【作者】 张新涛；

【导师】 孔宪刚；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2013， 博士

【摘要】 新城疫（Newcastle disease, ND）是由新城疫病毒（Newcastle disease virus, NDV）引起的急性、高度接触性的禽类传染病，给世界养禽业造成极大的经济损失。目前，疫苗接种仍然是预防和控制该病普遍采用的方法。NDV4是广泛应用于东南亚和非洲等热带亚热带地区的一种耐热疫苗株，具有耐热性和免疫持续期长等优点。NDV4-C株是V4疫苗株在SPF鸡胚上连续传代筛选获得的一株耐热无毒株，目前NDV4毒株的研究相对滞后，一个主要的原因是反向遗传操作系统平台尚未建立，相应的耐热性和免疫原性研究也鲜有报道。为此，本研究以NDV4-C株为研究对象，在对NDV4-C株全基因组序列系统解析的基础上，首次建立了分别以T7启动子和CMV启动子的NDV4-C株反向遗传操作系统，拯救病毒rNDV4-C （T7）和rNDV4-C （CMV）的耐热性和生物学特性均与亲本毒株相似。同时，为了评估NDV4-C株作为载体的可行性，构建了表达增强型绿色荧光蛋白的重组新城疫病毒rNDV4-eGFP，其在SPF鸡胚上生长特性与亲本病毒相似，重组病毒能够稳定表达增强型绿色荧光蛋白至少15代，结果表明，rNDV4-C具有作为病毒载体应用的潜力。聚合酶蛋白对副粘病毒的耐热性作用已有报道，为了调查聚合酶蛋白对NDV4-C株耐热性的作用影响，我们分别构建了以耐热株NDV4-C和不耐热株LaSota为骨架替换聚合酶基因的嵌合病毒株rNDV4-La-L和rLaSota-V4-L，测定其耐热性和病毒生物学特性，结果表明以NDV4-C为骨架的嵌合病毒耐热性降低，生长滴度低于NDV4-C株的生长滴度。反之，以LaSota为骨架嵌合NDV4-C株聚合酶基因的嵌合病毒耐热性有一定程度的提高，生长滴度也较LaSota的滴度升高。此外，用表达荧光素酶的微基因组系统检测聚合酶蛋白活性，结果表明NDV4-C株的聚合酶蛋白具有更高的活性。因此聚合酶蛋白影响了NDV4-C的耐热性。当前的NDV疫苗免疫可有效控制新城疫疫情，但不能够阻止新城疫病毒的排毒、传播和感染。V4疫苗株与LaSota、Clone30和Hitchner B1等疫苗株相比，免疫诱导的体液免疫抗体水平低，但粘膜免疫水平较高。为了探讨能否在保持NDV4-C耐热性的基础上提高其免疫应答水平，在前两项工作的基础上，我们构建并拯救了以NDV4-C为骨架嵌合当前流行的基因Ⅶ型强毒株Go/CH/HLJ/LL01/08（LL01/08）囊膜蛋白的两株嵌合病毒rNDV4-HNLL01和rNDV4-MuF/HNLL01。相对于亲本病毒LL01/08，嵌合病毒保持了低毒力，生长略有延迟，耐热性略有下降但保持了耐热性。病毒株rNDV4-C、rNDV4-HNLL01、rNDV4-MuF/HNLL01和疫苗株LaSota以1×10⁶EID₅₀剂量滴鼻免疫两周龄SPF鸡，能够诱导不同程度的体液免疫和粘膜免疫水平，免疫两周后以1×10⁶EID₅₀的剂量强毒株LL01/08攻毒。动物实验结果表明，嵌合病毒诱导产生的IgG抗体水平并没有升高，但在血清和十二指肠产生的IgA抗体水平较亲本病毒rNDV4-C显著提高。嵌合病毒能够完全保护SPF鸡抵抗致死剂量强毒的攻击，更重要地是嵌合病毒rNDV4-MuF/HNLL01免疫组的排毒量较NDV4-C免疫组有显著的下降。结果表明嵌合病毒rNDV4-MuF/HNLL01能够作为防控当前流行NDV的候选疫苗株。更多还原

