Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)

Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.

PLASMID VACCINE ENCODING HN GENE FROM NEWCASTLE DISEASE VIRUS HAS MARKED ANTITUMORAL EFFECT IN VITRO

Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

把 ZJI 紧张基于纽卡斯尔疾病病毒(NDV ) 的完全的染色体顺序，七份教材被设计为构造 plasmid pNDV/ZJI 放大 cDNA 碎片，它包含了 NDV ZJI 紧张的全身的 cDNA。与三助手 plasmids， pCIneoNP， pCIneoP 和 pCIneoL， pNDV/ZJI 当时是进表示 T7 RNA 聚合酶的 BSR-T7/5 房间的 cotransfected。在进受胎的鸡肉的 transfected 房间文化上层清液的接种以后，从 specific-pathogen-free (SPF ) 的鸡蛋结队，传染 NDV ZJI 紧张成功地被救。格林荧光灯蛋白质(GFP ) 基因被放大并且插入了到 NDV 全身的 cDNA 产生标注 GFP 的 recombinant plasmid pNDV/ZJIGFP。在进 BSR-T7/5 房间的结果的 plasmid 和三支持 plasmids 的 cotransfection 以后， recombinant NDV， NDV/ZJIGFP，被救。特定的绿荧光在 BSR-T7/5 和鸡胚胎成纤维细胞(CEF ) 房间 48h 被观察感染以后，显示 GFP 基因被表示在一相对高级。NDV/ZJIGFP 被 oculonasal 线路接种进 10-day-old SPF 鸡。四天感染以后的、强壮的绿荧光能在肾和 tracheae 被检测，显示标注 GFP 的 NDV 能是的 recombinant 为 NDV 传播和致病的分析的一个很有用的工具。关键词纽卡斯尔疾病病毒(NDV )- 格林荧光灯蛋白质(GFP )- 营救 - 表示 CLC 数字 S831.7 基础条款：给中国(No.30630048 )

Application of Newcastle disease virus in the treatment of colorectal cancer

Colorectal cancer (CRC) is one of the main reasons of tumor-related deaths worldwide.At present,the main treatment is surgery,but the results are unsatisfactory,and the prognosis is poor.The majority of patients die due to liver or lung metastasis or recurrence.In recent years,great progress has been made in the field of tumor gene therapy,providing a new treatment for combating CRC.As oncolytic viruses selectively replicate almost exclusively in the cytoplasm of tumor cells and do not require integration into the host genome,they are safer,more effective and more attractive as oncolytic agents.Newcastle disease virus (NDV) is a natural RNA oncolytic virus.After NDV selectively infects tumor cells,the immune response induced by NDV’s envelope protein and intracellular factors can effectively kill the tumor without affecting normal cells.Reverse genetic techniques make NDV a vector for gene therapy.Arming the virus by inserting various exogenous genes or using NDV in combination with immunotherapy can also improve the anti-CRC capacity of NDV,and good results have been achieved in animal models and clinical treatment trials.This article reviews the molecular biological characteristics and oncolytic mechanism of NDV and discusses in vitro and in vivo experiments on NDV anti-CRC capacity and clinical treatment.In conclusion,NDV is an excellent candidate for cancer treatment,but more preclinical studies and clinical trials are needed to ensure its safety and efficacy.

Biological Characterization and Whole Genome Sequencing of a Genotype VII Newcastle Disease Virus Strain

