新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

新城疫病毒分离株HN和P基因分子进化及其相关研究

为探讨新城疫病毒(Newcastle disease virus,NDV)血凝素-神经氨酸酶(HN)和磷蛋白(P)基因遗传特性以及相互关系,将1997～2005年间国内分离到12株NDV毒株,分别进行HN和P基因克隆测序,结合15个已发表的国内外不同时期的NDV毒株HN和P基因,计算所有毒株HN和P基因的不同核苷酸和氨基酸片段进化距离,利用统计软件进行了不同片段间进化距离的方差分析,HN或P基因核苷酸进化距离与毒株分离时间、HN或P基因片段与其全长间以及HN和P基因全长间的相关分析.统计分析显示:NDV HN或P基因不同核苷酸和氨基酸序列片段变异程度不一样;不同毒株间HN或P基因片段与其全长间以及HN和P基因全长间无论是核苷酸还是氨基酸遗传变异高度相关.以上说明,NDV HN和P基因虽以不同的方式进化,但是HN和P基因遗传变异的趋势是相同的.HN和P基因的变异与分离时间有一定的联系.     

新城疫病毒HN和F基因遗传变异相关性的研究

选取国内1999~2005年发生的NDV毒株,经CEF蚀斑纯化和SPF鸡胚增殖,对其融合蛋白(F)和血凝素-神经氨酸酶(HN)基因分别进行克隆测序,结合在GenBank中发表的具有F和HN基因的NDV序列,利用DNAStar软件,对其不同毒株的F或HN基因片段和全长、F和HN基因全长分别进行遗传变异的研究,利用统计学软件SPSS8.0进行同源性相关分析。结果表明:不同NDV毒株F或HN基因片段与其全长之间,核甘酸r≥0.973,氨基酸:0.911≤r≤0.968,遗传变异高度相关,但F与HN基因全长之间核甘酸的遗传变异呈现弱相关(r=0.312)。国内NDV野毒株之间HN核甘酸高度同源(同源率97%以上),而与La Sota同源率仅为79.2%~80.7%,且显示出明显的地域性。     

新城疫病毒HN基因的遗传变异与HI相关性的研究

选取国内1999~2004年分离的新城疫野毒10株,经CEF蚀斑纯化和SPF鸡胚增殖,对其血凝素-神经氨酸酶(HN)基因分别进行克隆和测序,结合在GenBank中发表的LaSota、F48E9和Clone30等参考序列,对其氨基酸序列进行遗传变异分析,绘制系统发育树。利用SPF鸡在隔离器中分别制备上述NDV毒株的单因子阳性血清,进行血凝抑制(HI)交叉试验,计算NDV不同株之间的HI相关系数(r)。利用统计学软件SPSS8.0对NDV不同株之间的HN氨基酸同源率和HI相关系数(r)进行相关比较。结果表明:NDV野毒间氨基酸高度同源,同源性为96.5%~99.8%,而与LaSota、F48E9和Clone30同源率仅为87.4%~89.9%;所有野毒均缺乏1个潜在的糖基化位点;HN基因的氨基酸同源性与HI相关系数显著相关(P<0.01,r=0.55)。     

