解脱时间超过120h,血凝素热稳定性56℃ 5min 仍具有血凝活性。系统发育进化树分析表明,NDV长春株与La Sota株亲缘关系最近,为弱毒株。参考多株NDV HN基因序列设计了1对特异性引物,应用RT-PCR一次性扩增出NDV长春株的全长HN基因。将该HN基因插入pKS（-）后,进行了序列测定。序列分析表明,该HN基因苷酸长度为1731bp,编码577个氨基酸,序列中有5个糖基化位点,12个半胱氨酸残基,与国外发表的NDV毒株序列相符,核苷酸同源性为88.1%～98.8%,推导的氨基酸同源性为91.1%～98.8%。     

Contribution of mutation I142M in fusion protein and Q44R in matrix protein of Newcastle disease virus to virulence in ducks

Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported. We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021). The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.     

10株新城疫病毒分离F基因的克隆及遗传变异分析

对10株具有一定代表性的NDV分离株的F基因进行RT-PCR扩增和序列测定,核苷酸序列及其推导的氨基酸序列比较结果表明:F基因核苷酸序列的同源性为93.6%,推导氨基酸序列同源性为95.39%;根据F基因裂解位点的氨基酸序列推测,其中2株属于弱毒株,8株属于强毒株,该结果与致病性试验测定的结果完全相符;不同年代、不同宿主分离株的F基因序列一致,高度保守.通过BLAST SEARCH比较,8株强毒株与广东鹅分离株GDGO(Y97)高度同源,处于进化树的同一分支.2株弱毒分离株与La Sota疫苗株仅有1～4个氨基酸改变,推测可能是免疫或散播La Sota疫苗株.抗原性指数分析表明HI分离株有三处明显变异,抗原位点推测分析表明H分离株比F48株和La Sota多出6个抗原位点,而Liu株抗原位点在446位后缺失.     

新城疫病毒F48E9株HN基因核苷酸序列

本研究采用 Ｓａｎｇｅｒ＇ｓ 双脱氧末端终止法测定了新城疫病毒国内标准强毒株 Ｆ４８ Ｅ９ 血凝素神经氨酸酶基因 ｃ Ｄ Ｎ Ａ 的核苷酸序列,推导出其氨基酸序列。 Ｆ４８ Ｅ９ Ｈ Ｎ 基因编码区全长为 １７１３ｂｐ,单一的开放阅读框编码５７１ 个氨基酸的糖蛋白。含有６ 个潜在的与天门冬酰胺（ Ｎ）连接的复合寡聚糖和高甘露糖寡聚糖结合位点,其中有 ５ 个簇集在蛋白分子的 Ｃ末端一半内,有１３ 个半胱氨酸残基。发现６ 个 Ｆ４８ Ｅ９ 特有的氨基酸残基,３７ 位 Ｆ（ Ｌ）、３８ 位 Ｓ（ Ａ）、６０ 位 Ｌ（ Ｐ）、１０５ 位 Ｓ（ Ｎ）、４４３ 位 Ａ（ Ｔ）、５７１ 位 Ｉ（ Ｖ）,这些位点除了 ６０ 位的 Ｐ有两株为 Ｓ以外,其它的在 １３ 株已知序列中均为高度保守序列。     

Isolation,Identification of Four Newcastle Disease Virus Strains and Study on Their Biological Characteristics and Genetic Characteristics

In order to study the relationship and evolution between the latest Newcastle disease virus (NDV) epidemic strain and the vaccine strain (Mukteswar,La Sota and Clone 30 strains),four NDV epidemic strains were initially isolated and identified from immunized chicken groups in Guangdong,Guangxi and Hainan provinces. Fusion (F) gene and hemaglutinin neuraminidase (HN) gene of four isolated stains were amplified,cloned and sequenced by RT-PCR method,and the homology analysis of amino acid sequences deduced by F and HN genes of NDV and other strains published in GenBank were studied, the phylogenetic tree were build,and the pathogenicity index of each strain (EID₅₀,MDT, ICPI and IVPI) were determined. The results showed that the EID₅₀,MDT,ICPI and IVPI of the HN-08 strain were 10^-8.37/0.2 mL,58.5 h,1.78 and 2.45,that of the XX-08 strain were 10^-6.50/0.2 mL,75.0 h,1.61 and 2.41,that of the YS-09 strain were 10^-7.75/0.2 mL,64.5 h,1.71 and 2.38,and of the LF-09 strain were 10^-7.60/0.2 mL,63.8 h,1.84 and 2.38.In this experiment,the similarity of amino acid sequences of F and HN genes was over 95.8% among four strains of NDV isolated from chicken,the similarity of four NDV strains with chicken epidemic strains (GX11/03 and GM strains),vaccine strains (Mukteswar,La Sota and Clone 30 strains) and standard virulent strain (F₄₈E₉ strain) were from 96.8% to 98.6%,86.7% to 90.6% and 88.4% to 91.3%,respectively. The results showed that four NDV epidemic strains were virulent strains,which were closely related to the GX11/03 and GM strains that were popular in China in recent years,but relatively far from the traditional vaccine strains.     