[Objective] The aim of this paper was to study the genotype, pathogenicity and nucleotide difference between Newcastle disease virus(NDV) isolates and traditional NDV vaccine strain(La Sota). [Method] A suspected NDV strain was isolated from a chicken farm. The isolate was preliminarily determined by HA and HI tests. A pair of primers was designed based on the partial sequence of NDV F gene published in GenBank(accession No. JF950510.1). F gene was amplified by RT-PCR, cloned and sequenced. The sequencing result was compared with the F gene sequences published in GenBank, and the phylogenetic tree was constructed to analyze the genotypes. The pathogenicity of the virus was determined by mean death time(MDT) of chicken embryos, intracerebral pathogenicity index(ICPI) of one-day-old chicks and intravenous pathogenicity index(IVPI) of six-week-old chickens, respectively. Based on the NDV genome sequence published in GenBank(accession No. JF950510.1), nine pairs of primers were designed to amplify the genome sequence of the isolate, and its structure was analyzed. [Result] The length of F gene was about 500 bp, and a NDV strain of genotype VII was isolated. The MDT, ICPI and IVPI were 52.8 h, 1.675 and 2.46, respectively, indicating the isolate was a virulent strain. The whole genome sequence analysis results showed that the full genome length of the isolate was 15 192 bp, which had 6 more nu-cleotides than that of La Sota strain, and the homology between the two strains was 82.8%. [Conclusion] A virulent NDV strain of genotype Ⅶ was isolated, with low homology to La Sota strain in nucleotide sequence.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

Phylogenetic Analysis of HN Gene of Eight Pigeon NDV Isolates in Guangxi

[Objective] The paper was to provide a basis for scientific prevention and control of pigeon Newcastle disease（ND）.[Method] The HN gene of eight pigeon NDV strains isolated from different pigeon farms in Guangxi were amplified by RT-PCR,sequenced and analyzed.The molecular evolution characteristics of HN gene of pigeon NDV isolates in Guangxi was discussed.[Result] The nucleotide sequence length of HN gene of the eight NDV isolates was 1 716 bp,encoding 571 amino acids.They belonged to virulent group C,and the gene length characteristic of HN gene accorded with virulent strain.Analysis of nucleotide homologies indicated that the eight NDV isolates shared higher homology with genotype VIb,ranging from 90.4% to 99.5%.Phylogenetic tree analysis demonstrated that the genetic relationship between the eight NDV strains in Gangxi and the NDV isolates from Guangxi,Guangdong,Jilin,Liaoning,Yunnan and Heilongjiang during 2011 and 2013 was close.They were located in the same cladogram branch.[Conclusion] We assume that the eight pigeon NDV isolates in Guangxi all belong to the gene class II genotype VI b NDV.

不同时期NDV地方株F基因的克隆及序列分析与基因型研究

利用RT-PCR技术对1998-2002年闯洛阳地区新城疫典型发病禽群中所分离到的15个毒株的F基因主要功能区片段进行了克隆和序列分析，并根据其47～435位核苷酸序列构建了系统进化树，确定了其毒力强弱和基因型。结果显示，依据核苷酸和推导的氨基酸同源性的不同，15个地方毒株可分为3群，群内各毒株同源性较高，群间差异较大，未发现有因分离禽品种不同所引起的明显的株间差异。其中第1群9株，涉及到各时间段分离的毒株和所有分离禽品种，属典型的基因Ⅶ型强毒株，高度集中于进化树的基因Ⅶ型C亚型区；第2群4株，属传统的基因Ⅵ型强毒株。分散于进化树的基因Ⅵ型区；第3群2株，属古典基因Ⅱ型的弱毒株，分散于进化树的基因Ⅱ型区。结果表明，近年来该地区流行的新城疫病毒以新的基因Ⅶ型为主，同时兼有传统的基因Ⅵ型和古老的基因Ⅱ型；并提示基因Ⅶ型各毒株在该地区流行的时间较短，株间差异不大。

单表达和共表达NDVF和HN基因重组鸡痘病毒活疫苗的免疫效力比较试验

为评价母源抗体对抗新城疫病毒(NDV)重组鸡痘病毒(rFPV)活疫苗免疫效力的影响,本研究用单表达、共表达NDV基因VII型病毒ZJ1株F、HN基因的rFPV活疫苗rFPV-12LSF、rFPV-12LSHN、rFPV-12LSFHN和油乳剂灭活疫苗分别免疫14日龄商品蛋鸡,rFPV疫苗的免疫剂量均为2×104pfu,免疫后21d分别用106ELD50NDV不同强毒株攻毒。其中F48E8株攻毒的免疫保护率分别为30.3%、73.2%、41.1%;而ZJ1株攻毒的免疫保护率分别为89.3%和35.8%、67.9%、78.6%、100%。用表达鸡IL-2基因的rFPV-12LSIL-2与rFPV-12LSHN联合免疫没有提高rFPV-12LSHN疫苗的免疫保护力。试验结果表明,单表达NDV HN基因的rFPV-12LSHN可作为NDV活疫苗候选株,为NDV重组鸡痘病毒活疫苗的进一步应用性开发奠定了基础。