中国禽源新城疫病毒(NDV)流行株F和HN基因的遗传演化和变异频率

【目的】新城疫(ND)是中国流行最严重的疫病之一,对家禽业可造成巨大的经济损失,疫苗防控是控制ND的重要措施。新城疫病毒(NDV)流行株的遗传演化一直是研究NDV的焦点。本文利用分子信息学手段,通过比较近20年间NDV流行株不同基因型F和HN基因的分子特征和遗传变异频率,解析免疫压力下NDV的演化规律。【方法】利用Lasergene 7.1和MEGA5.1软件,选取本实验室89株NDV分离株,结合从Gen Bank下载的364株NDV流行株以及15株NDV经典毒株的基因序列,对其进行系统发育、分子特征和替代频率分析。【结果】系统发育表明,NDV已经演化为15个基因型。一致性比较显示,NDV流行株相同基因型之间核苷酸(氨基酸)高度同源,而不同基因型之间差异较大且存在明显的氨基酸变异积累。NDV基因型的分布与时间、地域密切相关,VII d亚型为中国NDV优势流行株。为评估NDV变异的频率,以Go/GD/QY/1997株(中国较早发生的基因VII亚型)为参照,1997-2015年间NDV的F/HN基因的年平均核苷酸(氨基酸)替代率为2.31×10~(-3)(2.26×10~(-3))/3.37×10~(-3)(2.35×10~(-3))。其中,1997-2001年(未使用基因VII型疫苗)F/HN基因核苷酸年平均替代率为4.72×10~(-3)/8.28×10~(-3);2002-2015年(疫苗使用后)为1.6×10~(-3)/1.84×10~(-3),显示出基因VII型疫苗在控制NDV变异速度方面具有明显的效果。【结论】生物信息学分析证实:研制出与NDV流行毒株相匹配的新型疫苗是控制当前NDV变异的关键。     

新城疫分离毒HN蛋白的抗原性初步分析及分子特性研究

运用针对NDV囊膜糖蛋白(HN)的单克隆抗体(MAbs),对2005～2006年间自我国江苏和广西部分地区的20株NDV分离株进行排谱试验,初步分析了不同毒株之间HN蛋白的抗原表位差异;并应用RT-PCR技术成功扩增了其HN基因整个编码区,经克隆、测序最终获得13株鸡源NDV与7株鹅源NDV HN基因的编码区序列,分析测定核苷酸序列及推导的氨基酸序列,并将鹅源NDV与鸡源NDV相应序列进行了比较.结果单抗排谱试验表明,20株NDV分离株之间HN蛋白的抗原表位存在差异;测序结果表明,测定的HN基因的编码区长度皆为1716nt编码571个氨基酸;分离株中18株基因Ⅶ型NDV分离株之间HN基因编码区核苷酸序列具有较高的同源性,达94.8%～100%;与近几年国内流行的其它基因Ⅶ型NDV之间的核苷酸序列同源性为92.1%～99.6%.对其推导的HN蛋白一级结构中潜在的糖基化位点及HN蛋白细胞受体结合相关区域的氨基酸序列等进行了比较分析.结果显示,单抗排谱差异显著株在部分氨基酸位点发生了突变;同时揭示我国部分地区同期流行的鹅源NDV与鸡源NDV HN基因之间具有较近的亲缘关系.     

一株基因Ⅱ型新城疫强毒株的分子特性

从患病肉鸡群分离到一株新城疫病毒(NewcastleDiseasevirus,NDV)SQZ04。经蚀斑纯化后接种40日龄SPF鸡可诱发典型病变。经蚀斑纯化前和后的MDT为50·5h和51·2h,ICPI为2·0和1·92,IVPI为2·8和2·68,表明属强毒株。但F基因分型表明SQZ04属基因Ⅱ型,而且其与已知基因Ⅱ型的疫苗株LaSota、B1和Texas48的同源性分别为99·3%、98·7%和96·9%,显著高于与基因Ⅶ或Ⅸ型强毒株的同源性88·3%~88·6%或91·3%~92·1%。这是国内第一株属于基因Ⅱ型的NDV强毒株。SQZ04F多肽氨基酸裂解位点的序列为111GGRQGRL117,与弱毒株序列完全相同,这也是国内外首次报道具有这一氨基酸序列的强毒野毒株。然而,SQZ04株与其他已知强毒株的HN氨基酸同源性高达95·3%~97·3%,显著高于与弱毒株LaSota等的同源性87·8%~89·5%。     

