鸭星状病毒的遗传变异性和抗原性及感染鸭肝脏的转录组分析
发布时间：2019-02-24 14:03
【摘要】：鸭病毒性肝炎(Duck viral hepatitis,DVH)是危害养鸭业的重要疾病,鸭星状病毒1型(Duck astrovirus,DAstV-1)和 2型(Duck astrovirus2,DAstV-2)是DVH 的两种病原,其中 DAstV-1 已在我国多个地区流行。为了解我国DAstV的分子流行病学特点,开展了分子检测工作,并对检出的新DAstV进行了基因组鉴定、致病性和传播途径分析。2012～2014年,从9个地区采集678份样品,从94份(13.9%)样品中检测到DAstV。在阳性样品所含毒株中,48株(51.1%)为DAstV-1,4株(4.3%)为DAstV-2,33株(35.1%)为新DAstV(称为DAstV-3),2株(2.1%)可能属于鸡星状病毒,7株(7.4%)与针尾鸭源星状病毒具有相近的遗传演化关系。结果表明,我国鸭群中流行多种星状病毒。对4株DAstV-2和1株DAstV-3进行了基因组鉴定。序列分析结果显示,DAstV-2和DAstV-3的基因组长度分别为7319nt和7463 nt,基因组结构均符合禽星状病毒的特点。在衣壳蛋白区,DAstV-2和DAstV-3之间的遗传距离为0.612,它们与其他禽星状病毒种的遗传距离分别为0.579～0.721和0.577～0.744,据此可将DAstV-2和DAstV-3鉴定为禽星状病毒属内的两个新病毒种。用3个ORF进行遗传演化分析,结果显示,DAstV-2在遗传演化过程中可能发生过重组,这种现象可影响到禽星状病毒的准确分类。在评估DAstV-2对雏鸭的致病性时,发现刚出壳的雏鸭携带DAstV-3,因此,围绕DAstV-3进行了分子检测。结果显示,从DAstV-3阳性场采样,46%的种鸭新鲜粪便、60%的种蛋、83.9%的死胚和60%的雏鸭呈DAstV-3阳性。从其他6个地区不同孵化场采集130枚出壳前死亡鸭胚,27.7%的死胚为DAstV-3阳性,说明DAstV-3可通过水平和垂直传播途径传播。为观察DAstV-3与孵化中鸭胚死亡的相关性,用鸭胚进行了病毒分离和传代。结果显示,感染鸭胚的尿囊膜增厚、发育受阻,部分鸭胚死亡。用10枚鸭胚进行致病性试验,结果显示,8枚在孵化后期死亡,2枚可孵出雏鸭,但携带病毒。结果表明,DAstV-3是导致孵化中鸭胚死亡的因素之一。DAstV-1是主要流行的鸭星状病毒,研发DAstV-1的疫苗具有重要意义,但DAstV-1的培养较为困难,增加了常规疫苗的研发难度。本研究用杆状病毒表达系统表达了 DAstV-1的衣壳蛋白,并评估了其免疫原性。结果显示,分别在1日龄和14日龄免疫,可刺激雏鸭产生较高水平的抗体。在24日龄接种强毒,免疫组和对照组分别有30%和80%的鸭呈现肝脏出血。结果表明,免疫重组衣壳蛋白可在一定程度上保护雏鸭抵抗强毒感染。根据衣壳蛋白线性B细胞表位预测结果,合成12条多肽。间接ELISA和间接免疫荧光染色的结果显示,DAstV-1抗血清能特异性识别12条多肽,但重组衣壳蛋白的抗血清仅识别3条多肽(A1、A5和A6),4条多肽(A1、A4、A5和A6)的抗血清可与重组衣壳蛋白发生反应。据此鉴定出DAstV-1衣壳蛋白的可能的3个线性B细胞表位,分别位于7～22位、166～179位和199～213位。迄今为止,对DAstV-1致病机理的研究尚属空白。因此,用转录组学技术分析了感染DAstV-1的雏鸭肝脏,共筛选出2377个差异表达基因,其中38.75%为表达上调基因,主要为与免疫应答相关基因,61.25%为表达下调基因,主要为凝血因子和生长代谢相关基因。差异表达基因显著富集到36个生物学过程、18个分子功能和5个细胞组分相关的GO类别,以及与代谢和机体免疫应答相关的43个信号通路。研究结果为进一步研究DAstV-1的致病机制提供了线索。
[Abstract]:Duck viral hepatitis (DVH) is an important disease that is harmful to the duck industry, and the duck-like virus type 1 (Duckstrovirus, DAstV-1) and type 2 (Duckbavirus2, DAstV-2) are two pathogens of DVH, among which, DAstV-1 has been popular in many regions of China. In order to understand the molecular epidemiological characteristics of DASTV in China, the molecular detection was carried out, and the new DASTV was identified, the pathogenicity and the transmission route were analyzed. In the period of 2012 ~ 2014, 678 samples were collected from 9 regions and the DAstV was detected from 94 (13. 9%) samples. Among the strains of the positive samples, 48 (51.1%) were DAstV-1, 4 (4.3%) were DAstV-2, 33 (31.1%) were the new DASTV (known as DstV-3), 2 (2.1%) may belong to the chicken star-like virus, and 7 (7. 4%) had similar genetic evolution relation with the star-like virus of the tail of the needle. The results show that there are many star-like viruses in the canard of China. 4 strains of DASTV-2 and 1 strain of DstV-3 were identified. The results of sequence analysis show that the genome length of DAstV-2 and DAstV-3 is 7319nt and 7463nt, respectively, and the genomic structure is in accordance with the characteristics of avian star virus. The genetic distance between the capsid protein region, the DAstV-2 and the DAstV-3 is 0.612, and the genetic distance of the DAstV-2 and the DAstV-3 is 0. 579-0. 721 and 0. 577-0.744, respectively, and the DAstV-2 and the DAstV-3 can be identified as two new viruses in the genus star-like virus. Genetic evolution analysis was carried out with three ORFs, and the results showed that, in the course of the genetic evolution, DAstV-2 could be recombined, which could affect the accurate classification of the avian star-like virus. In the assessment of the pathogenicity of the DodstV-2 to the ducklings, the DDAV-3 was found to be carried in the hatchling of the shell, and therefore, the molecular detection was carried out around the DAstV-3. The results showed that 46% of the duck's fresh feces, 60% of the eggs, 83.9% of the dead embryos and 60% of the ducklings were positive for DAstV-3 from the DAstV-3 positive field. After 130 out-of-shell dead duck embryos were collected from different hatching fields in other 6 regions, 27. 