新城疫病毒F_(48)E_8株HN基因的克隆和序列分析及与国外NDV HN基因的同源性比较

目的 克隆ＮＤＶ Ｆ４８Ｅ８株ＨＮ基因，与国外ＮＤＶ ＨＮ基因进行比较分析。方法 提取 ＲＮＡ，应用 ＲＴ－ＰＣＲ一次性扩增出ＮＤＶ Ｆ４８Ｅ８株的全长ＨＮ基因，将该ＨＮ基因插入ｐＫＳ（－）后进行克隆并测序。结果 ＮＤＶ Ｆ４８Ｅ８株的ＨＮ基因核苷酸长度为１７１３ｂｐ，编码５７１个氨基酸，有５个糖基化位点，１２个极保守的半胱氨酸残基，与 国外发表的ＮＤＶ序列相符。结论ＮＤＶＦ１８Ｅ８株ＨＮ蛋白基因与国外发表的ＮＤＶ ＨＮ蛋白基因核苷酸同源性在 ８３．３％～８９．２％之间，推导的氨基酸同源性在８７．６％－９１．１％之间。

NDV F基因真核表达质粒的构建及其与鸡IL-18基因联合免疫的初步研究

为了探索鸡新城疫病毒(NDV)F蛋白的免疫原性及鸡白细胞介素-18(IL-18)的免疫增强作用,通过PCR和酶切方法将NDV F基因克隆至真核表达载体pcDNA3.1(+),构建真核表达质粒pcDNA-F。用质粒pcDNA-F单独或与鸡IL-18重组真核表达质粒pIL-18联合免疫14日龄SPF鸡。首免后每周采取外周血及外周抗凝血,应用ELISA和MTT法分别检测免疫鸡的体液免疫及细胞免疫水平。二免后第14 d用NDV强毒F48E9株进行攻击。结果表明,质粒pcDNA-F与pIL-18联合免疫显著提高鸡体的细胞免疫水平,并且提供了50%免疫保护,这为ND的防治开辟了新的途径。

藏鸡NDV的分离鉴定及F基因的遗传进化分析

【目的】探讨藏鸡NDV的毒力和F基因的遗传进化特征。【方法】从采集的藏鸡病料中分离鉴定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方法克隆藏鸡NDV分离株F基因,测序后进行序列分析,并进行进化分析。【结果】共分离鉴定出F5、FX10、F20、F74、F75、F17、F72、FH2等8株NDV,其中F17、F72、FH2为强毒株,其余5株为弱毒株。8株病毒F基因ORF均为1 662bp,编码553个氨基酸,强毒株ORF编码产物含有12个半胱氨酸残基,弱毒株有13个半胱氨酸残基,比强毒株在27位(强毒株C27变为R27)多了1个半胱氨酸残基;有6个潜在的糖基化位点。分离的8株NDV与Gen-Bank中发表的20株NDV参考毒株比较结果表明,F蛋白氨基酸序列变异位点较多,主要集中在8-30,97-124,45,385-386,402-403,479-494,509-520位氨基酸处,强毒株AA替代较集中,而弱毒株保守区较多且AA替代较分散。分离NDV株之间F基因编码区核苷酸序列同源性为83.5%～99.9%,其中强毒株之间同源性为95.2%～99.4%,弱毒株之间同源性为99.4%～99.9%。分离NDV株之间F基因编码的氨基酸同源性为87.5%～99.5%,其中强毒株之间同源性为95.3%～98.2%,弱毒株之间同源性为98.6%～99.5%。进化分析显示,3株强毒株均为基因Ⅶ型,5株弱毒株均为基因Ⅱ型。【结论】分离了8株藏鸡NDV,其中3株为强毒株,属于基因Ⅶ型;5株为弱毒株,属于基因Ⅱ型。