4株NDV流行株的分离鉴定、生物学特性及遗传学特性的研究

为研究近年国内的新城疫病毒（Newcastle disease virus,NDV）流行株与传统疫苗株（Mukteswar、La Sota及Clone 30）的亲缘关系和遗传演化情况,试验从广东、广西和海南地区患病的免疫鸡群病料中分离鉴定得到4株NDV流行株。通过RT-PCR技术对各毒株的F和HN基因进行扩增、克隆与序列分析,然后与GenBank上发表的NDV序列进行同源性比对、遗传进化树分析,并测定各毒株致病指数。结果显示,本试验中HN-08株的鸡胚半数感染量（EID₅₀）、平均死亡时间（MDT）、脑内致病指数（ICPI）和静脉致病指数（IVPI）分别为10^-8.37/0.2 mL、58.5 h、1.78和2.45,XX-08株分别为10^-6.50/0.2 mL、75.0 h、1.61和2.41,YS-09株分别为10^-7.75/0.2 mL、64.5 h、1.71和2.38,LF-09株分别为10^-7.60/0.2 mL、63.8 h、1.84和2.38。4株鸡源NDV流行株彼此间F和HN基因推导的氨基酸序列同源性在95.8%以上,与鸡源流行株GX11/03、GM株的同源性为96.8%~98.6%,而疫苗株Mukteswar、La Sota及Clone 30株的同源性为86.7%~90.6%,与标准强毒株F₄₈E₉的同源性为88.4%~91.3%。表明4株NDV流行株均为强毒株,与近年来国内流行的GX11/03、GM株有较近的亲缘关系,而与传统疫苗株的关系相对较远。     

鸡新城疫病毒河北分离株F基因的分子特性分析

通过毒力测定、RT-PCR及F基因的序列测定与遗传进化分析,对2005-2008年从河北省部分地区的发病鸡群中分离到的10株新城疫病毒（NDV）进行了研究。各分离株经典毒力测定结果显示：MDT在37.6～54.4h之间,ICPI在1.71～2.0之间,IVPI值在2.16～2.8之间,均为新城疫病毒强毒株特征。F基因的序列测定表明,分离株之间的核苷酸序列具有77.4%～98.0%的同源性,与疫苗株Lasota的同源性为87.0%～98.9%,与国内标准强毒株F48E9同源性为89.7%～98.9%。推导其氨基酸序列分析表明,8个分离株的F蛋白的裂解位点氨基酸组成为112 R-R-Q-K-R-F117,具有NDV强毒株特征,与毒力测定结果相符,2个分离株的F蛋白的裂解位点氨基酸组成为112 G-R-Q-G-R-L117,与弱毒株特征相符。F基因分型和同源性比较显示：目前河北新城疫的流行以基因Ⅶ型为主（占70%）,同时兼有基因Ⅱ型（占20%）和基因Ⅸ型（占10%）。     

新城疫病毒山东分离株ShD-5-06 F和HN基因序列分析

从山东济南某非典型新城疫发病鸡群中分离到一株新城疫病毒株（ShD-5—06）,研究其生物学特性表明,该病毒具有新城疫强毒株的一些特征。从该分离株扩增出其F和HN基因,并与标准株进行同源性比较,为探讨NDV是否发生变异提供理论依据。本试验通过RT—PCR法特异性地扩增出F和HN基因全基因序列,并对其与已经发表的序列进行核苷酸序列测定和分析。结果表明,ShD-5—06株的F和HN基因开放性阅读框架（ORF）为1662bp和1716bp,分别编码489个和571个氨基酸。与国外发表的部分新城疫病毒强毒株和弱毒株之间相应序列进行比较,F基因核苷酸序列的同源性在84．1％～88．7％之间,氨基酸同源性在88．1％～93．3％之间;HN基因核苷酸序列的同源性在82．19,5～87．4％之间,氨基酸同源性在88．6％～90．9%之间;F蛋白裂解位点区（112～117）氨基酸组成与强毒株一致,说明NDV山东分离株（ShD-5—06）为新城疫强毒株。     

