本文選題：新城疫病毒 + 致病性　； 參考：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：新城疫(Newcastle disease, ND)是危害世界養(yǎng)禽業(yè)的一種重要傳染性疾病,其病原為新城疫病毒(Newcastle disease virus, NDV)。根據(jù)遺傳進(jìn)化距離,NDV可分為Class Ⅰ和Class Ⅱ兩大分支,目前已知的Class Ⅰ NDV自然分離株均為弱毒株。本實(shí)驗(yàn)室保存的一株無毒力鴨源Class Ⅰ NDV Duck/JS/10 (HQ008337),將該株病毒經(jīng)氣囊連續(xù)傳代10次后得到了變異株JS10-A10。經(jīng)測定該變異株的MDT、ICPI、IVPI等毒力指標(biāo)均符合強(qiáng)毒標(biāo)準(zhǔn),其F蛋白裂解位點(diǎn)亦變?yōu)?12KRQKRF117。動(dòng)物試驗(yàn)發(fā)現(xiàn),JS10-A10通過肌肉注射或靜脈注射途徑對雞的致死率均為100%,而通過滴鼻、點(diǎn)眼自然感染的途徑卻不能致死雞,這與已知的新城疫傳統(tǒng)強(qiáng)毒株的特性嚴(yán)重不符。為了解析該變異強(qiáng)毒株與傳統(tǒng)強(qiáng)毒株的致病性差異,我們選擇了NDV國際標(biāo)準(zhǔn)強(qiáng)毒株Herts/33與變異株JS10-A10分別從體內(nèi)、體外試驗(yàn)以及生物信息學(xué)分析對兩者的致病力進(jìn)行了系統(tǒng)的分析和比較。1.兩株新城疫病毒強(qiáng)毒細(xì)胞感染的比較試驗(yàn)分別使用JS10-A10和Herts/33在體外感染DF-1和脾淋巴細(xì)胞,比較了這兩株強(qiáng)毒在細(xì)胞水平上的致病性。在不同時(shí)間點(diǎn)分別檢測兩株病毒感染細(xì)胞后上清的病毒滴度,結(jié)果表明,Herts/33的滴度要比JS10-A10稍高,但經(jīng)統(tǒng)計(jì)后無明顯差異。兩株病毒感染脾淋巴細(xì)胞后,分別在6h、12h、18h和24h收集細(xì)胞樣品,檢測細(xì)胞因子(IFN α、IFN β、IFN γ、IL-1β、IL-6和IL-18)的表達(dá)情況。結(jié)果表明,JS10-A10和Herts/33在不同時(shí)間點(diǎn)誘導(dǎo)脾淋巴細(xì)胞表達(dá)細(xì)胞因子的變化趨勢基本一致,在mRNA的轉(zhuǎn)錄水平上無顯著差異。同時(shí),JS10-A10和Herts/33誘導(dǎo)DF-1細(xì)胞以及脾淋巴細(xì)胞產(chǎn)生的凋亡水平均無顯著差異。此外,兩株病毒引起的細(xì)胞病變程度也進(jìn)一步表明兩者在細(xì)胞水平的致病力相似。2.兩株新城疫病毒強(qiáng)毒感染SPF雞的致病力差異分析JS10-A10和Herts/33分別通過滴鼻點(diǎn)眼和肌肉注射感染4周齡SPF雞,根據(jù)不同組織的病毒載量、病變程度、細(xì)胞因子表達(dá)量以及機(jī)體的排毒和抗體水平,比較兩株強(qiáng)毒株對雞致病力差異。JS10-A10滴鼻點(diǎn)眼感染組在第5天病毒滴度最高(103-104 ELD50),但第7天開始病毒在組織的分布逐漸降低直至消失；而JS10-A10肌肉注射組和Herts/33感染組感染后病毒滴度不斷增加,直至雞死亡,且病毒量可達(dá)到106ELD50。JS10-A10和Herts/33通過滴鼻點(diǎn)眼途徑感染雞,取其脾、肺、腸、和氣管制作病理切片。