【作者】 李敏；

【导师】 韦平；

【作者基本信息】 广西大学 ， 兽医学， 2021， 硕士

【摘要】 网状内皮组织增殖病(reticuloendothelisis,RE)是由网状内皮组织增殖病病毒(reticuloendotheliosis virus,REV)导致的一种主要侵害鸡、火鸡、鸭和其他禽类的病理综合征。REV属于反转录病毒,是引起家禽发生肿瘤性疾病的病原之一,其传播方式主要为水平传播、垂直传播和疫苗传播。REV引起的免疫抑制一方面可以导致机体抵抗力下降,容易继发其他疾病,另外一方面可导致疫苗免疫应答水平降低,严重者会造成疫苗免疫失效,给养禽业带来更大经济损失。REV在自然条件下极少会单独引起鸡群发病,主要以混合感染的形式出现。课题对2018年-2020年临床疑似肿瘤病病例采集样品进行细胞培养病毒分离,并通过PCR和间接免疫荧光试验(immunofluorescence assay,IFA)进行鉴定,结果成功分离到5株REV分离株GX18NNR1、GX19YLR1、GX19NNR1、GD20R1和GX20YLR1;对分离株的全基因组进行扩增、序列测定及分析,结果显示其全基因组长度为8284 bp-8416 bp。全基因组序列比对发现,分离株GX20YLR1与REV美国鸭源经典毒株SNV相似性最高(nt:99%,aa:96.5%),而与疫苗分离株FA的相似性最低(nt:90.5%,aa:88.4%);其余4株与参考株104865的相似性最高(nt>99.8%,aa>97.4%),与ATCC-VR775的相似性最低(nt<91.6%,aa<87.1%);各段基因序列的对比显示,REV较为保守,其中以pol最为保守。基于REV全基因及分段基因的序列构建遗传进化树分析发现,REV分为3个大的进化分支,即REV的三个亚型(Ⅰ型、Ⅱ型和Ⅲ型),4个分离株GX18NNR1、GX19YLR1、GX19NNR1和GD20R1属于REVⅢ型,为全球范围的主要流行基因型,而分离株GX20YLR1则属于REVⅡ型。基于贝叶斯(Bayesian)方法对REV毒株进行时间、种群动态、地理和宿主四个方面的分析,发现REV在中国的遗传多样性变化呈现一个先升后趋平缓的态势,且其近十年来的遗传多样性并没有明显的增加。优势基因Ⅲ型毒株早期是由美国传入中国,其中黑龙江是中国大陆最先开始出现的省份,并于20世纪90年代开始在中国流行。证实鸡是REV的自然宿主,其他宿主的感染最可能来源于鸡。本课题应用常规的鸡胚成纤维细胞(chicken embryo fibroblast,CEF)与实验室常用细胞系DF1对REV进行了生长曲线的试验。结果发现REV在细胞中的病毒载量与其感染时间均成正相关,其中DF1感染组的病毒增殖速率较为平缓,其病毒载量在4 dpi(days post-infection,dpi)开始到达平台期,5 dpi达到最高水平。CEF感染组的病毒增殖速率较快,并且在3 dpi开始到达平台期,到6 dpi达到最高水平。CEF感染组的病毒载量显著高于相同时间点的DF1感染组。课题从广西三家地方黄鸡种鸡公司采集种鸡血清样品584份,应用EILISA试剂盒检出361份REV抗体阳性样品,平均阳性率为61.82%,其中DGGX11公司的阳性率最高(84.15%),DGGX22公司的阳性率最低(8.89%);并随机收集同群181份抗体阳性和206份抗体阴性鸡的外周血淋巴细胞和血浆,接种CEF进行细胞培养分离病毒,通过PCR检测,结果均未分离到REV。课题成功分离了到5株REV,通过病毒全基因组序列的测定与分析,确定了我国流行的主要基因型,并揭示了我国REV的来源、传播路径及其时间,发现REV的基因组较为保守,近十年来并没有明显的遗传多样性变化。确定了REV培养的最佳细胞和最适合的培养时间。证明了种鸡的REV抗体阳性与病毒的存在并无相关性。更多还原

