我国地方品系鸡群中ALV的流行病学调查及ALV-K生物学特性的研究

发布时间：2018-05-09 20:24

本文选题：禽白血病病毒 + 遗传多样性　；参考：《山东农业大学》2017年博士论文

【摘要】：禽白血病病毒(Avian leukosis viruses,ALV)属于反转录病毒科(Retroviridae)α反转录病毒属(Alpharetrovirus)的一类病毒。ALV感染不仅诱发多种肿瘤性疾病,还可引起大多数感染鸡的亚临床症状,如产蛋下降、免疫抑制或生长迟缓。根据病毒中和试验和gp85的特性,迄今为止将ALV分为A-J十个亚群,以及新鉴定的K亚群。自20世纪90年代以来,禽白血病特别是J亚群禽白血病病毒(ALV-J)给我国的养鸡业造成了重大的经济损失,严重危害我国白羽肉鸡、蛋用型鸡、黄羽肉鸡和固有的地方品种鸡等各种类型鸡的健康发展。先天垂直感染是ALV的重要传播方式。由于宿主对ALV难以产生有效免疫应答反应,加之ALV基因组变异频率较快,至今为止,尚未有商品化的疫苗可用。由于ALV亚群的多样性,感染和传播的复杂性,给我国的养鸡业造成了重大的经济损失,本研究对我国不同鸡群进行了ALV的流行病学调查,并对公鸡精液在ALV传播中作用进行了系统的研究;通过研究不同佐剂及免疫增强剂对ALV-J gp85重组蛋白诱导抗体的产生,从而加速和辅助我国ALV的净化;对新分离到的ALV-K全基因组做了系统的分析,并研究了ALV-K的原型株JS11C1对SPF鸡的致病性;本研究利用反向遗传操作技术,构建JS11C1株的感染性克隆,并在此基础上首次构建了env(J)-LTR(K)和env(K)-LTR(J)的嵌合感染性克隆,借以探究env基因在ALV-K的致病性中所发挥的作用。1.我国不同鸡群中ALV的流行病学调查及其准种分析本研究对我国12个不同地方品系鸡群中ALV进行了分离鉴定,共分离鉴定到14株ALV,对这些分离毒株gp85基因进行了克隆测序和亚型鉴定,结果显示有2株ALV-A,1株ALV-B,8株ALV-J和3株ALV-K。其中,2012年从山东某地方品系鸡群发生疑似为血管瘤的肿瘤病例中分离到5株J亚型ALV-J,定名为SDLY1201-SDLY1205。分别扩增五株ALV-J的gp85基因,并对五个毒株准种多样性进行分析和比较。结果显示:五个毒株其gp85核苷酸长度分别为921bp、921bp、924bp、918bp、912bp,其不同克隆之间的氨基酸同源性分比为99.3%-100%、99.3%-100%、99.4%-100%、98.4%-100%、99.0%-100%,其中序列完全相同的克隆比例分别为13/20、17/20、17/20、9/18、16/19,代表了五个毒株中优势准种的比例,对五个毒珠优势准种的gp85比较显示其氨基酸同源性为89.2%-92.5%。本研究证实即使在同一时期同一鸡群感染的不同个体中,ALV-J野毒株准种也存在巨大的差异,特别是其中优势准种发生了改变。2.公鸡精液在ALV传播中的作用长期以来,对ALV感染鸡群过程中公鸡的作用特别是对后代的传播作用一直没有得到明确的阐述。本研究通过鸡胚静脉接种J亚群禽白血病病毒(ALV-J),获得持续病毒血症的公鸡,并将这些公鸡的精液人工授精给产蛋期的母鸡,1周后收集种蛋并孵化出雏鸡。结果显示,两只受精的母鸡在4-5w时产生ALV-J的抗体,在6只母鸡的泄殖腔中检测到p27抗原,母鸡产的26个鸡蛋中检测到3个蛋清P27为阳性。此外,对12只母鸡进行病毒分离时发现,在授精后6w均没有检测到病毒血症的存在。然而,1周龄时从34只后代的雏鸡中的1只雏鸡中分离到ALV-J,其与从公鸡精液样品中分离到的ALV-J的gp85基因的序列同源性为98.4%-99.2%。我们的研究表明,ALV-J可以通过公鸡的精液的人工授精感染母鸡并且垂直传播给子代的雏鸡。这一发现对于改进和完善种鸡场ALV净化程序有重要的指导作用。3.不同佐剂及免疫增强剂对ALV-J gp85重组蛋白诱导抗体产生的研究原核表达的ALV-J gp85囊膜蛋白能够诱导SPF鸡产生抗体,但抗体阳性率和抗体水平均较低,本研究观察了免疫增强剂松花粉多糖(TPPPS)联合不同佐剂(CPG、YF01)对增强ALV-J gp85囊膜蛋白免疫后抗体水平的影响。结果显示,单一gp85重组蛋白只能诱导少数鸡产生抗体,且产生的抗体效价较低、维持时间较短;而YF01或Cp G佐剂分别联合重组蛋白能够使大部分免疫鸡产生抗体,且效价较高,当两种佐剂再联合使用免疫增强剂泰山松花粉多糖后能够使产生的高效价抗体维持时间更长。对ALV-J抗体水平较高的4组收集种蛋进行孵化检测母源抗体,人工攻毒后观察母源抗体对雏鸡的保护效果。结果发现发现添加免疫增强剂TPPPS组产生的母源抗体水平较没有添加组高,且对雏鸡具有较高的保护效力,与对母体的使用效果一致。通过本研究我们可以发现泰山松花粉多糖联合使用YF01和CPG佐剂可以增强gp85重组蛋白的免疫原性,产生较好的免疫保护作用,这将为开发更加有效的ALV-J亚单位疫苗提供有力实验依据,高效亚单位疫苗的研制也将为J亚群禽白血病的净化提供更大的帮助。4.K亚群ALV的全基因组分析及其致病性研究本研究从我国地方品系鸡群中分离鉴定到了3株ALV-K,并对其做了全基因组分析,结果表明,这3株ALV-K与之前已发表的ALV-K毒株位于一个单独的分支,与鸡群中其他已知亚群的ALV均不在同一分支中。其中,与SDAUAK-11、SDAUAK-12和SDAUAK-13的全基因序列同源性最高的分别为中国广东分离株GD14LZ,GDFX0601,江苏分离株JS11C1,与其它ALV-K毒株的同源性分别为90.8%-97.7%,90.8%-97.7%,91.2%-97.3%。为研究ALV-K的致病性,将2011年分离自中国某地方品系鸡的ALV-K原型株JS11C1株分别采用卵黄囊接种、鸡胚静脉接种和1日龄腹腔接种三种方式感染SPF鸡,系统记录分析了感染鸡群的病毒血症和抗体产生动态、对感染鸡群体重增重的抑制以及感染鸡群对疫苗免疫应答的影响等方面,以对JS11C1的致病性进行全面评估。结果表明,当通过卵黄囊接种和鸡胚静脉接种时,JS11C1可以诱发感染鸡的持续病毒血症,抑制感染鸡群的体重增重和对疫苗的免疫应答,并可诱发一定比例的淋巴细胞肿瘤。相对于其他亚群ALV,JS11C1的传播效率和致病性相对较弱。本研究系统研究了JS11C1株对SPF鸡的致病性,为下一步研究其致病机制和科学防控奠定了基础。5.env基因在ALV-K致病作用中的研究在所有亚群ALV中,ALV-J致病性最强,而ALV-K与ALV-A、B等类似。为了阐明env基因和LTR片段在ALV-J与ALV-K致病性差异的作用,本研究构建和比较了ALV-J野毒株SDAU1005和ALV-K野毒株JS11C1及其重组嵌合病毒renv(J)-LTR(K)和renv(K)-LTR(J)的感染性克隆的生物学特性。在这4个感染性克隆病毒中,r SDAU1005(ALV-J)在DF-1细胞上的复制能力最强,r JS11C1最弱,而2个重组病毒介于二者之间,其强度次序r SDAU1005renv(K)-LTR(J)renv(J)-LTR(K)r JS11C1。此外,在与致病性相关的对增重、对法氏囊和胸腺发育及疫苗免疫后抗体反应的抑制作用上,这一比较结果表明,env基因和LTR片段在影响ALV-J与ALV-K之间致病性差异中起着重要作用,其中env基因的作用似乎大于LTR片段。本研究发现ALV-K在细胞上的复制水平显著低于ALV-J及其他亚群,这也为改进净化程序中的检测方法和判定标准提供了新的科学数据。