7% of the dead embryos were DAstV-3 positive, indicating that the DAstV-3 could be spread through the horizontal and vertical propagation routes. In order to observe the correlation between DstV-3 and the death of duck embryo in hatching, the virus isolation and passage were carried out with duck embryo. The results showed that the allantoic membrane of the infected duck embryo was thickened, the development was blocked, and some of the duck embryos died. The pathogenic test was carried out with 10 duck embryos. The results showed that 8 of them died in the later stage of incubation, and 2 of them were hatched, but the virus was carried. The results showed that DASTV-3 was one of the factors leading to the death of the duck embryo. DASTV-1 is the main popular duck-star virus, and it is of great significance to develop the DASTV-1 vaccine, but the culture of the DAstV-1 is more difficult, and the development difficulty of the conventional vaccine is increased. This study used a baculovirus expression system to express the capsid protein of DASTV-1 and to evaluate its immunogenicity. The results showed that, at the age of 1 and 14, the chickens can be stimulated to produce higher levels of antibodies. In 24-day-old, 30% and 80% of the ducks with strong toxicity, 30% and 80% of the control group showed liver bleeding. The results showed that the recombinant capsid protein could protect the ducklings to a certain extent to resist the virulent infection. 12 polypeptides were synthesized according to the predicted results of the capsid protein linear B cell table. The results of indirect ELISA and indirect immunofluorescence staining showed that the antisera of the recombinant capsid protein only identified three polypeptides (A1, A5, and A6), and the antiserum of the four polypeptides (A1, A4, A5, and A6) could react with the recombinant capsid protein. The possible three linear B-cell table bits of the DDAV-1 capsid protein were identified, which were located in 7-22, 166-179 and 199-213 positions, respectively. To date, the research on the pathogenesis of DASTV-1 is a blank. In this study, 2377 differentially expressed genes were screened by transcriptome technology, of which 2377 differentially expressed genes were selected, of which 38. 75% were the expression up-regulated genes, mainly related to the immune response, and 61.25% were down-regulated genes, mainly related to the blood coagulation factor and the growth metabolism. The differentially expressed genes were significantly enriched in 36 biological processes, 18 molecular functions and 5 cell components-related GO categories, as well as 43 signal pathways associated with metabolism and body immune response. The results of the study provide a clue for further study of the pathogenesis of DASTV-1.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.32
本文编号：2429611
[Abstract]:Duck viral hepatitis (DVH) is an important disease that is harmful to the duck industry, and the duck-like virus type 1 (Duckstrovirus, DAstV-1) and type 2 (Duckbavirus2, DAstV-2) are two pathogens of DVH, among which, DAstV-1 has been popular in many regions of China. In order to understand the molecular epidemiological characteristics of DASTV in China, the molecular detection was carried out, and the new DASTV was identified, the pathogenicity and the transmission route were analyzed. In the period of 2012 ~ 2014, 678 samples were collected from 9 regions and the DAstV was detected from 94 (13. 9%) samples. Among the strains of the positive samples, 48 (51.1%) were DAstV-1, 4 (4.3%) were DAstV-2, 33 (31.1%) were the new DASTV (known as DstV-3), 2 (2.1%) may belong to the chicken star-like virus, and 7 (7. 4%) had similar genetic evolution relation with the star-like virus of the tail of the needle. The results show that there are many star-like viruses in the canard of China. 4 strains of DASTV-2 and 1 strain of DstV-3 were identified. The results of sequence analysis show that the genome length of DAstV-2 and DAstV-3 is 7319nt and 7463nt, respectively, and the genomic structure is in accordance with the characteristics of avian star virus. The genetic distance between the capsid protein region, the DAstV-2 and the DAstV-3 is 0.612, and the genetic distance of the DAstV-2 and the DAstV-3 is 0. 579-0. 721 and 0. 