鸭新城疫病毒山东分离株的生物学特性与F基因遗传进化分析

从山东规模化鸭场采集棉拭子样品并接种SPF鸡胚分离鉴定NDV,通过测定鸡胚平均致死时间(MDT)和1日龄雏鸡脑内接种致死指数(ICPI)来确定分离株的毒力,采用RT-PCR方法对分离株F基因进行测序并进行遗传进化分析。结果显示,从正常鸭群棉拭子中分离到4株NDV,F蛋白裂解位点的氨基酸序列表明4个分离株中2株氨基酸序列为112 ERQERL117,1株为112 GKQGRL117,符合弱毒株序列特征;1个分离株为112 RRQKRL117,符合强毒株序列特征。系统进化分析表明,其中2株属于ClassⅠ分支、2株属于ClassⅡ分支,ClassⅡ分支中的2个分离株1个为基因Ⅶ型强毒株,1个属于基因Ⅰ型弱毒株,与Queensland V4等毒株的同源性较高。     

6株鸽源新城疫病毒广西分离株F基因的遗传演化分析

应用RT-PCR方法对分离自广西不同鸽场的6株鸽源新城疫病毒F基因进行扩增、序列测定和分析。结果显示:只有1株分离株F基因的ORF全长为1 659 bp,编码552个氨基酸,其他5株分离株F基因的ORF全长均为1 662 bp,编码553个氨基酸,6株分离株的F0蛋白裂解位点的序列均为112RRQKRF117,符合强毒株的序列结构特性;核苷酸同源性比较显示,6株分离株与NDV基因Ⅵ型中的Ⅵb亚型同源性最高,为91.3%~99.3%(与比利时分离株11(JX901124)的同源性最高),与其他基因Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅶ、Ⅷ、Ⅸ型毒株的同源性在81.3%~89.2%。与经典强毒株F48E9相比,核苷酸序列同源性为83.6%~84.8%,氨基酸序列同源性为89.2%~92.4%;与Ⅰ型疫苗株V4、I-2相比,核苷酸同源性分别为83.6%~85.1%和82.6%~84.2%,氨基酸序列同源性分别为88.1%~91.3%和87.7%~90.8%;与Ⅱ型疫苗株La Sota和B1相比,核苷酸同源性分别为81.7%~83.9%和82.0%~83.9%,氨基酸序列同源性分别为86.6%~89.9%和86.6%~89.9%。遗传进化分析显示,6株广西鸽源分离株与2011年比利时分离株11(JX901124)和中国毒株SDS、LLN713、BJP13、LJL404、P4的遗传关系最为接近,位于同一进化树分支上,属于基因Ⅵb亚型。     

山东新城疫病毒分离株F基因的遗传进化分析

应用特异性引物从山东某发病鸡场病鸡体内分离到的命名为JN09的新城疫病毒(Newcastle Disease Virus,NDV)中扩增其F基因,预计扩增片段大小为1 700 bp. RT-PCR扩增出的目的片段大小与预计扩增片段大小相符合,测序结果表明扩增片段与新城疫病毒F基因序列相符.对其F基因进行遗传进化分析,并结合山东地区近几年来的分离毒株的相关信息,着重分析该地区近年来新城疫病毒 F基因的变异情况.     

山东新城疫病毒分离株F基因的遗传进化分析

应用特异性引物从山东某发病鸡场病鸡体内分离到的命名为JN09的新城疫病毒（Newcastle Disease Virus,NDV）中扩增其F基因,预计扩增片段大小为1 700 bp。RT-PCR扩增出的目的片段大小与预计扩增片段大小相符合,测序结果表明扩增片段与新城疫病毒F基因序列相符。对其F基因进行遗传进化分析,并结合山东地区近几年来的分离毒株的相关信息,着重分析该地区近年来新城疫病毒F基因的变异情况。     

新城疫病毒基因组与基因演化

新城疫在世界范围内出现了4次大流行,造成了极大的经济损失和贸易影响。虽然新城疫病毒仅有1个血清型,但不同毒株生物学特性和毒力相差很大。近来,病毒宿主谱扩大,开始感染水禽,造成鹅的大规模发病。经典的新城疫病毒基因组长度为15 186个核苷酸(nt),如La Sota和V4毒株等,而我室报道的鹅源毒株ZJ1全长为15 192 nt。我们对基因组进行比对分析,发现新城疫病毒是在基因组水平上整体演化的。     

一株新城疫病毒广东分离株的遗传关系分析

根据新城疫病毒已报道的基因序列设计了一对引物,扩增F基因3 ′端374 bp片段,包括F基因信号肽序列和裂解位点.经过RT-PCR、PCR产物测序和序列分析,表明该毒株为基因Ⅶ型强毒株,和中国动物卫生与流行病学中心2009年分离到的毒株NDV09-038相似性最高,可达98％.基因进化树分析表明该毒株与常用疫苗株亲源关系较远,与I系疫苗株Mukteswar相似性为81.4％,与Herts' 33的相似性为84.5％.氨基酸序列分析表明该毒株为典型的强毒株.     