新城疫分离毒HN蛋白的抗原性初步分析及分子特性研究

运用针对NDV囊膜糖蛋白(HN)的单克隆抗体(MAbs), 对2005~2006年间自我国江苏和广西部分地区的20株NDV分离株进行排谱试验, 初步分析了不同毒株之间HN蛋白的抗原表位差异; 并应用RT-PCR技术成功扩增了其HN基因整个编码区, 经克隆、测序最终获得13株鸡源NDV与7株鹅源NDV HN基因的编码区序列, 分析测定核苷酸序列及推导的氨基酸序列, 并将 鹅源NDV与鸡源NDV相应序列进行了比较。结果单抗排谱试验表明, 20株NDV分离株之间 HN蛋白的抗原表位存在差异; 测序结果表明, 测定的HN基因的编码区长度皆为1716nt编码571个氨基酸; 分离株中18株基因Ⅶ型NDV分离株之间HN基因编码区核苷酸序列具有较高的同 源性,达94.8%~100%; 与近几年国内流行的其它基因Ⅶ型NDV之间的核苷酸序列同源性 为92.1%~99.6%。对其推导的HN蛋白一级结构中潜在的糖基化位点及HN蛋白细胞受体结合相关区域的氨基酸序列等进行了比较分析。结果显示, 单抗排谱差异显著株在部分氨基酸位点发生了突变; 同时揭示我国部分地区同期流行的鹅源NDV与鸡源NDV HN基因之间具有较近的亲缘关系。     

4株鹅源新城疫病毒融合蛋白基因的克隆及序列分析

测定了4株鹅源新城疫病毒(NDV)融合蛋白(F)基因5’端1700核苷酸片段的序列,并由此推导了F蛋白氨基酸序列,并对鹅源NDV的基因型分类地位进行探讨。结果表明,4株病毒F基因的同源性大于97%,与DNV标准强毒株F48E8 F基因的同源性为860%～868%,F基因转录起始序列及起始密码子位置与已知NDV完全相同;F蛋白具有和已知NDV相似的各种功能区,F蛋白前体F0裂解位点附近的氨基酸序列为112RRQKRF117,符合NDV强毒株的特征。对F基因第334～1682位核苷酸之间3种限制性内切酶HinfⅠ、BstoⅠ\,\%Rsa\%Ⅰ酶切图谱的分析表明,4株病毒的基因型与文献报道的I～Ⅷ型有明显差异。     

新城疫病毒分离株的生物学特性鉴定及F蛋白基因序列分析

通过部分生物学特性鉴定、RT-PCR及F基因的序列测定与遗传进化分析,对2005~2006年从我国江苏省和广西省部分地区的发病鸡群和鹅群中分离到的20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45.3h~58.2h之间,ICPI在1.61~2.00之间,均为新城疫病毒强毒株特征。血凝解脱及血凝素热稳定性试验显示:各分离株的血凝解脱时间短,血凝素热稳定性较差,符合NDV强毒株的特征。F基因的序列测定表明,分离株之间的核苷酸序列具有79.7%~100%的同源性,与疫苗株LaSota的同源性为78.1%~83.4%;与国内标准强毒株F48E8同源性为80.2%~90.1%。推导其氨基酸序列分析表明,各分离株的F蛋白的裂解位点氨基酸组成为112R-R-Q-R/K-R-F117,具有NDV强毒株特征,与毒力测定结果相符。根据序列所绘制系统进化发生树,表明20株NDV分离株中有18株为基因Ⅶd型,2株为基因Ⅲ型。     

Monoclonal antibodies to hemagglutinin-neuraminidase and fusion glycoproteins of Newcastle disease virus: relationship between glycosylation and reactivity.

Eighteen hybridoma lines obtained by immunization of mice with Newcastle disease virus (NDV) lentogenic strain La Sota or velogenic strain Italien produced hemagglutinating monoclonal antibodies. The 18 monoclones were divided into four groups according to their reactivity toward native hemagglutinin neuraminidase protein (HN), nonglycosylated HN precursor, and heat-denatured HN blotted on nitrocellulose membranes. Only group II reagents were reactive toward their targets in all conditions tested. They were considered sequence-specific antibodies. Group I antibodies did not require glycosylation but lacked reactivity towards the denatured glycosylated antigen. Monoclonal antibodies from group III recognized only the native HN. Group IV was made up of a single monoclone that lacked reactivity with NDV Italien but recognized the La Sota strain in hemagglutination inhibition and enzyme-linked immunosorbent assays. Five hybridoma lines produced monoclonal antibodies which neutralized viral infectivity but failed to inhibit hemagglutination. One monoclonal antibody obtained after immunization of mice with NDV La Sota showed a low neutralization index versus NDV Italien. Four monoclonal antibodies derived from mice immunized with NDV Italien showed higher neutralization indices towards this strain. Neither the denatured F protein nor its nonglycosylated precursor was reacted against by the five monoclonal antibodies.     