577-0.744, respectively, and the DAstV-2 and the DAstV-3 can be identified as two new viruses in the genus star-like virus. Genetic evolution analysis was carried out with three ORFs, and the results showed that, in the course of the genetic evolution, DAstV-2 could be recombined, which could affect the accurate classification of the avian star-like virus. In the assessment of the pathogenicity of the DodstV-2 to the ducklings, the DDAV-3 was found to be carried in the hatchling of the shell, and therefore, the molecular detection was carried out around the DAstV-3. The results showed that 46% of the duck's fresh feces, 60% of the eggs, 83.9% of the dead embryos and 60% of the ducklings were positive for DAstV-3 from the DAstV-3 positive field. After 130 out-of-shell dead duck embryos were collected from different hatching fields in other 6 regions, 27. 7% of the dead embryos were DAstV-3 positive, indicating that the DAstV-3 could be spread through the horizontal and vertical propagation routes. In order to observe the correlation between DstV-3 and the death of duck embryo in hatching, the virus isolation and passage were carried out with duck embryo. The results showed that the allantoic membrane of the infected duck embryo was thickened, the development was blocked, and some of the duck embryos died. The pathogenic test was carried out with 10 duck embryos. The results showed that 8 of them died in the later stage of incubation, and 2 of them were hatched, but the virus was carried. The results showed that DASTV-3 was one of the factors leading to the death of the duck embryo. DASTV-1 is the main popular duck-star virus, and it is of great significance to develop the DASTV-1 vaccine, but the culture of the DAstV-1 is more difficult, and the development difficulty of the conventional vaccine is increased. This study used a baculovirus expression system to express the capsid protein of DASTV-1 and to evaluate its immunogenicity. The results showed that, at the age of 1 and 14, the chickens can be stimulated to produce higher levels of antibodies. In 24-day-old, 30% and 80% of the ducks with strong toxicity, 30% and 80% of the control group showed liver bleeding. The results showed that the recombinant capsid protein could protect the ducklings to a certain extent to resist the virulent infection. 12 polypeptides were synthesized according to the predicted results of the capsid protein linear B cell table. The results of indirect ELISA and indirect immunofluorescence staining showed that the antisera of the recombinant capsid protein only identified three polypeptides (A1, A5, and A6), and the antiserum of the four polypeptides (A1, A4, A5, and A6) could react with the recombinant capsid protein. The possible three linear B-cell table bits of the DDAV-1 capsid protein were identified, which were located in 7-22, 166-179 and 199-213 positions, respectively. To date, the research on the pathogenesis of DASTV-1 is a blank. In this study, 2377 differentially expressed genes were screened by transcriptome technology, of which 2377 differentially expressed genes were selected, of which 38. 75% were the expression up-regulated genes, mainly related to the immune response, and 61.25% were down-regulated genes, mainly related to the blood coagulation factor and the growth metabolism. The differentially expressed genes were significantly enriched in 36 biological processes, 18 molecular functions and 5 cell components-related GO categories, as well as 43 signal pathways associated with metabolism and body immune response. The results of the study provide a clue for further study of the pathogenesis of DASTV-1.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.32
【参考文献】
相关期刊论文 前5条
1 马凡舒;张蕾;王洋;孙彦刚;闫喜军;;B细胞抗原表位预测方法的研究进展[J];中国畜牧兽医;2016年01期
2 文雪霞;陈化兰;熊永忠;王秀荣;;抗原表位鉴定方法的研究进展[J];中国畜牧兽医;2012年07期
3 张中旺;张永光;;抗原表位的研究方法及口蹄疫病毒抗原表位的研究进展[J];微生物学通报;2008年08期
4 胡奇林,陈少莺,林锋强,程晓霞,林天龙,江斌,陈仕龙,程由铨,李怡英,朱小丽;番鸭呼肠孤病毒的鉴定[J];病毒学报;2004年03期
5 吴玉章,刘茂昌,贾正才,邹丽云;HBV新型免疫原的设计、合成及免疫原性研究[J];第三军医大学学报;2000年10期
相关博士学位论文 前3条
1 王笑言;鸭产蛋下降相关新病毒的分子鉴定与生物学特性研究[D];中国农业大学;2015年
2 丛锋;IBV感染鸡肾脏和脾脏转录组学分析及鸡IFITM1抑制IBV和NDV复制机制的研究[D];中国农业科学院;2015年
3 廖勤丰;新发现的水禽星状病毒、微RNA病毒和嵌杯病毒的分子检测与鉴定[D];中国农业大学;2014年
，本文编号：2429611
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2429611.html