[Abstract]:Avian leukemic virus (Avian leukosis viruses, ALV) belongs to the retrovirus family (Retroviridae) alpha retrovirus genus (Alpharetrovirus), a class of viral.ALV infection not only induces a variety of tumor diseases, but also causes subclinical symptoms of most infected chickens, such as egg drop, immunosuppression or growth retardation. According to the virus neutralization test And the characteristics of gp85 have so far divided ALV into ten subgroups of A-J and a newly identified K subgroup. Since 1990s, avian leukaemia, especially the J subgroup of avian leukemic virus (ALV-J), has caused major economic losses to the poultry industry in our country, seriously endangering China's white feathered chicken, egg type chicken, Huang Yu broiler and inherent local breed chicken. The healthy development of various types of chickens. Congenital vertical infection is an important mode of transmission of ALV. Because the host is difficult to produce effective immune response to ALV, and the mutation frequency of the ALV genome is fast, so far, no commercialized vaccine is available. Because of the diversity of the ALV subgroup, the complexity of infection and transmission, the chicken industry is made in our country. It has been a major economic loss. This study conducted a ALV epidemiological survey on different chicken groups in China, and systematically studied the role of the cock semen in the transmission of ALV. Through the study of different adjuvants and immune enhancers, the production of antibodies induced by the recombinant protein of ALV-J gp85 was studied, thus accelerating and assisting the purification of ALV in China; The whole genome of ALV-K has been systematically analyzed, and the pathogenicity of ALV-K's prototype strain JS11C1 on SPF chicken is studied. In this study, the infective clones of the JS11C1 strain were constructed by using the reverse genetic manipulation technique, and on this basis, a chimeric infection clone of env (J) -LTR (K) and env (K) -LTR was constructed for the first time. The role of the disease in the epidemiological investigation of ALV in different chicken groups in China and its quasi species analysis, the ALV was isolated and identified in 12 different local chicken groups, 14 strains of ALV were isolated and identified, and the gp85 gene of these isolates was cloned, sequenced and identified. The results showed that 2 strains of ALV-A, 1 ALV-B, 8, 8, 8, 8. Strain ALV-J and 3 strains of ALV-K., 5 J subtypes ALV-J were isolated from a local strain of chicken group in Shandong in 2012. The gp85 gene of five strains of ALV-J was amplified by SDLY1201-SDLY1205., and the ratio of the five strains of quasi species diversity was analyzed and compared. The results showed that the gp85 nucleotide length of five strains. 