两株新城疫病毒强毒致病性的比较研究

发布时间：2018-05-12 14:54

本文选题：新城疫病毒 + 致病性　；参考：《扬州大学》2015年硕士论文

【摘要】：新城疫(Newcastle disease, ND)是危害世界养禽业的一种重要传染性疾病,其病原为新城疫病毒(Newcastle disease virus, NDV)。根据遗传进化距离,NDV可分为Class Ⅰ和Class Ⅱ两大分支,目前已知的Class Ⅰ NDV自然分离株均为弱毒株。本实验室保存的一株无毒力鸭源Class Ⅰ NDV Duck/JS/10 (HQ008337),将该株病毒经气囊连续传代10次后得到了变异株JS10-A10。经测定该变异株的MDT、ICPI、IVPI等毒力指标均符合强毒标准,其F蛋白裂解位点亦变为112KRQKRF117。动物试验发现,JS10-A10通过肌肉注射或静脉注射途径对鸡的致死率均为100%,而通过滴鼻、点眼自然感染的途径却不能致死鸡,这与已知的新城疫传统强毒株的特性严重不符。为了解析该变异强毒株与传统强毒株的致病性差异,我们选择了NDV国际标准强毒株Herts/33与变异株JS10-A10分别从体内、体外试验以及生物信息学分析对两者的致病力进行了系统的分析和比较。1.两株新城疫病毒强毒细胞感染的比较试验分别使用JS10-A10和Herts/33在体外感染DF-1和脾淋巴细胞,比较了这两株强毒在细胞水平上的致病性。在不同时间点分别检测两株病毒感染细胞后上清的病毒滴度,结果表明,Herts/33的滴度要比JS10-A10稍高,但经统计后无明显差异。两株病毒感染脾淋巴细胞后,分别在6h、12h、18h和24h收集细胞样品,检测细胞因子(IFN α、IFN β、IFN γ、IL-1β、IL-6和IL-18)的表达情况。结果表明,JS10-A10和Herts/33在不同时间点诱导脾淋巴细胞表达细胞因子的变化趋势基本一致,在mRNA的转录水平上无显著差异。同时,JS10-A10和Herts/33诱导DF-1细胞以及脾淋巴细胞产生的凋亡水平均无显著差异。此外,两株病毒引起的细胞病变程度也进一步表明两者在细胞水平的致病力相似。2.两株新城疫病毒强毒感染SPF鸡的致病力差异分析JS10-A10和Herts/33分别通过滴鼻点眼和肌肉注射感染4周龄SPF鸡,根据不同组织的病毒载量、病变程度、细胞因子表达量以及机体的排毒和抗体水平,比较两株强毒株对鸡致病力差异。JS10-A10滴鼻点眼感染组在第5天病毒滴度最高(103-104 ELD50),但第7天开始病毒在组织的分布逐渐降低直至消失；而JS10-A10肌肉注射组和Herts/33感染组感染后病毒滴度不断增加,直至鸡死亡,且病毒量可达到106ELD50。JS10-A10和Herts/33通过滴鼻点眼途径感染鸡,取其脾、肺、肠、和气管制作病理切片。结果表明,JS10-A10感染鸡只有肺脏组织切片稍微出血,而Herts/33感染鸡脾、肺、肠和气管都有显著病变,尤其肺脏大片出血。排毒结果显示JS10-A10滴鼻点眼组在感染后第5天喉气管、泄殖腔棉拭子病毒量最高,分别为101.8和101.6TCID50;而经同样途径攻毒的Herts/33在相同时间点的病毒量分别为103.5和103.9TCID50。抗体水平检测显示JS10-A10滴鼻点眼组在第4天抗体中和效价为24.5,之后逐渐升高,在第12天达到27.96；Herts/33滴鼻点眼组在第3天出现抗体中和效价的峰值(22),之后降低直至死亡。细胞因子表达量检测显示JS10-A10滴鼻点眼组能在短时间内诱导较高水平的细胞因子,包括IFN α、IFN β、IFN γ、IL-1β、IL-6、IL-18、Mx和PKR；而Herts/33滴鼻点眼组诱导的细胞因子表达水平显著低于JS10-A10,且表达量峰值出现的时间较晚。总之,JS10-A10通过滴鼻点眼能诱导机体在较早的时间产生高水平的细胞因子,以及较高水平的病毒中和抗体效价,有效的抑制了病毒在体内组织的扩散和复制,直至将病毒清除。3.两株新城疫病毒强毒生物信息学分析及初步验证体内试验表明,JS10-A10滴鼻点眼途径感染鸡,其脏器的病毒含量比较低,但诱导的细胞因子水平比较高。我们猜测：两株强毒①F蛋白的差异导致病毒在体内复制过程中毒力发生改变；②HN蛋白的差异导致病毒与受体结合的能力不同；③V蛋白的差异导致病毒拮抗细胞因子(尤其干扰素)的能力不同。针对以上推测,我们应用DNAStar、MEGA、Clustal等软件以及在线工具ESPript 3.x、 SWISS-MODEL,比对了JS10-A10和Herts/33的F、HN、V蛋白的氨基酸序列和空间结构,并绘制遗传进化树,初步分析了两株强毒株在遗传信息学上的差异。结果表明,两株病毒的F基因裂解位点均为强毒特征,JS10-A10F蛋白第25位氨基酸不是半胱氨酸(C),而是丙氨酸(A); Herts/33 HN蛋白第520位为碱性氨基酸R,而JS10-A10为S。此外JS10-A10 HN蛋白比Herts/33多14个氨基酸,这可能影响病毒与受体的结合能力；V蛋白氨基酸序列差异最大,而NDV的V蛋白具有拮抗干扰素的功能,这可能是导致两株病毒诱导机体细胞因子(尤其干扰素)表达量差异的原因。通过遗传信息学分析,本实验进一步探究了两株病毒HN蛋白受体结合能力的差异,结果表明Herts/33受体结合能力远远强于JS10-A10,这也是导致两株病毒致病力差异显著的原因。根据以上研究,我们初步确定了两株病毒HN蛋白与受体结合能力存在差异。
[Abstract]:Newcastle disease (ND) is an important infectious disease that endangers the poultry industry in the world. The pathogen is Newcastle disease virus (Newcastle disease virus, NDV). According to the genetic evolution distance, NDV can be divided into two branches of Class I and Class II. At present, all known Class NDV natural isolates are all weak strains. Class I NDV Duck/JS/10 (HQ008337) was derived from the nontoxic strain of duck. After 10 consecutive passages of the strain of the virus, the mutant strain JS10-A10. was obtained. The MDT, ICPI, IVPI and other toxicity indexes of the mutant strain were all consistent with the strong toxicity standard. The F protein lysis site also changed into 112KRQKRF117. animal test, JS10-A10 by intramuscular injection or vein. The lethal rate of the injecting route to the chicken