坦布苏病毒SDS-70株弱毒苗的制备及安全性与免疫效力的初步评价

发布时间：2018-05-26 16:09

本文选题：坦布苏病毒感染 + 弱毒疫苗　；参考：《山东农业大学》2017年硕士论文

【摘要】：鸭坦布苏病毒感染(TMUV infection)是2010年我国新发的一种急性传染性疾病。TMUV属于黄病毒科,黄病毒属,恩塔亚病毒群。TMUV可感染危害多个品种的鸭,主要造成5-15日龄的雏鸭食欲减退、生长缓慢和神经症状,死淘率可达5%-10%,育成鸭感染后临床症状轻微,死亡率较低,产蛋鸭的产蛋率大幅下降,产蛋率从90%降至10%以下,该病的发生给我国养禽业造成了巨大的经济损失。免疫预防是防控TMUV感染的重要措施之一,本研究利用实验室分离到的TMUV SDS株进行传代致弱,利用致弱毒株SDS-70株对雏鸭进行免疫,对其免疫保护效果进行了评价。本研究对从山东省某种鸭场分离的一株TMUV,采用SPF鸭胚和SPF鸡胚进行交替传代成功致弱该毒株。对获得的不同代次的传代病毒进行致弱效果分析,对不同代次的弱毒株进行免疫效果比对,结果显示SDS-70具有较好的免疫原性。对筛选到的SDS-70传代毒株进行免疫效力的评价。7日龄樱桃谷鸭(无TMUV母源抗体),每只腿肌注射102.46EID50的SDS-70的弱毒疫苗,20日龄进行第2次免疫,在2免后一周,利用同源强毒SDS-10对免疫组和对照组分别进行攻毒,攻毒剂量为1mL(102.9ELD50)。免疫后,观察各组精神状态并记录;定期采血检测抗体水平;定期采集免疫组与同居感染组的泄殖腔棉试子,利用荧光定量RT-PCR方法进行排毒情况的检测;定期剖杀,采集各组织器官进行带毒情况检测与病理组织学研究。结果免疫组和对照组均精神状态良好,无明显的临床症状和病理变化,无发病和死亡情况。病理组织学检测结果显示,脾脏出现出血,后期出血减轻,心肌出现轻微水肿现象,其余各组织器官均无明显的病理学损伤,结果表明SDS-70对雏鸭无致病性,免疫性和安全性良好。攻毒试验结果显示SDS-70免疫过的樱桃谷鸭对强毒SDS-10的攻击具有100%的临床保护力。对SDS-70弱毒疫苗进行纯净性检测,包括支原体、细菌和外源病毒的检测,检测结果均为阴性,说明该弱毒疫苗纯净。将SDS-70在鸭体内连续传代5次,各代次的试验鸭均无临床症状,精神良好,剖检内脏组织器官无病理变化,收集各个代次的病料进行RT-PCR检测,结果为阳性,表明SDS-70具有良好的稳定性,体内传代后毒力无返强情况。对20、40、60、80和100代次的传代毒株进行全基因组序列分析,与SDS原始毒株全基因组序列相比较,结果20、40、60、80和100代5个代次的传代病毒株共出现了33碱基突变,包括18个导致氨基酸发生变化的位点和15个中性突变点。对E蛋白氨基酸序列上的N-连接的潜在糖基化位点进行预测,根据预测结果可以看出,在SDS-80代次时在E基因上出现了一个糖基化位点的缺失,根据氨基酸变化分析可知,该糖基化位点的缺失是由于E蛋白154位上N变为H,改变了糖基化位点的固有结构,导致该位点消失。据此推测病毒毒力的改变可能是碱基的突变所导致的。综上所述,SDS-70弱毒疫苗株对樱桃谷鸭安全性、纯净性与免疫效力较好,并且毒力稳定,可以作为TMUV弱毒疫苗候选株。
[Abstract]:Duck tanboa virus infection (TMUV infection) is a new acute infectious disease in China in 2010,.TMUV belongs to the family of the yellow virus family, the yellow virus and the ntaya virus group.TMUV can infect many varieties of ducks, which mainly cause anorexia, slow growth and neurological symptoms of 5-15 days old ducks, and the death rate can reach 5%-10%. The clinical symptoms are mild, the mortality rate is low, the laying rate of laying ducks has fallen sharply, the egg production rate has dropped from 90% to less than 10%. The occurrence of the disease has caused great economic loss to the poultry industry in China. Immune prevention is one of the important measures to prevent and control TMUV infection. This study uses the TMUV SDS strains isolated from the laboratory to make a weak passage and make use of the weak poison. The strain SDS-70 strain was immune to the ducklings, and the effect of their immune protection was evaluated. In this study, a strain of TMUV isolated from a duck farm in Shandong Province, using SPF duck embryo and SPF chicken embryo, was successfully induced by alternate passages. The results showed that SDS-70 had good immunogenicity. The evaluation of the immune efficacy of the screened SDS-70 passages was.7 day old Cherry Valley ducks (no TMUV mother antibody), 102.46EID50 SDS-70 vaccine of 102.46EID50 in each leg muscle, and second times of immunization at 20 days of age, and the same homologous strong toxic SDS-10 was used in the immune group one week after 2 Exempts. In the control group, the attack dose was 1mL (102.9ELD50). After immunization, the mental state of each group was observed and recorded; the level of antibody was detected regularly; the cloaca cotton specimens of the immune group and the cohabiting infection group were collected regularly, and the fluorescence quantitative RT-PCR method was used to detect the detoxification. Results the immune group and the histopathological study showed that both the immune group and the control group had good mental state, no obvious clinical symptoms and pathological changes, no disease and death. The results of histopathological detection showed that the spleen appeared bleeding, late bleeding, slight edema in the myocardium, and no obvious pathological pathology in the other tissues and organs. The results showed that SDS-70 had no pathogenicity, immunity and safety. The results of the attack test showed that the SDS-70 vaccinated Cherry Valley Duck had 100% clinical protection against the attack of strong toxic SDS-10. The purity test of the SDS-70 attenuated vaccine, including the detection of mycoplasma, fine bacteria and exogenous virus, was negative. It showed that the vaccine was pure. SDS-70 was passed on the duck for 5 times in the duck. All the ducks in each generation had no clinical symptoms, the spirit was good, and the visceral organs were examined without pathological changes. The samples of each generation were collected for RT-PCR detection, and the results were positive, indicating that the SDS-70 had good stability and the virulence of the body after the passage was not strong. The whole genome sequence analysis of 20,40,60,80 and 100 generation passages was carried out and compared with the whole genome sequence of the original SDS strain. The results showed that the 33 base mutations in the 20,40,60,80 and the 100 generation of 5 generations of the generation virus strains, including 18 loci causing the change of amino acids and 15 neutral points of mutation, and N on the amino acid sequence of the E protein. - the potential glycosylation site of the connection was predicted. According to the predicted results, a glycosylated site was absent at the SDS-80 generation. According to the analysis of amino acid changes, the deletion of the glycosylation site was due to the change of the intrinsic structure of the glycosylation site in the 154 position of the E protein, which changed the structure of the glycosylation site, resulting in the elimination of the site. It is concluded that the changes in virulence of the virus may be caused by the mutation of the base. To sum up, the SDS-70 attenuated vaccine strain is safe, pure and immune to Cherry Valley duck, and has a stable virulence. It can be used as a candidate for TMUV attenuated vaccine.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

【参考文献】