海安地区鸡新城疫病毒分离株的遗传进化分析

2011-2012年,从来海安县兽医站门诊就诊的20个疑似新城疫发病鸡群中分离到8株有血凝性的病毒,其中5株病毒能够凝集鸡的红细胞,并且血凝性可以被NDV阳性血清所抑制,MDT在49-60.8小时之间,经鉴定为NDV强毒株。新城疫是危害养禽业的重要疫病之一[1],由新城疫病毒（NDV）强毒株引起,NDV是有囊膜的单股、负链、不分节段RNA病毒,融合（F）蛋白是位于病毒囊膜表面的一种糖蛋白,在病毒感染过程中介导病毒囊膜与宿主细胞膜的融合,F基因前375个核苷酸序列变异度较大[2],可以反映NDV毒株的遗传进化规律。     

一株鸵鸟胚源新城疫病毒生物学特征和遗传发生分析

从死亡的鸵鸟胚胎中分离到1株新城疫病毒毒株,经毒力检测为强毒株,鸡胚平均死亡时间为45h,IVPI为2.90,IPCI为1.88.RT-PCR扩增出融合蛋白(F)基因,测定核苷酸序列并进行遗传发生分析和酶切位点分析.对基因编码的氨基酸序列进行了推导,该毒株的蛋白酶裂解激活位点附近的氨基酸序列为112R(K)RQKR↓F117,为强毒特征序列,然而该毒株并未引起该鸵鸟群发病.根据绘制的系统进化发生树和F基因上3种限制性内切酶(RE)位点分布,确定该毒株均为基因Ⅶ型.该病毒与在我国鸡群、鹅群同期流行的毒株有很高的同源性.     

一株新城疫病毒鹧鸪分离株分子生物学特性的研究

本研究从一例鹧鸪暴发新城疫的病例中成功分离出一株新城疫病毒(NDV)。运用RT-PCR扩增了该分离株F基因的重要功能片段(约535kb),并进行了序列测定。序列分析表明其在F蛋白裂解位点的氨基酸顺序为112R-R-Q-K-R-F117,与NDV的强毒株特征相符。通过构建NDV的F基因遗传进化树,发现该分离株在分类地位上属于基因Ⅶ型NDV,与同期我国普遍流行的基因Ⅶ型NDV在遗传进化上极为相似,且具有较高的同源性,表明它们可能具有共同的来源。     

鸭源新城疫病毒强毒株对鸭的致病性

对鸭源新城疫病毒（NDV）强毒株JSD0812对鸭的致病性进行了研究。结果显示,对于15日龄雏鸭,JSD0812株病毒尿囊液的半数致死剂量（LD50）为101mL。15,30,45,60。110日龄鸭肌肉注射感染试验结果表明,所有日龄鸭感染JSD0812株后均可发病,但死亡率随日龄的增长而下降,110日龄鸭感染后未见死亡。鸭感染后可经咽喉和/或泄殖腔短期排毒,感染鸭组织器官中病毒分离率较低。     

异源新城疫病毒感染对雏鸭组织抗氧化酶活性的影响

选择隔离饲养至20日龄的健康雏鸭120只,随机分为4组,Ⅰ组为对照组,皮下注射0.5mL/只灭菌生理盐水,Ⅱ、Ⅲ、Ⅳ组分别皮下注射0.5mL/只1∶5稀释的鸡源、鹅源和鸭源新城疫病毒SPF胚液,并分别置于25℃隔离环境下饲养与观察。分别于感染后4、8、12、16d每组随机抽取雏鸭7只颈静脉放血致死,采集心、肝、脾、肺及肾组织,制成10%的组织匀浆液,测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)及谷胱甘肽过氧化物酶(GSH-Px)的活性或含量,探讨异源新城疫病毒感染对雏鸭组织自由基代谢的影响。结果表明,感染异源新城疫病毒的雏鸭组织SOD、CAT、GSH-Px活性和MDA含量变化不同,感染鸭源和鹅源新城疫病毒的雏鸭组织抗氧化酶的活性变化明显,而感染鸡源新城疫病毒的则变化不明显。这说明不同来源的新城疫病毒对雏鸭的感染性有一定差异。     

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as ¹¹²RRQKRF¹¹⁷ at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif ¹¹²RRQKRF¹¹⁷ in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs (¹¹²KRQKRF¹¹⁷, ¹¹²RRQRRF¹¹⁷ and ¹¹²RRRKRF¹¹⁷) but not an avirulence motif (¹¹²GRQGRL¹¹⁷) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.     

