鴨星狀病毒的遺傳變異性和抗原性及感染鴨肝臟的轉(zhuǎn)錄組分析
發(fā)布時(shí)間：2019-02-24 14:03
【摘要】：鴨病毒性肝炎(Duck viral hepatitis,DVH)是危害養(yǎng)鴨業(yè)的重要疾病,鴨星狀病毒1型(Duck astrovirus,DAstV-1)和 2型(Duck astrovirus2,DAstV-2)是DVH 的兩種病原,其中 DAstV-1 已在我國多個(gè)地區(qū)流行。為了解我國DAstV的分子流行病學(xué)特點(diǎn),開展了分子檢測(cè)工作,并對(duì)檢出的新DAstV進(jìn)行了基因組鑒定、致病性和傳播途徑分析。2012～2014年,從9個(gè)地區(qū)采集678份樣品,從94份(13.9%)樣品中檢測(cè)到DAstV。在陽性樣品所含毒株中,48株(51.1%)為DAstV-1,4株(4.3%)為DAstV-2,33株(35.1%)為新DAstV(稱為DAstV-3),2株(2.1%)可能屬于雞星狀病毒,7株(7.4%)與針尾鴨源星狀病毒具有相近的遺傳演化關(guān)系。結(jié)果表明,我國鴨群中流行多種星狀病毒。對(duì)4株DAstV-2和1株DAstV-3進(jìn)行了基因組鑒定。序列分析結(jié)果顯示,DAstV-2和DAstV-3的基因組長度分別為7319nt和7463 nt,基因組結(jié)構(gòu)均符合禽星狀病毒的特點(diǎn)。在衣殼蛋白區(qū),DAstV-2和DAstV-3之間的遺傳距離為0.612,它們與其他禽星狀病毒種的遺傳距離分別為0.579～0.721和0.577～0.744,據(jù)此可將DAstV-2和DAstV-3鑒定為禽星狀病毒屬內(nèi)的兩個(gè)新病毒種。用3個(gè)ORF進(jìn)行遺傳演化分析,結(jié)果顯示,DAstV-2在遺傳演化過程中可能發(fā)生過重組,這種現(xiàn)象可影響到禽星狀病毒的準(zhǔn)確分類。在評(píng)估DAstV-2對(duì)雛鴨的致病性時(shí),發(fā)現(xiàn)剛出殼的雛鴨攜帶DAstV-3,因此,圍繞DAstV-3進(jìn)行了分子檢測(cè)。結(jié)果顯示,從DAstV-3陽性場(chǎng)采樣,46%的種鴨新鮮糞便、60%的種蛋、83.9%的死胚和60%的雛鴨呈DAstV-3陽性。從其他6個(gè)地區(qū)不同孵化場(chǎng)采集130枚出殼前死亡鴨胚,27.7%的死胚為DAstV-3陽性,說明DAstV-3可通過水平和垂直傳播途徑傳播。為觀察DAstV-3與孵化中鴨胚死亡的相關(guān)性,用鴨胚進(jìn)行了病毒分離和傳代。結(jié)果顯示,感染鴨胚的尿囊膜增厚、發(fā)育受阻,部分鴨胚死亡。用10枚鴨胚進(jìn)行致病性試驗(yàn),結(jié)果顯示,8枚在孵化后期死亡,2枚可孵出雛鴨,但攜帶病毒。結(jié)果表明,DAstV-3是導(dǎo)致孵化中鴨胚死亡的因素之一。DAstV-1是主要流行的鴨星狀病毒,研發(fā)DAstV-1的疫苗具有重要意義,但DAstV-1的培養(yǎng)較為困難,增加了常規(guī)疫苗的研發(fā)難度。本研究用桿狀病毒表達(dá)系統(tǒng)表達(dá)了 DAstV-1的衣殼蛋白,并評(píng)估了其免疫原性。結(jié)果顯示,分別在1日齡和14日齡免疫,可刺激雛鴨產(chǎn)生較高水平的抗體。在24日齡接種強(qiáng)毒,免疫組和對(duì)照組分別有30%和80%的鴨呈現(xiàn)肝臟出血。結(jié)果表明,免疫重組衣殼蛋白可在一定程度上保護(hù)雛鴨抵抗強(qiáng)毒感染。根據(jù)衣殼蛋白線性B細(xì)胞表位預(yù)測(cè)結(jié)果,合成12條多肽。間接ELISA和間接免疫熒光染色的結(jié)果顯示,DAstV-1抗血清能特異性識(shí)別12條多肽,但重組衣殼蛋白的抗血清僅識(shí)別3條多肽(A1、A5和A6),4條多肽(A1、A4、A5和A6)的抗血清可與重組衣殼蛋白發(fā)生反應(yīng)。據(jù)此鑒定出DAstV-1衣殼蛋白的可能的3個(gè)線性B細(xì)胞表位,分別位于7～22位、166～179位和199～213位。迄今為止,對(duì)DAstV-1致病機(jī)理的研究尚屬空白。因此,用轉(zhuǎn)錄組學(xué)技術(shù)分析了感染DAstV-1的雛鴨肝臟,共篩選出2377個(gè)差異表達(dá)基因,其中38.75%為表達(dá)上調(diào)基因,主要為與免疫應(yīng)答相關(guān)基因,61.25%為表達(dá)下調(diào)基因,主要為凝血因子和生長代謝相關(guān)基因。差異表達(dá)基因顯著富集到36個(gè)生物學(xué)過程、18個(gè)分子功能和5個(gè)細(xì)胞組分相關(guān)的GO類別,以及與代謝和機(jī)體免疫應(yīng)答相關(guān)的43個(gè)信號(hào)通路。研究結(jié)果為進(jìn)一步研究DAstV-1的致病機(jī)制提供了線索。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.32
本文編號(hào)：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.32
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 馬凡舒;張蕾;王洋;孫彥剛;閆喜軍;;B細(xì)胞抗原表位預(yù)測(cè)方法的研究進(jìn)展[J];中國畜牧獸醫(yī);2016年01期
2 文雪霞;陳化蘭;熊永忠;王秀榮;;抗原表位鑒定方法的研究進(jìn)展[J];中國畜牧獸醫(yī);2012年07期
3 張中旺;張永光;;抗原表位的研究方法及口蹄疫病毒抗原表位的研究進(jìn)展[J];微生物學(xué)通報(bào);2008年08期
4 胡奇林,陳少鶯,林鋒強(qiáng),程曉霞,林天龍,江斌,陳仕龍,程由銓,李怡英,朱小麗;番鴨呼腸孤病毒的鑒定[J];病毒學(xué)報(bào);2004年03期
5 吳玉章,劉茂昌,賈正才,鄒麗云;HBV新型免疫原的設(shè)計(jì)、合成及免疫原性研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2000年10期
相關(guān)博士學(xué)位論文 前3條
1 王笑言;鴨產(chǎn)蛋下降相關(guān)新病毒的分子鑒定與生物學(xué)特性研究[D];中國農(nóng)業(yè)大學(xué);2015年
2 叢鋒;IBV感染雞腎臟和脾臟轉(zhuǎn)錄組學(xué)分析及雞IFITM1抑制IBV和NDV復(fù)制機(jī)制的研究[D];中國農(nóng)業(yè)科學(xué)院;2015年
3 廖勤豐;新發(fā)現(xiàn)的水禽星狀病毒、微RNA病毒和嵌杯病毒的分子檢測(cè)與鑒定[D];中國農(nóng)業(yè)大學(xué);2014年
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