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.

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.

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.

6株鸽源新城疫病毒广东分离株F、HN基因的克隆及遗传进化分析

为研究广东省鸽源新城疫病毒(NDV)的遗传进化规律,试验于2021—2022年从广东6家疑似感染NDV的鸽场采集病鸽临床样品,进行RT-PCR诊断、病毒分离鉴定和动物回归试验,并对分离毒株进行生物学毒力测定以及F基因、HN基因的同源性和遗传进化分析。结果显示:共分离到6株鸽源NDV,对幼鸽进行分组攻毒14 d后,发病率均为100%,死亡率为60%~80%;6株毒株的F蛋白裂解位点氨基酸序列为112R-R-Q-K-R-F117/112R-R-R-K-R-F117,具有NDV强毒株的典型分子特征,和分离毒株ICPI的测定结果共同表明6株毒株均为强毒株;6株毒株均为NDV的ClassⅡ基因Ⅵ.2.1.1.2.2型;与传统疫苗株La Sota的同源性较低,且F、HN蛋白氨基酸出现多处位点变异。研究表明,广东省鸽源NDV流行以Class Ⅱ基因Ⅵ.2.1.1.2.2型为主,常用的疫苗株La Sota可能对广东地区流行的鸽源NDV保护效果不佳。

基于F基因的鸽新城疫病毒分子特征分析

试验旨在了解鸽新城疫病毒流行株的分子特征,利用MEGA7.0、Lasergene7.0软件对2020—2022年新疆和田地区规模化养鸽场流行病学调查阳性样品进行了鸽新城疫病毒F基因的测序和序列分析。结果显示,6株F基因属于Ⅵ.2.1.1.2.2基因亚型,F蛋白裂解位点序列均为112R-R-Q-K-R-F117,与基因Ⅱ型疫苗株La Sota株、HB1株、VG/GA株、Clone30株及基因Ⅲ型Mukteswarz株的核苷酸同源性为83.5%~85.7%,与甘肃、宁夏鸽新城疫核苷酸同源性为97.3%~99.5%。6株F蛋白融合肽区均有两处氨基酸突变(V121I、A132S),HRa区均有1处氨基酸突变(V179I),NDV/PIGEON/XJHT/802/2022/F在HRb、HRc和跨膜区还有1~2个氨基酸突变。研究表明,该地区鸽新城疫病毒遗传相对稳定,变异持续存在,定期监测与分析鸽新城疫病毒分子流行特征十分必要。

鸡TIFA和TRAF6蛋白的分子特征及其基因在免疫器官中的表达特性

【目的】明确鸡肿瘤坏死因子受体相关因子相互作用的具有叉形头相关结构域蛋白(TIFA)和肿瘤坏死因子受体相关因子6(TRAF6)的分子特征及其基因在鸡免疫器官中的表达特性,为后续深入研究TIFA和TRAF6相互作用调控家禽病毒NDV复制的作用机制提供理论依据。【方法】采用生物信息学软件结合实时荧光定量PCR(RT-qPCR)方法,通过分析鸡TIFA和TRAF6蛋白的理化性质、二级结构、三级结构、氨基酸同源性和结构域保守性及其在1~6月龄白来航鸡和新城疫病毒(NDV)感染SPF鸡后第3~5天的免疫器官(脾脏、胸腺和法氏囊),分别检测鸡免疫器官中TIFA和TRAF6基因的表达水平,进而研究鸡TIFA和TRAF6蛋白的分子特征及其基因在免疫器官中的表达特性。【结果】鸡TIFA和TRAF6蛋白分别含有187和545个氨基酸残基,相对分子量约为21.8和61.9 kDa,其二级结构均主要由无规则卷曲和α-螺旋组成。氨基酸同源性分析结果表明,鸡与火鸡TIFA和TRAF6蛋白的氨基酸同源性最高,分别为97.3%和98.2%,而与猪TIFA及小鼠TRAF6蛋白的氨基酸同源性最低,分别为51.1%和74.7%。同时,鸡TIFA蛋白的FHA结构域及TRAF6蛋白的RING、Zinc finger和MATH结构域均在禽类中保守性高。RT-qPCR分析结果表明,鸡TIFA和TRAF6基因在1~6月龄鸡免疫器官中均存在表达,但在脾脏中的相对表达量最高(47.94和10.84),而在法氏囊中最低(8.08和2.01),其中,TIFA基因在1月龄鸡免疫器官中表达量最高(22.15),在6月龄时最低(1.11),呈先减后增再减趋势,整体表达趋势呈倒“N”型模式;TRAF6基因在4月龄鸡免疫器官中表达量最高(7.13),在5月龄时最低(1.05),呈先降后升再降再升趋势,整体表达趋势呈“W”型模式。在NDV感染SPF鸡后的免疫器官中,TIFA和TRAF6基因的相对表达量均显著高于对照,其中,TIFA基因在第3天的鸡脾脏、第4天的鸡胸腺和法氏囊中的表达量最高,而TRAF6基因则在第3天的鸡脾脏、第5天的鸡胸腺和法氏囊中的表达量最高。【结论】鸡TIFA和TRAF6蛋白在禽类中的保守性高,两者在不同月龄鸡和NDV感染鸡的免疫器官中呈不同的表达水平和表达变化趋势,其参与的先天性免疫应答可能在抵抗NDV感染过程中发挥一定作用。