截短NDVF蛋白的原核表达

根据NDV LaSota株F基因已知的抗原表位,对F蛋白进行分段表达。应用RT-PCR方法分段扩增F基因,并将其克隆到pET30a(+)原核表达载体上,得到重组质粒pET30-F780和pET30-F760,将质粒导入BL21(ED3)感受态中,经IPTG诱导表达。表达的重组蛋白通过SDS-PAGE和Westem-blotting方法进行鉴定。表达的两段蛋白大小约为31.1 kDa和27.9 kDa,与预期的蛋白分子量大小相符。Western blot分析表明重组蛋白可以和NDV抗体发生特异性反应。成功构建了原核表达质粒pETF780和pET-F760,并获得了高效表达,通过Western blot分析表明重组蛋白具有良好的免疫反应性。

含NDV F48E9株F基因的MDV CVI988株转移质粒载体的真核表达

为进一步提高重组病毒的表达水平,用构建的含gB启动子和NDV F48E9株F基因的MDV CVI988株转移质粒载体pUS10F转染293T细胞,通过PCR和接免疫荧光(IFA)法研究了其在真核中的表达,并确定最佳的转染条件。结果表明,真核细胞中带有F基因,转移质粒载体已经进入细胞DNA;转染转移质粒载体 pUS10F后的293 T细胞所表达的NDV F基因产物抗原性较好,能够与抗NDV的标准血清反应。确定的最佳转染条件为:DNA 3μg／mL,转染后48 h外源基因在细胞中的表达量最高,在6孔板和96孔板中细胞数量分别以 2×105／孔和1×101／孔的转染效果最好。

真核表达NDV F基因重组质粒的构建及其在CEF细胞中的转染

通过PCR方法自含NDVZF基因的克隆质粒中扩增NDVF基因 ,将其与真核表达载体pcDNA3..1/V5_His_TOPO重组。经核酸内切酶酶切 ,阳性克隆鉴定准确无误 ,核酸序列测定结果与原始基因比较 ,同源性大于 99% ,启始密码和终止密码未出现变异 ,将该重组质粒命名为pcDNANDVZF。将pcDNANDVZF在脂质体作用下转染CEF细胞 ,用间接免疫荧光试验检测 ,结果证明pcDNANDVZF可在CEF细胞中大量表达F蛋白。

NDV江苏分离株F蛋白潜在抗原表位分析

从可塑性、亲水性、表面可能性、抗原性、二级结构等5项参数指标来综合预测1株基因Ⅶ型新城疫病毒（Newcastle disease virus,NDV）,NDV JSXZ株F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基因序列进行分析。综合预测显示：NDV JSXZ株F蛋白预测抗原表位有23处;La Sota F蛋白预测抗原表位分别有19处。与La Sota F蛋白相比,NDV JSXZ毒株在222~228、260~267、308~314、401~413位氨基酸处多出4处抗原表位,而在29~37、104~113、482~488位氨基酸有3处抗原表位不同。其中29~37、104~113aa这2处抗原表位位于裂解位点之前,可能对病毒蛋白水解酶的敏感性、毒力等造成影响。应用Protein-TM-Plot软件预测到NDV JSXZ株F蛋白有3个跨膜区域,分别位于9~27,115~143,497~525位氨基酸处,与La Sota株的三个跨膜区位置接近。结果提示,NDV JSXZ株F蛋白与La Sota株F蛋白相比,可能具有相近或更强的抗原性。

鸡源NDV WF00C株F基因的测序与蛋白特性分析

用鸡源新城疫病毒凤阳分离株WF00C对9-10日龄SPF鸡胚的尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF00C病毒的F基因,获得了1条1.7kb的特异性条带。用PCR产物直接测序。测序结果表明,扩增片段大小为1782bp,含有1个1662bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF00C株与国内外其他NDV F基因的同源性为84.8%-97.5%,其中与国内标准强毒株F48E9的同源性为87.1%,说明WF00C与国内外的传统毒株有较大变异。与Taiwan95株和J株的同源性为94.1%和97.5%,说明WF00C与Taiwan95株和J株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-Gln-Lys-Arg-Phe117,表明为NDV的强毒株。蛋白疏水性和抗原性分析表明与标准强毒株相比没有太大的变异。