新城疫病毒糖蛋白致病作用的研究

本实验以分子生物学和现代免疫学方法,对新城疫病毒（ＮＤＶ）中国毒株Ｆ４８Ｅ９和ＬａＳｏｔａ等毒株的糖蛋白的致病作用进行了探讨。结果表明,ＮＤＶ Ｆ和ＨＮ蛋白为糖蛋白,强弱毒株之间Ｆ蛋白裂解位点的氨基酸序列有明显的不同,并决定了毒株的毒力,但ＮＤＶ的致病作用尚需ＨＮ、Ｆ蛋白的协同作用,单一的Ｆ或ＨＮ蛋白不能构成ＮＤＶ的致病作用。     

新城疫病毒西藏分离株HN基因克隆与序列分析

为确定西藏新城疫病毒(Newcastle disease virus,NDV)分离株HN基因结构特征及其与已知毒株的遗传相关性,本试验应用RT-PCR技术对西藏NDV分离株HN基因进行扩增,然后克隆至pMD18-T载体测序,并与国内外代表性毒株和当前疫苗株进行比对及系统发育分析。结果表明,NDV分离株HN基因片段长度为1734 bp, 编码577个氨基酸;推导的氨基酸序列均有5个糖基化位点, XZ10和XZ17有12个半胱氨酸残基,XZ20只有11个半胱氨酸残基。NDV西藏分离株HN基因与国内代表性毒株核苷酸同源性在81.1%~93.0%之间,氨基酸同源性在81.1%~93.6%之间;与疫苗株核苷酸同源性在87.8%~98.7%之间,氨基酸同源性在88.0%~98.9%之间。系统进化分析结果表明,NDV西藏分离株HN基因均属于Ⅱ型。     

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens,and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site ¹¹²RRRKGF¹¹⁷ and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.     

1株鸭源Ⅰ类新城疫病毒分离株F和HN基因的分子特性分析

采用RT-PCR技术对Ⅰ类新城疫病毒(NDV)09-014分离株完整的融合蛋白(F)基因和血凝素-神经氨酸酶(HN)基因进行了扩增和遗传进化分析。F基因的序列测定结果表明:该分离株F基因全长为1 792 bp,可编码553个氨基酸,裂解位点的氨基酸组成为112E-R-Q-E-R-L117,具有典型的新城疫弱毒株特征。同源性分析表明本分离株的F基因与Ⅰ类新城疫病毒代表毒株之间核苷酸的同源性为93%～95.2%,而与Ⅱ类新城疫病毒代表毒株的同源性较低,介于70.6%～72.4%。HN基因的序列测定结果表明:HN基因全长2 001 bp,可编码616个氨基酸,同源性分析表明本分离株的HN基因与Ⅰ类新城疫病毒代表毒株之间核苷酸的同源性在92.7%～94.7%之间,而与Ⅱ类新城疫病毒同源性较低,为70.7%～71.5%。根据完整的F基因和HN基因构建的遗传进化树均表明:本分离株在分类地位上属于Ⅰ类新城疫病毒基因3型,因此Ⅰ类新城疫病毒的F基因和HN基因具有相似的进化速率。     

Deduced Amino Acid Sequences Surrounding the Fusion Glycoprotein Cleavage Site and of the Carboxyl-terminus of Haemagglutinin–Neuraminidase Protein of the Avirulent Thermostable Vaccine Strain I-2 of <Emphasis Type="Italic">Newcastle disease virus</Emphasis>

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F₀ cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain I-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain I-2 had a sequence motif of ¹¹² RKQGRLIG¹¹⁹, consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain I-2 had a 7-amino-acid extension (VEILKDGVREARSSR. This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain I-2 confirmed its avirulent nature and its Australian origin.     

新城疫病毒囊膜糖蛋白生物学特性的研究进展

新城疫是当今全球范围内最严重的禽类传染病之一。其病原新城疫病毒是单股负链RNA病毒,编码NP、P、M、F、HN和L 6种结构蛋白,其中最主要的是位于囊膜上的F和HN两种糖蛋白。F蛋白具有使病毒囊膜与宿主细胞膜融合,致使病毒穿入宿主细胞膜的作用,是决定病毒毒力的关键因子;HN蛋白具有血凝素和神经氨酸酶两种活性,这两种活性对于NDV侵染细胞具有重要的作用。这两种糖蛋白不仅对NDV的毒力及致病性方面起着决定性作用,而且也是诱导产生保护性抗体的蛋白。作者主要对这两种蛋白的结构与功能作一概述。     