Phylogenetic analysis of Newcastle disease viruses isolated from wild birds in the Poyang Lake region of China

Newcastle disease virus (NDV) causes a highly contagious viral disease in poultry and wild birds, and it can cause significant economic loss worldwide. Eight viral strains were isolated by inoculating embryonated chicken eggs from the Poyang Lake region of China with swab samples. All eight of the NDV isolates were identified as class I genotype 3 strains, but they diverged notablely from class II viruses. Further analysis revealed that all eight NDV isolates were lentogenic strains containing the ¹¹²ERQER↓L¹¹⁷ motif at the F protein cleavage site. The strains were highly identical and were more species specific (chicken and waterfowl) than site specific (Nanchang and Duchang regions). The close phylogenetic proximity of these isolates indicates that viral transmission may happen between poultry and wild birds. Our study demonstrates that lentogenic class I NDVs exist in clinically healthy wild waterfowl and poultry within the Poyang Lake region. Active surveillance of these viruses to determine their evolution and origin is one of the most realistic strategies for preventing and controlling NDV outbreaks.     

3株新城疫山东分离株HN基因全序列测定和分析

用设计的特异性引物,通过RT-PCR法对1999和2004年间分离自山东省的具有一定代表性的3株鸡新城疫病毒(ShD-2-04,ShD-3-99,ShD-5-04)进行HN基因的扩增和克隆,并对其进行核苷酸序列测定和分析。结果表明:3个毒株HN基因开放性阅读框架(ORF)均为1716bp,编码571个氨基酸,属强毒的C群;3株山东分离毒的核苷酸同源性为98.2%~98.8%,氨基酸同源性为98.4%~98.8%,其中ShD-2-04和ShD-5-04核苷酸及氨基酸同源性均为最高,达到99.8%和99.8%;将3株山东分离毒株与国内外标准强毒株及弱毒株的HN基因核苷酸及氨基酸同源性分别进行比较:3毒株与国内外标准毒株HN的核苷酸同源性为82.7%~87.2%,氨基酸同源性为87.6%~90.0%,表明已发生一定的变异。     

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.     

一株野鸭源新城疫病毒F基因的克隆及序列分析

根据GenBank中新城疫病毒(Newcastle disease virus,NDV)的F基因序列设计了1对特异性引物,成功地对其中一株野鸭源NDV的F基因进行了克隆、测序,并推导其氨基酸序列,选择F基因部分片段绘制了遗传进化树。F基因全长1 662 bp,单一的阅读框架编码553个氨基酸,构成的F蛋白上有13个Cys残基位点和6个潜在的糖基化位点,其裂解位点的氨基酸序列为112R-R-Q-K-116R-117F。遗传进化分析表明,该NDV分离株为基因Ⅶ型NDV,与近年来我国家禽中所报道的NDV的基因型相一致。     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.     

人工感染种鸭生殖系统中新城疫病毒的检测

本研究旨在探明鸭源新城疫病毒(NDV)通过人工感染后在种鸭生殖系统中分布。应用本实验室分离的一株经鸭胚传递的NDV SDFCH株,对42只无NDV感染的28周龄樱桃谷种鸭进行人工感染试验,试验分母鸭接种强毒组,公鸭接种强毒组和对照组。强毒接种4 d后,每隔2 d每组各迫杀2只,采集母鸭卵巢、卵泡、输卵管组织,公鸭采集睾丸、输精管进行病毒分离鉴定,并利用RT-PCR方法进行病毒F基因的扩增与同源性分析。同时,以NDV F蛋白的单克隆抗体(MAb)为一抗,建立间接免疫荧光检测方法检测输卵管组织中NDV及病毒在输卵管的分布情况。结果表明采用RT-PCR方法能够在种鸭生殖系统各个组织中检测并重新分离到NDV;输卵管子宫部和蛋白分泌部纤毛柱状上皮的纤毛细胞和分泌细胞内有阳性荧光信号存在。结果表明人工感染的种鸭卵巢、卵泡、睾丸、输精管以及输卵管子宫部和蛋白分泌部纤毛柱状上皮的纤毛细胞和分泌细胞中均有NDV分布,病毒能否随蛋白分泌、精液的排泄进入种蛋传递给雏鸭还有待于进一步研究。     

1株鸽新城疫病毒F和HN蛋白的潜在抗原表位分析

从亲水性、抗原性、可塑性、表面可能性、二级结构等5项参数指标来综合预测1株鸽新城疫病毒（Newcastledisease virus,NDV）JN08株F和HN蛋白的潜在B细胞抗原表位,并比较其与疫苗株La Sota抗原表位的差异,同时对F和HN基因序列进行分析。综合预测显示：JN08株F和HN蛋白预测抗原表位分别有21,26处;La Sota F和HN蛋白预测抗原表位分别有19,25处。就F蛋白而言,与La Sota相比JN08毒株在82～87、99～102、411～412aa多出3处抗原表位,而在450～455aa缺失1处抗原表位。其中82～87、99～102aa这2处抗原表位位于裂解位点之前,是否对病毒的抗原性、蛋白水解酶的敏感性、毒力等造成影响还有待于进一步研究。就HN蛋白而言,JN08毒株与La Sota相比在128～132aa多出1处抗原表位,此表位位于HN蛋白的柄状部,该部分存在促进F蛋白融合活性的区域,JN08毒株在此区域多出1处抗原表位可能会对F蛋白的融合活性有一定的影响。