NDV F与ChIL-18融合基因“自杀性”质粒的构建

根据GeneBank公布的鸡新城疫病毒(NDV)Lasota株F基因和鸡源性白细胞介素18(ChIL-18)基因序列分别设计特异性引物,且在鸡NDV F基因引物上游引入BamHⅠ位点、下游引入SalⅠ位点,在Ch IL-18基因引物上游引入SalⅠ位点、下游引入HindⅢ、BamHⅠ位点。以鸡新城疫病毒Lasota株RNA为模板RT-PCR扩增其F基因,将F基因插入pMD18-T的BamHⅠ与SalⅠ位点中。同时扩增上游带有SalⅠ位点、下游带有HindⅢ、BamHⅠ位点的ChIL-18基因,并将其插入到含有F基因的pMD18-T质粒的SalⅠ、HindⅢ位点之间,从而使NDV F基因与ChIL-18基因形成具有一个完整阅读框的融合基因,然后将融合基因片段定向克隆到pSFV单一启动子(CMV)下游,构建成F-ChIL-18融合基因重组"自杀性"质粒pSFV-F-ChIL-18。

NDV江苏分离株F基因的克隆与序列分析

为比较NDV江苏分离株的遗传变异特征,应用RT-PCR扩增出新城疫病毒(NDV)江苏徐州株(JSXZ)、江苏宿州株(JSSZ)、江苏连云港株(JSLYG)和江苏淮安株(JSHA)的融合蛋白F基因的全长核苷酸,将其分别克隆至pMD18-T载体中。将获得的阳性重组质粒进行序列测定后,推导出其氨基酸序列,进行分析和比较。结果表明,所获得的4个分离株F基因完整的开放阅读框长度为1662bp,共编码F蛋白的553个氨基酸;4个毒株与常用疫苗株之间核苷酸序列同源性只有69.3%~80.8%。JSXZ株和JSSZ株F基因的裂解位点为112-RRQK/RRF-117,符合强毒株裂解区氨基酸组成的特征。以1bp^374bp的核苷酸序列绘制系统发育树分析表明,JSXZ和JSSZ株NDV为基因Ⅷ型,JSLYG和JSHA株NDV为基因Ⅸ型。

TLR4^-/- DF1细胞系感染NDV后免疫机制的初步研究

为初步探究鸡TLR4在抗病毒感染天然免疫中发挥的作用,本研究利用CRISPR/Cas9基因编辑技术针对鸡TLR4基因的功能域,设计、构建了CRISPR/Cas9双质粒表达系统,转染鸡DF1细胞;经流式细胞仪分选、测序及western blot鉴定筛选得到TLR4^-/- DF1细胞系;利用新城疫病毒(NDV)感染TLR4^-/- DF1细胞系,采用绝对荧光定量PCR检测NDV拷贝数,相对荧光定量PCR检测TLR4下游接头蛋白、细胞因子及干扰素基因的转录水平。结果显示:与野生型DF1细胞相比,NDV感染6 h、16 h和36 h的TLR4^-/- DF1细胞中病毒基因拷贝数显著升高(p<0.05),16 h升高了2.95倍,感染6 h、16 h和24 h下游接头蛋白MyD88基因转录水平下降,感染16 h促炎因子IL-1β下降了48.5%(p<0.01),IL-8下降了62.5%(p<0.01),I型干扰素转录水平下降。以上结果表明:NDV感染诱导的天然免疫应答可能是通过MyD88途径诱导高水平促炎性因子和I型干扰素(IFN-α、IFN-β)的表达,从而抑制NDV的增殖,TLR4在NDV感染引起的天然免疫应答和疾病防控研究中具有重要意义。同时,本研究TLR4^-/- DF1细胞系的建立可用于病毒引起的炎性因子风暴及天然免疫抗病毒作用的探究。