【Abstract】 Newcastle disease (ND), caused by Newcastle disease virus (NDV), is an acute and highlycontagious disease which affects many domestic and wild avian species and causes severe economiclosses to the poultry industry in the world. Currently, vaccination remains a primary strategy for NDcontrol.NDV4vaccine strain is being widely used in the tropical and sub-tropical countries of SoutheastAsia and Africa due to its thermostability and long-lasting immunity. NDV4-C is an avirulent andthermostable strain which is screened by passaging in specific-pathogen-free (SPF) chicken eggs. Todate, the reverse genetics of NDV4strain is unavailable, which accounts for the limited research ofNDV4strain. Based on the systemic sequence analysis of NDV4-C, in this study, we established thereverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery ofNDV4-C was achieved by using either T7RNA polymerase or cellular RNA polymerase II to drivetranscription of the full-length virus antigenome from cloned cDNA. The recovered viruses rNDV4-C(T7) and rNDV4-C (CMV) showed similar thermostability and biological properties as the parentalstrain NDV4-C. The potential of rNDV4-C (T7) to serve as a viral vector was assessed by generating arecombinant virus, rNDV4-eGFP, which expressed enhanced green fluorescent protein (eGFP). TherNDV4-eGFP could stably carry and express eGFP for at least fifteen passages. These results indicatedthat the rNDV4-C (T7) has the potential to serve as a live vaccine vector.It is reported that the large protein is associated with the temperature-sensitive in theparamyxovirus. Here, we engineered these chimeras rNDV4-La-L and rLaSota-V4-L by exchanging theL gene between the thermostable NDV4-C strain and the thermolabile LaSota strain to evaluate the roleof large protein in NDV4-C thermostability. Thermostability of chimeric viruses was examined bydetermining the infectious titre after incubation for different periods of time at55°C. The characteristicof chimeric viruses was also evaluated in embryonated SPF chickens and eggs. Chimera rNDV4-La-Lposses decreased thermostability and lower viral titre, and rather than vise versa, suggesting that the Lgene contributes to the thermostability of NDV4-C. Furthermore, the in vitro replication assay with aminigenome system expressing luciferase showed that the L activity of NDV4-C was higher than that ofLaSota, which contributes to the thermostability of NDV.Routine and/or intensive vaccinations have been shown to be effective in preventing disease andmortality but not in preventing viral shedding, transmission, and infection, NDV4vaccine inducedlower humoral immune response antibody level than the LaSota, Clone30, and Hitchner B1, but couldinduce good mucosal immune response. We engineered two chimeras, rNDV4-HNLL01andrNDV4-MuF/HNLL01, which combined the thermostability of the NDV4-C strain with the goodimmunogenicity of the velogenic sub-genotype VIId Go/CH/HLJ/LL01/08(LL01/08) strain.Thermostability of these chimeras was confirmed by determining the infectious titre after incubation for different periods of time at55°C. Both chimeras fulfilled the criteria for lentogenic virus.Two-week-old SPF chickens, which were inoculated by the oculonasal route with each of thesechimeras, the rNDV4-C strain, or the widely used NDV vaccine strain LaSota, respectively, produceddifferent levels of humoral IgG and mucosal IgA antibodies. The IgG antibody level induced by LaSotawas higher than that induced by the other groups, while these chimeras induced higher levels of IgAantibody than that of rNDV4-C in sera and dodecadactylon. The animal experiments demonstrated thatall vaccinated groups were fully protected from morbidity and mortality. Importantly, bothrNDV4-MuF/HNLL01-and LaSota-vaccinated chickens showed a significant reduction in viralshedding in the cloaca compared to chickens of the other vaccinated groups. In summary, our datasuggest that the lentogenic and thermostable rNDV4-MuF/HNLL01strain, which induces enhancedmucosal immune response, could be a promising vaccine candidate for the control of current NDepidemics.更多还原