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.

Epitope variation in the Newcastle disease virus HN gene under antibody immune selective pressure in cell culture

The influence of antibody immune selective pressure on Newcastle disease virus (NDV) HN and F gene mutations was studied in cell cultures.NDV field strain TZ060107 was inoculated into chicken embryo fibroblast cells and continuously passaged with (group A) or without (group B) anti-NDV monospecific serum.Each group contained three independent passage series.HN and F genes were amplified and sequenced for the 10th,20th,30th,40th and 50th generations of each serial passage,and compared with the original strain.The results demonstrated that increased HN gene mutations were observed in group A with the antibody than in group B without the antibody.The nonsynonymous (NS) to synonymous (S) mutations ratio was 6 for group A,significantly higher than 3.4 in group B.In group A with the antibody,there were five stable NS mutations in HN gene,three of which (related to aa#353,521 and 568) were related to known epitopes.There were two stable NS mutations in F gene in group A,but no stable NS mutations in group B.The NS/S ratios of F gene were less than 2.5 for both groups A and B.Our results suggested that the antibody strongly influenced HN gene mutations,while the F gene was less influenced by the same antibody.

免疫鸡血清新城疫病毒抗体水平与攻毒保护效果的相关性研究

为了明确不同血清新城疫病毒(NDV)抗体水平对鸡的保护效果,本研究采用重组NDV灭活疫苗(A-Ⅶ株)对鸡进行免疫,通过交叉血凝抑制(HI)试验比较A-Ⅶ株与La Sota株间的血清学差异,并采用NDV标准强毒株F_(48)E_(8)和基因Ⅶ型强毒株JSC0804对不同抗体水平免疫鸡进行了攻毒保护试验。结果显示:抗A-Ⅶ株血清用A-Ⅶ株抗原测得的平均HI抗体效价显著高于La Sota株抗原(P<0.01),约高1.5log2,而抗La Sota株血清用La Sota株抗原测得的平均HI抗体效价稍高于A-Ⅶ株抗原,但无显著差异(P>0.05),表明2种疫苗株存在一定的血清学差异。在试验条件下,所有免疫鸡经点眼滴鼻攻毒后均未表现明显临床症状,但存在不同程度的排毒现象,排毒率总体上随抗体效价升高呈逐渐下降趋势。A-Ⅶ株疫苗免疫鸡血清HI抗体效价分别达≥13log_(2)和≥14log_(2)时可完全阻止F_(48)E_8株和JSC0804株排毒。免疫鸡排毒一般仅限于攻毒后5 d内。本研究阐明了免疫鸡的抗体水平和免疫保护效果的相关性,为新城疫免疫控制提供了依据。

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LaSota同源率仅为79．2％-80．7％，且显示出明显的地域性。

10株鸭源新城疫病毒的分离鉴定及F基因遗传进化分析

为调查山东省鸭群中新城疫病毒(NDV)的流行情况,对2007—2009年山东不同地区发病鸭群进行了NDV的分离鉴定,并对分离的10株鸭源NDV进行了生物学特性和遗传特性研究。结果显示:10株鸭源NDV的致病指数MDT和ICPI测定结果分别为50.4～60.0和1.66～1.78,表明10株分离株均属强毒株。致病性试验显示,3株代表毒株对不同日龄雏鸭均具有较强的致病性,发病率和死亡率分别达70%～100%和20%～70%。对F基因序列分析表明,相对于基因Ⅰ～Ⅵ型NDV,10株鸭源NDVF蛋白存在较大变异,这些变异主要集中在1—30、101—124、479—494和509—516 aa处,且在52、71、176、272、314、402和489位出现了与近几年流行毒株一致的氨基酸替代。核苷酸同源性分析结果表明,10株鸭源NDV之间同源性在95.8%～99.9%,与2000年以来国内外分离的基因Ⅶ型NDV同源性较高,在95.7%～99.8%,与传统LaSota、F48E9同源性较低,为83.8%～90.9%。遗传进化分析结果显示分离毒株均属于基因Ⅶd型。此外,10株鸭源NDV还具有大多数鹅源NDV所特有的973位和1 249位RsaⅠ位点。上述结果表明基因Ⅶd型NDV是造成我国鸭群近年来发生新城疫的主要病原,鸭源NDV很可能源于早期的鹅源NDV。