was 100%, but the way of natural infection by the nose drops could not kill the chicken, which was not consistent with the known characteristics of the traditional strong strain of Newcastle disease. In order to analyze the difference of the virulence between the strong strain and the traditional virulent strain, we chose the NDV international standard strong strain Herts/33 and the variant strain JS10-A10 In vivo, in vitro and bioinformatics analysis, the pathogenicity of both.1. and two strains of Newcastle disease virus was systematically compared and compared with JS10-A10 and Herts/33 in the infection of DF-1 and splenic lymphocytes in vitro, and the pathogenicity of these two strains at the cell level was compared. The virus titer of the supernatant of two virus infected cells was detected at the time point. The results showed that the titer of Herts/33 was slightly higher than that of JS10-A10, but no significant difference was found after statistics. Two strains of virus infected splenic lymphocytes and collected cell samples in 6h, 12h, 18h and 24h respectively, and detected the table of cytokines (IFN alpha, IFN beta, IFN gamma, IL-1 beta, IL-6, IL-18). The results showed that the changes in the expression of cytokines expressed by JS10-A10 and Herts/33 at different time points were basically consistent, and there was no significant difference at the transcriptional level of mRNA. At the same time, there was no significant difference between the JS10-A10 and Herts/33 induced DF-1 cells and the apoptotic water produced by the splenic lymphocytes. In addition, two strains of virus caused by virus. The degree of cytopathic disease further indicated that the pathogenicity difference between the.2. two strains of Newcastle disease virus (NDV) and SPF chicken was similar to the pathogenicity of the two cells. JS10-A10 and Herts/33 were infected by JS10-A10 and Herts/33 respectively by instillation and intramuscular injection of SPF chickens, according to the viral load, degree of disease, the expression of cytokines and the body. The difference of virulence and antibody level between two strains of virulence and two strains of virulence was compared to the fifth day virus titer (103-104 ELD50), but at the beginning of the seventh day the distribution of the virus in the tissue decreased gradually and disappeared, while the titer of the JS10-A10 muscle injection group and the Herts/33 infection group increased continuously until the chickens were infected. Death, and the virus can reach 106ELD50.JS10-A10 and Herts/33 through the nose drops through the infection of the chicken, take its spleen, lung, intestines, and trachea to make pathological sections. The results show that only the lung tissue section of JS10-A10 infected chicken is slightly bleeding, and Herts/33 infection of the chicken spleen, lung, intestines and trachea, especially lung massive bleeding. Detoxification results. The number of JS10-A10 in the larynx and trachea was the highest in the fifth days after infection, and the amount of the cloaca swab swab was the highest, 101.8 and 101.6TCID50, respectively, while the virus volume of the Herts/33 at the same