10株新城疫病毒广西分离株HN蛋白基因的克隆与序列分析

根据基因库(GenBank)新城疫病毒(NDV)的HN基因序列设计了2对特异性引物,应用RT-PCR技术对广西在2000~2003年暴发新城疫的鸡群中分离的10株NDV毒株的HN基因进行了扩增,扩增产物克隆并测序,拼接出10个NDV广西分离株的HN基因全序列,其序列全长均为1 713 bp,编码571个氨基酸,均有13个半胱氨酸残基。其中GX8/03有6个糖基化位点,而GX2/00、GX6/02、GX7/02和GX5/00有5个糖基化位点,GX1/00、GX3/00、GX4/00和GX9/03有4个糖基化位点。除GX5/00和GX10/03分离株外,其他8个NDV分离株在HN基因抗原位点Ⅰ发生变异,即347位由谷氨酸(E)被甘氨酸(G)替代,GX8/03分离株在HN基因抗原位点Ⅱ的495位由赖氨酸(K)替代谷氨酸(E)。与11株已发表的NDV HN基因全序列相比较,其核苷酸同源性在79.6%~97.9%之间,推导的氨基酸同源性在87.2%~98.1%之间。     

新城疫病毒弱毒骨架表达强毒F基因的重组病毒生物学特性研究

为了解目前中国新城疫病毒（Newcastle disease virus,NDV）优势基因VIId型的毒力机制,应用反向遗传技术将我国优势流行基因VIId型强毒株I4的F基因替换弱毒LX的F基因,获得表达NDV强毒株I4F基因的重组病毒NDV/LX-If。测定重组病毒的致病指数和组织分布,结果发现,重组病毒NDV/LX-If毒力比骨架病毒有了显著的提高。NDV/LX-If的鸡胚平均致死时间（mean death time,MDT）为56 h,雏鸡脑内接种致病指数（intracebral pathogenicity index,ICPI）为1.49,属于中等毒力,毒力比亲本病毒毒力低,但都能使自然途径感染的鸡100%死亡,同时获得了亲本毒株的组织嗜性。可见,新城疫病毒F基因是毒力和组织嗜性的主要决定因素,但是其它基因对新城疫病毒的毒力也可以产生影响。     

2005-2006年流行的致病性鹅新城疫病毒的分子流行病学特征

为了对能导致鹅临诊疾病的新城疫病毒(Newcastle disease virus,NDV)进行监测,2005-2006年从江苏、安徽2省不同地区的发病鹅群分离、鉴定了10株NDV,并对其中8株病毒的部分生物学特性及分子生物学特征进行了研究.鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内接种致病指数(ICPI)测定结果表明8株病毒皆为NDV强毒株.对融合蛋白(F)和血凝素-神经氨酸酶(HN)基因序列的分析发现,8株病毒中有7株病毒在基因型分类上属于基因Ⅶd亚型,并与1997-2001年间流行的对鹅具有高度致病性的NDV高度同源(95.5%～99.8%),而且这7株病毒的F、HN蛋白具有出现于2001年以前流行的致病性鹅NDV的绝大部分特征性氨基酸,进一步说明这些特征性氨基酸非常保守,可以作为对鹅具有高度致病性的NDV的分子标志.     

Characterization of pigeon-origin Newcastle disease virus isolated in China

Fourteen pigeon-origin Newcastle disease virus (NDV) isolates were obtained from sick pigeons in China between 1996 and 2005. The mean death time (MDT) of embryonated eggs and the intracerebral pathogenicity indices (ICPI) were tested to determine the virulence of the field isolates. The result indicated that most isolates were proved to be mesogenic (MDT 60-90 hr and ICPI > 1.2). The main function regions of F protein gene of the isolates were amplified and sequenced for phylogenetic and residue substitutive analysis. The fusion protein cleavage site sequences of most isolates had multiple basic amino acids R/KRQKRF at positions 112-116 and a phenyl alanine at position 117, characteristic of velogenic isolates. In the phylogenetic tree, the majority of the isolates were clustered into a single genetic lineage, termed genotype VIb, and were typical pigeon paramyxovirus type 1, whereas a small number of recent isolates (three strains) were grouped into genotype VIId, a predominant genotype responsible for most Newcastle disease outbreaks in chickens and geese since the end of last century. One isolate, PK9901, was proved to be a lentogenic strain, of genotype II NDV, to which the vaccine strain La Sota belongs.