新城疫不同毒株交叉鸡胚中和指数及其与F和HN基因变异的相关性

选取13株国内2001～2004年分离的新城疫流行病毒(Newcastle disease virus,NDV),经蚀斑纯化,克隆其融合蛋白(F)和血凝素.神经氨酸酶(HN)基因,结合疫苗株La Sota、Clone30和国内标准强毒株F_(48)E_9等的基因序列,进行遗传变异分析。利用纯化的病毒制备特异阳性血清,进行鸡胚交叉中和试验,确定不同NDV毒株之间的抗原相关性,并与NDV不同毒株之间的HN和F基因核苷酸(氨基酸)同源性进行相关比较。结果表明:病毒中和指数与HN基因的核苷酸(氨基酸)同源性显著相关(P<0.01,r=0.35),与F基因呈弱相关(P<0.05,r=0.20),而与F基因前374bp的核甘酸同源性不相关。这表明,NDV的分子变异已经对NDV的抗原性变异产生了影响,研制新型的疫苗成为必然。

鸡IL-2在新城疫病毒F基因疫苗免疫中的作用

利用国内首次克隆成功的鸡 IL- 2基因真核表达质粒与新城疫病毒 (NDV) D2 6株 F基因真核质粒 (F基因疫苗 )联合免疫 SPF雏鸡 ,观察了其诱导的免疫应答及免疫保护作用 ,以及不同免疫方式对其免疫作用的影响。结果证实了国内克隆鸡 IL- 2基因的免疫增强作用 ,以及作为免疫佐剂应用于禽类基因免疫的可行性 ,为鸡 IL- 2这一新型佐剂的实际应用提供了重要的试验依据。

新城疫病毒(长春株)F蛋白基因的克隆和序列分析

新城疫病毒（ＮＤＶ）长春株在鸡胚增殖后纯化，提取ＲＮＡ，然后利用特异性引物，经ＲＴ－ＰＣＲ一次性扩增出了ＮＤＶ长春株的全长Ｆ基因。将该Ｆ基因插入ｐＫＳ（－）后，进行了序列测定。序列分析表明，该Ｆ基因核苷酸长度为１７５８ｂｐ，编码５５３个氨基酸，序列中有６个糖基化位点，１３个半胱氨酸残基，裂解位点区（１１２～１１７）氨基酸序列为Ｇｌｙ－Ａｒｇ－Ｇｌｎ－Ｇｌｙ－Ａｒｇ－Ｌｅｕ，与所有弱毒株在这一区域的序列（Ｇｌｙ－Ａｒｇ／Ｌｙｓ－Ｇｌｎ－Ｇｌｙ／Ｓｅｒ－Ａｒｇ－Ｌｅｕ）相符，证明长春株为弱毒株。同源性分析表明，长春株Ｆ基因与目前国外发表的其他ＮＤＶＦ基因相比，核苷酸序列同源性在８８％～９９％之间。

新城疫病毒F基因在新型杆状病毒表达系统中的表达及重组病毒的免疫原性

将新城疫病毒 (四平株 ) F基因插入到 p Fast Bac 质粒中 ,构建了重组质粒 p FF。 p FF转化大肠杆菌 DH10 Bac后 ,F基因在助手质粒 (helper plasmid)提供转座酶的情况下 ,通过转座进入 Bacmid,构建了重组 Bacmid(re- Bacmid)。在含有 X- gal/ IPTG、庆大霉素、卡那霉素和四环素的琼脂平板上 ,筛选白色菌落后 ,提取 re- Bacm id转染 Sf- 9昆虫细胞。在昆虫细胞内 ,re- Bacm id经复制、表达、装配 ,形成重组杆状病毒 ,并表达 F蛋白。经 SDS- PAGE和 Western- blot分析 ,表达的 F蛋白的分子大小为 6 30 0 0 ,并证明在昆虫细胞内表达的蛋白能部分糖基化。表达的 F蛋白约占细胞总蛋白的 10 %。动物试验证明 。