【Abstract】 Reticuloendotheliosis(RE) is a pathological syndrome caused by reticuloendotheliosis virus(REV),which mainly affects chickens,turkeys,ducks and other birds.REV belongs to retrovirus,which is one of the pathogens that cause tumorous diseases in poultry.Its transmission modes mainly include horizontal transmission,vertical transmission and vaccine transmission.On the one hand,the immune suppression caused by REV can result in a decline in the diseases resistance of the host,which can easily lead to the secondary infections of other diseases.On the other hand,it can result in a reduction in the level of the vaccination response and that may cause vaccine breaks,in severe cases and bring greater economic losses to the poultry industry.REV rarely causes the clinical disease alone under natural conditions,mainly in the form of co-infections.In the study,samples collected from clinical suspected tumor disease cases during 2018-2020,were used for the virus isolation via the cell cultures,and the subsequently detected by PCR and immunofluorescent assay(IFA).And totally5 REV strains were successfully isolated and named as GX18NNR1,GX19YLR1,GX19NNR1,GD20R1 and GX20YLR1,respectively.The full-genome sequences analysis was performed on these isolates.The results showed that the full-genome of these 5 isolates were 8284 bp-8416 bp in length and the isolate GX20YLR1 had the highest similarity with the REV American duck-derived classic strain SNV(nt: 99%,aa: 96.5%),and had the least similarity with the vaccine isolate FA(nt: 90.5%,aa: 88.4%).The remaining four strains had the highest similarity with the reference strain 104865(nt>99.8%,aa>97.4%),and had the least similarity with the ATCC-VR775 strain(nt<91.6%,aa<87.1%;The comparison of gene sequences showed that each gene segment of REV was relatively conservative and the pol gene is the most conservative.Based on the sequence analysis of the full-genome and the gene segments of REV,REV strain could be classified into three major evolutionary branches,namely REV subtypes I,II and III respectively.The four isolates GX18NNR1,GX19YLR1,GX19NNR1 and GD20R1 belong to REV subtype III,which was the major epidemic genotypes worldwide,and the isolate GX20YLR1 belongs to REV subtype II.Based on the Bayesian method to analyze REV strains in terms of time,population dynamics,geography and host,it was found that the genetic diversity of REV in China showed a trend of rising first and then leveling off,and its genetic diversity has not been increased significantly in the past ten years.The REV subtype III was introduced into China from the United States in the early stage,and Heilongjiang was the first province to be detected in China’s mainland,and it became popular over China from the 1990 s.It was confirmed that chicken was the natural host of REV,and the infection of other birds was most likely to be originated from chicken.In this study,the growth curve of REV was tested by the culturing on the conventional chicken embryo fibroblasts(CEF)and the common laboratory cell line DF1,respevtively.The results showed that the viral load of REV in the cell cultures was positively correlated with its infection time.The viral propagation of the DF1 culture group was relatively slow,and the viral load began to reach a plateau at 4 dpi and the highest level at 5 days post-infection(dpi).The CEF culture group had a faster speed of the virus propagation,and reached a plateau at 3 dpi and the highest level at 6 dpi.The viral load of the CEF culture group was significantly higher than that of the DF1 culture group at all of the same time points.In the study,584 serum samples were collected from three local Yellow-chicken breeding companies in Guangxi and were detected by using the REV antibody detection EILISA kit.361 of the samples were positive and showed a mean positive rate of 61.82%,of which company DGGX11 had the highest positive rate of 84.15%,while company DGGX22 had the lowest of8.89%;Peripheral blood lymphocytes and plasma were collected from 181 antibody positive and 206 antibody negative chickens,and inoculated on to the CEF cell cultures for the isolation of REV,and the results of the PCR detection showed that no REV was isolated.In summary,5 REV strains were successfully isolated from the clinical cases,and the subtypes prevailing in China were identified by the full-genome sequencing and analysis.And the origin,the transmission route and the time of REV in China have also been revealed.The genome of REV is relatively conservative,and there has not obviously been change in the genetic diversity during the past decade.The optimal cells and the optimal culture time for REV were determined.It was proved that there was no correlation between the positive of the serum antibody and the existence of REV.更多还原