921bp, 921bp, 924bp, 918bp, 912bp, the amino acid homology ratio between the different clones is 99.3%-100%, 99.3%-100%, 99.4%-100%, 98.4%-100%, 99.0%-100%, and the proportion of the identical sequences is 13/20,17/20,17/20,9/18,16/19, representing the proportion of the dominant species in the five strains, and the g of the five virulent bead predominant species. P85 showed that its amino acid homology was 89.2%-92.5%.. This study confirmed that even among the different individuals infected with the same chicken group at the same time, there were significant differences in the ALV-J wild strains, especially the dominant species changed the role of the.2. Rooster semen in the ALV transmission for a long time, for the rooster in the ALV infected chicken group. The effect of the effect, especially on the transmission of offspring, has not been clearly stated. This study was inoculated with the J subgroup of avian leukemic virus (ALV-J) from the chicken embryo vein to obtain the rooster of continuous viremia, and the semen of the cocks was artificially inseminated to the hens at the egg laying period. After 1 weeks, the eggs were collected and hatched out of the chicks. The results showed that two fertilized chickens were fertilized. The hens produced ALV-J antibodies at 4-5W, detected p27 antigen in the cloaca of 6 hens, and 3 egg white P27 were positive in 26 hen's eggs. Furthermore, when the virus was separated from 12 hens, none of the virus was detected in 6W after the insemination. However, 1 of the 34 chicks were 1 weeks old. ALV-J is isolated from the chicks, and the sequence homology of the gp85 gene of ALV-J isolated from the sample of the cock semen is 98.4%-99.2%.. Our study showed that ALV-J could infect hens through the seminal seminal insemination of the cock and propagate the chicks vertically to the progeny of the chicken. This discovery has a great effect on improving and improving the ALV purification procedure of the chicken farm. The effect of different adjuvant and immuno enhancers on the production of ALV-J gp85 recombinant protein induced antibodies by.3. and ALV-J gp85 capsule protein can induce antibody in SPF chicken, but the antibody positive rate and antibody level are low. This study observed the combination of immune enhancement agent pine pollen polysaccharide (TPPPS) combined with different adjuvant (CPG, YF01). The results showed that the antibody level of the ALV-J gp85 capsule protein was enhanced. The results showed that the single gp85 recombinant protein could only induce a few chickens to produce antibodies, and the antibody titer produced was lower and the maintenance time was shorter, while the combination of YF01 or Cp G adjuvant combined with recombinant protein could make most of the immunized chickens produce antibodies, and the titer was higher when two adjuvants were reproduced. The combined use of the immunosuppressive agent of Taishan pine pollen polysaccharide can keep the high effective antibody for longer. 4 groups of eggs collected from the high level of ALV-J antibody were incubated to detect the mother source antibody, and the protective effect of the mother antibody on the chicken was observed after the attack. The results showed that the parent source produced by the immune enhancement agent TPPPS group was found. In this study we can find that the combination of Taishan pine pollen polysaccharide and the use of YF01 and CPG adjuvant can enhance the immunogenicity of the gp85 recombinant protein and produce a better immune protective effect, which will be more effective for the development of A. The LV-J subunit