鸡痘病毒282E4株表达载体的构建及新城疫病毒F蛋白的表达

将鸡痘病毒 2 82 E4株 TK基因和 1 .5kb B片段 2个非必需区克隆后 ,分别在 TK基因 Nco 位点和 B片段 Eco R 位点插入复合启动子 (由牛痘病毒 A型包涵体启动子和 2 0个串联的痘苗病毒 ( VV)突变型 p7.5早期启动子串联组成 ) ,然后在复合启动子上游插入单一启动子 (人工合成的由 1 6个串联的 VV突变型p7.5早期启动子组成 )调控的 Lac Z基因或 GFP基因作为报告基因 ,构建成分别以 TK基因或 B片段为侧翼、以复合启动子调控目的基因的 2个以 Lac Z为报告基因的表达载体 p UTA-2 -1 6Lac Z和 p UBA-7-1 6Lac Z,2个以 GFP为报告基因的表达载体 p UTA-2 -F71 6和 p UBA-7-F71 6。在 TK基因 Nco 位点和 B片段 Eco R 位点插入单一启动子和单一启动子调控的 Lac Z基因 ,得到 2个分别以 TK基因或 B片段为侧翼、以单一启动子调控目的基因、以 Lac Z为报告基因的表达载体 p UT1 61 6Lac Z和 p UB1 61 6Lac Z。将构建的 6个表达载体分别转染 FPV HIPRA株感染的鸡胚成纤维 ( CEF)细胞 ,仅在转染以 TK基因为侧翼、以 Lac Z为报告基因的 2个表达载体 p UTA-2 -1 6Lac Z和 p UT1 61 6Lac Z收获的病毒中 ,在 X-gal存在下 ,筛选出了表达 Lac Z的蓝色重组病毒蚀斑 ,而转染以 B片段为侧翼的表达载体在同样条件下未筛选到蓝色病?

中国水禽新城疫病毒F和HN基因的变异趋势

选取中国1996-2005年的8株鹅、鸭等水禽NDV流行株,克隆其融合蛋白(F)和血凝素-神经氨酸酶(HN)基因,参考GenBank中具有F和HN核苷酸全长的16株国内外NDV代表株、16株国内水禽流行株以及12株只具有F基因的水禽毒株序列,参照传统F基因分型方法,分别对F和HN基因进行分型,同时进行相关比较、核苷酸和氨基酸同源性以及系统发育分析。结果表明:16株国内外代表株F和HN基因分型基本一致,但24株水禽NDV流行株中只有15株相同,符合率为62.5%,不同占37.5%(9/24)。系统发育和同源比较发现:水禽NDV流行株HN基因高度同源,同属Ⅶ型,而F基因则分属不同的基因型。通过对24株NDVF和HN基因氨基酸全长以及进行F基因分型的F基因47-420位核苷酸的同源性相关性比较证实:F基因前47-420位核苷酸的遗传变异与F基因氨基酸全长变异密切相关,r=0.916,但与HN基因的氨基酸变异不相关,显示出NDV在演变过程中F和HN基因具有相对的独立性。水禽NDV是家禽NDV的重要储存宿主,应高度重视水禽NDV的变异。

新城疫病毒(青岛株)F蛋白基因的克隆和序列分析

参考新城疫病毒 ( NDV)长春株的 F基因序列设计了 1对特异性引物 ,应用 RT-PCR对 NDV青岛株(野毒 )的 F基因进行了扩增 ,扩增产物克隆后测序。扩出的 F基因核苷酸长度为 792 bp,编码 2 6 1个氨基酸 ,包括完整的 F2片段和部分 F1片段。裂解位点区 ( 1 1 2～ 1 1 7aa)氨基酸序列为 Arg-Arg-Gln-Arg-Arg-Phe,与国外发表的强毒株序列相符 ,证明青岛株为强毒株。根据该基因推导的氨基酸序列 ,与国内外发表的 NDVF蛋白氨基酸序列相比 ,同源性为 88.1 %～ 94 .3%。

鸡新城疫病毒F基因高效真核表达质粒的构建

为进一步研究基因疫苗的免疫机制和基因疫苗的免疫效果 ,应用 DNA重组技术 ,根据载体 pc D-NA4 .0 / His Max的多克隆位点设计引物 ,对含有 NDV保护性抗原基因 F的质粒 PCR扩增。将得到的目的片段插入到载体的启动子下游 ,成功得到含有 NDV F基因的真核表达质粒载体 PC4 F的基因疫苗。