結(jié)果表明,JS10-A10感染雞只有肺臟組織切片稍微出血,而Herts/33感染雞脾、肺、腸和氣管都有顯著病變,尤其肺臟大片出血。排毒結(jié)果顯示JS10-A10滴鼻點(diǎn)眼組在感染后第5天喉氣管、泄殖腔棉拭子病毒量最高,分別為101.8和101.6TCID50;而經(jīng)同樣途徑攻毒的Herts/33在相同時(shí)間點(diǎn)的病毒量分別為103.5和103.9TCID50�？贵w水平檢測顯示JS10-A10滴鼻點(diǎn)眼組在第4天抗體中和效價(jià)為24.5,之后逐漸升高,在第12天達(dá)到27.96；Herts/33滴鼻點(diǎn)眼組在第3天出現(xiàn)抗體中和效價(jià)的峰值(22),之后降低直至死亡。細(xì)胞因子表達(dá)量檢測顯示JS10-A10滴鼻點(diǎn)眼組能在短時(shí)間內(nèi)誘導(dǎo)較高水平的細(xì)胞因子,包括IFN α、IFN β、IFN γ、IL-1β、IL-6、IL-18、Mx和PKR；而Herts/33滴鼻點(diǎn)眼組誘導(dǎo)的細(xì)胞因子表達(dá)水平顯著低于JS10-A10,且表達(dá)量峰值出現(xiàn)的時(shí)間較晚�？傊�,JS10-A10通過滴鼻點(diǎn)眼能誘導(dǎo)機(jī)體在較早的時(shí)間產(chǎn)生高水平的細(xì)胞因子,以及較高水平的病毒中和抗體效價(jià),有效的抑制了病毒在體內(nèi)組織的擴(kuò)散和復(fù)制,直至將病毒清除。3.兩株新城疫病毒強(qiáng)毒生物信息學(xué)分析及初步驗(yàn)證體內(nèi)試驗(yàn)表明,JS10-A10滴鼻點(diǎn)眼途徑感染雞,其臟器的病毒含量比較低,但誘導(dǎo)的細(xì)胞因子水平比較高。我們猜測：兩株強(qiáng)毒①F蛋白的差異導(dǎo)致病毒在體內(nèi)復(fù)制過程中毒力發(fā)生改變；②HN蛋白的差異導(dǎo)致病毒與受體結(jié)合的能力不同；③V蛋白的差異導(dǎo)致病毒拮抗細(xì)胞因子(尤其干擾素)的能力不同。針對以上推測,我們應(yīng)用DNAStar、MEGA、Clustal等軟件以及在線工具ESPript 3.x、 SWISS-MODEL,比對了JS10-A10和Herts/33的F、HN、V蛋白的氨基酸序列和空間結(jié)構(gòu),并繪制遺傳進(jìn)化樹,初步分析了兩株強(qiáng)毒株在遺傳信息學(xué)上的差異。結(jié)果表明,兩株病毒的F基因裂解位點(diǎn)均為強(qiáng)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位為堿性氨基酸R,而JS10-A10為S。此外JS10-A10 HN蛋白比Herts/33多14個(gè)氨基酸,這可能影響病毒與受體的結(jié)合能力；V蛋白氨基酸序列差異最大,而NDV的V蛋白具有拮抗干擾素的功能,這可能是導(dǎo)致兩株病毒誘導(dǎo)機(jī)體細(xì)胞因子(尤其干擾素)表達(dá)量差異的原因。通過遺傳信息學(xué)分析,本實(shí)驗(yàn)進(jìn)一步探究了兩株病毒HN蛋白受體結(jié)合能力的差異,結(jié)果表明Herts/33受體結(jié)合能力遠(yuǎn)遠(yuǎn)強(qiáng)于JS10-A10,這也是導(dǎo)致兩株病毒致病力差異顯著的原因。根據(jù)以上研究,我們初步確定了兩株病毒HN蛋白與受體結(jié)合能力存在差異。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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