vaccine provides a powerful experimental basis. The development of high efficiency subunit vaccine will also provide greater help for the purification of the J subgroup of avian leukosis. The whole genome analysis and pathogenicity of the.4.K subgroup ALV and its pathogenicity study, 3 strains of ALV-K were isolated and identified from the local chicken flock of our country, and the whole genome analysis was made. The 3 strains of ALV-K and the previously published ALV-K strains are located in a separate branch and are not in the same branch with the other known subgroups of the chicken group. Among them, the highest homology of the whole gene sequences of SDAUAK-11, SDAUAK-12 and SDAUAK-13 are GD14LZ, GDFX0601, Jiangsu isolates, and other ALV-K venom of Guangdong isolates in China. The homology of the plant was 90.8%-97.7%, 90.8%-97.7% and 91.2%-97.3%., respectively, to study the pathogenicity of ALV-K. In 2011, the JS11C1 strains of ALV-K prototype strains isolated from a local chicken in China were inoculated with yolk sac, chicken embryo vein inoculation and 1 day old abdominal inoculation were infected with three ways of SPF chickens. The virus infection of the infected chickens was recorded and analyzed systematically. The results showed that when the egg yolk sac was inoculated and the chicken embryo was inoculated, JS11C1 could induce the continuous viremia of the infected chicken and inhibit the weight of the infected chicken group. The results showed that JS11C1 was inoculated with the egg yolk sac and the chicken embryo vein. Weight gain and immune response to vaccines can induce a certain proportion of lymphocyte tumors. Relative to other subgroups ALV, the transmission efficiency and pathogenicity of JS11C1 is relatively weak. This study systematically studied the pathogenicity of JS11C1 strains to SPF chickens, and laid the foundation for the pathogenesis and scientific control of the.5.env gene for the pathogenesis of ALV-K in the next step. In all subgroup ALV, the pathogenicity of ALV-J is the strongest, while ALV-K is similar to ALV-A and B. In order to clarify the role of env gene and LTR fragment in the pathogenicity of ALV-J and ALV-K, this study constructs and compares the infectivity of SDAU1005 and ALV-K wild strains and their recombinant inlay viruses. In the 4 infectious clones, R SDAU1005 (ALV-J) has the strongest replicative ability on DF-1 cells and the weakest R JS11C1, while the 2 recombinant viruses are between two, and the intensity sequence R SDAU1005renv (K) -LTR (J) renv, in addition to the pathogenicity, and the development of the bursa and thymus. The results showed that the env gene and the LTR fragment played an important role in influencing the pathogenicity difference between ALV-J and ALV-K, in which the role of the env gene seemed to be greater than that of the LTR fragment. This study found that the level of ALV-K replication on the cell was significantly lower than that of ALV-J and other subgroups, which was also modified. The detection methods and criteria in the purification process provide new scientific data.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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