time point was 103.5 and the 103.9TCID50. antibody level, respectively, showed that the antibody neutralization titer was 24.5 in the JS10-A10 nose drops group at fourth days. After a gradual increase of 27.96 in twelfth days, the peak of antibody neutralization titer (22) appeared at the third day in the Herts/33 nose drops group (22) and then decreased until death. Cytokine expression detected that the JS10-A10 nasal drops could induce higher levels of cytokines in a short time, including IFN alpha, IFN beta, IFN gamma, IL-1 beta, IL-6, IL-18, Mx, and P. KR, while the expression level of cytokine induced by Herts/33 nasal drops was significantly lower than that of JS10-A10, and the peak time of the expression was late. In a word, JS10-A10 could induce the body to produce high level of cytokine in earlier time and high level of virus neutralization antibody titer, effectively inhibiting the virus in body. The diffusion and replication of internal tissue, until the virus scavenging.3. two strains of Newcastle disease virus bioinformatics analysis and preliminary verification in vivo test showed that JS10-A10 noses infected chickens, the virus content of their organs is low, but the induced cytokine level is higher. We guess: two strains of strong toxic F protein differences lead to disease Toxicity changes in the process of replication in the body; the difference in HN protein leads to the difference in the ability to combine the virus with the receptor; (3) the difference in the V protein leads to the difference in the ability of the virus to antagonize the cytokines (especially interferon). In view of the above, we use DNAStar, MEGA, Clustal and other software as well as the online tool ESPript 3.x, SWISS-MODEL, The amino acid sequence and spatial structure of F, HN, V protein of JS10-A10 and Herts/33 were compared and the genetic evolution tree was plotted. The difference of genetic informatics between two strains of virulence strains was preliminarily analyzed. The results showed that the F gene cracking loci of the two strains were all strongly toxic, and the twenty-fifth amino acids of JS10-A10F egg white were not cysteine (C), but alanine. A); Herts/33 HN protein 520th is an alkaline amino acid R, while JS10-A10 is S. and JS10-A10 HN protein is 14 more than Herts/33, which may affect the binding ability of the virus to the receptor; the V protein amino acid sequence has the greatest difference, while NDV V protein has the power to antagonize interferon, which may lead to two strains of virus induced cellular cause. The reason for the difference in the expression of the children (especially interferon). Through the genetic information analysis, this experiment further explored the difference in the binding capacity of the HN protein receptor of the two viruses. The results showed that the binding ability of the Herts/33 receptor is far stronger than that of JS10-A10. This is also the cause of the significant difference in the virulence of the two strains. The binding capacity of HN protein to two receptors was different.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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