Genomic characterisation of two virulent Newcastle disease viruses isolated from crested ibis (Nipponia nippon) in China

This paper describes the complete genomic sequences of two virulent Newcastle disease virus (NDV) isolates, Shaanxi06 (prevalent genotype VIId) and Shaanxi10 (novel sub-genotype VIi), from sick crested ibises. The genomes of both isolates were 15,192 nt long and consisted of six genes in the order of 3′-NP-P-M-F-HN-L-5′. The genomes of the two isolates were highly similar to other reference NDV strains. However, some unique features were found in the HN protein of Shaanxi06 and the F gene end of Shaanxi10. Shaanxi06 and Shaanxi10 shared the same virulent motif ^112 −R-R-Q-K-R-F^− 117 at the F protein cleavage site, which coincided with previous pathogenicity test results. Phylogenetic analysis revealed that both isolates were clustered within class II NDV, with Shaanxi06 in genotype VII and Shaanxi10 in genotype VI. Both isolates shared high homology with the prevalent genotype NDV strains that circulate in fowls and waterfowls. This study is the first to provide genomic information about a novel sub-genotype VIi NDV strain and another genotype VIId virus, which will be useful for subsequent investigations.     

三株新城疫病毒强毒株的生物学特性及全基因组序列分析

2009～2011年从北方发病鸡群和鸭群中分离出3株新城疫病毒（Newcastle disease virus,NDV）。通过致病性指数测定及交叉血凝抑制试验初步分析了3个毒株的毒力和相互之间的同源性。选取鸡源分离株SDLY01与新城疫疫苗株（LaSota）进行了交叉保护试验,选取鸭源毒株SD03对樱桃谷鸭进行攻毒实验,同时设计引物对3个毒株进行了全基因组测序,并与36株NDV参考株进行了分子进化分析。结果表明3个分离株F蛋白裂解位点的氨基酸序列均为112R-R-Q-K-R-F117符合强毒株的序列特征,并与致病性指数测定结果相符。交叉血凝抑制试验发现3个分离株与疫苗株LaSota 的抗原同源性较低为82.5%～89.4%,两个鸡源分离株间的抗原同源性为90%,而鸭源毒株SD03与鸡源毒株SDSG01同源性为100%。交叉保护试验和攻毒实验结果显示传统的LaSota疫苗能对SDLY01流行株提供100%免疫保护,但第5天仍检测到排毒;鸭源毒株SD03对樱桃谷鸭不致病,但能检出排毒,排毒期最长为5d。全基因组测序与分析表明3个毒株基因组长度均为15192bp,属于基因Ⅶd型毒株,与同期流行的鹅源及鸭源NDV毒株之间全基因组核苷酸序列具有高度的同源性,揭示鸭源、鹅源NDV与鸡源NDV在遗传学和流行病学上密切相关。     

LTR point mutations in the Tax-responsive elements of HTLV-1 isolates from HIV/HTLV-1-coinfected patients

Background

Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics.

Findings

Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes.

Conclusions

These above data suggested that the swine virus was NDV and it might be generated from La Sota.     

Construction and characterization of recombinant fowlpox virus co-expressing F and HN genes of newcastle disease virus and gB gene of infectious larygnotracheitis virus

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2), whereas the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ was characterized by Western blot (F and gB proteins) and indirect immunofluorescence tests (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10-kb gene fragment, could be expressed authentically and efficiently. Compared with the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in the cell culture of chicken embryo fibroblasts (CEF). Overall, this study suggests that FPV can be a useful live virus vector for the expression of multiforeign genes against multiple avian pathogens.