【作者】 孙静；

【导师】 鲁兴萌； 李建荣； 于涟；

【作者基本信息】 浙江大学 ， 预防兽医学， 2014， 博士

【摘要】 禽偏肺病毒(Avian metapneumovirus,aMPV),又称火鸡鼻气管炎病毒(Turkey rhinotracheitis virus, TRTV),早期称为禽肺病毒(Avian pneumovirus, APV),该病毒最早于1978年从南非发病火鸡中分离得到,之后在世界范围内蔓延传播。aMPV是副粘病毒科、肺病毒亚科、偏肺病毒属成员,有囊膜,属单节段负链RNA病毒。病毒mRNA的加工处理对病毒基因的表达与病毒基因组的复制至关重要,mRNA的加工处理过程包括加帽、甲基化和聚腺苷酸化。其中加帽和甲基化对mRNA的稳定、翻译及基因表达最为重要,这些加工过程需要一系列的酶促反应,AMPV复制过程中的上述一系列酶促反应均由其L蛋白负责完成。对于单股负链R.NA病毒而言,其L蛋白包括六个高度保守的功能域,分别为CRⅠ、CRⅡ、CRⅢ、 CRIV、 CRV和CRVI功能域。单股负链RNA病毒具有独特的mRNA甲基化机制。生物信息学分析显示,aMPV L蛋白CRVI功能域具有guanine-N-7 (G-N-7)和ribose2’-O(2’-O)甲基转移酶催化活性,这两个甲基转移酶活性位点共用一个甲基供体S-腺苷甲硫氨酸(SAM)结合位点。基于生物信息学分析,我们对aMPV L蛋白CRVI功能域预测的MTase催化位点及S-腺苷甲硫氨酸结合位点进行氨基酸定点突变,并运用反向遗传学系统,成功拯救了raMPV-G1696A、raMPV-G1700A、raMPV-N1701A和raMPV-D1755A四株aMPV科罗拉多株(Colorado)重组病毒。体外甲基化研究结果显示,四株重组病毒在2’-0位点的甲基化有明显的消减,但在G-N-7位点的甲基化却几乎未受影响。另外,进一步的两步甲基化试验证明G-N-7位点的甲基化先于2’-O位点的甲基化且前者对后者有促进作用。重组病毒蚀斑试验和生长特性分析表明,四株重组病毒在细胞上培养时高度致弱,且保持着良好的遗传稳定性。为进一步验证aMPV甲基转移酶缺陷株是否可以作为弱毒疫苗进行开发,本研究开展了重组病毒的动物试验研究。通过点眼、滴鼻途径将5×106PFU的重组病毒接种2周龄火鸡,研究重组病毒在火鸡内的致病性和免疫原性。结果显示,四株甲基缺陷性重组病毒在火鸡呼吸道内的复制能力均降低,并且可以诱发高水平的中和抗体,可以完全保护火鸡免受同源性野生型aMPV科罗拉多株(aMPV/CO)和异源性野生型aMPV明尼苏达株(aMPV/MN)的攻毒感染。总之,体外试验显示,缺少2’-O位点甲基化的重组禽偏肺病毒(raMPV),高度致弱且保持着良好的遗传稳定性,动物试验表明,raMPV对天然宿主火鸡具有很好的免疫原性。因此甲基转移酶缺陷型重组病毒可作为aMPV理想的疫苗候选株。此外,甲基化位点突变使raMPV致弱的策略可望用于其它禽类和人类副粘病毒疫苗的研制开发。更多还原

【Abstract】 Avian metapneumovirus (aMPV), previously known as Avian pneumovirus (APV), also called Turkey rhinotracheitis virus (TRTV) belongs to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. AMPV is an important pathogen that causes an acute respiratory disease in turkeys and is the etiological agent of "swollen head syndrome" in chickens. Since the first isolation of aMPV in South Africa in 1978, the virus has become prevalent worldwide. Avian metapneumovirus (aMPV) is non-segmented negative-sense RNA virus, which possess a unique mechanism for mRNA cap methylation. Messenger RNA processing is the essential issue in Human metapneumovirus (hMPV) and aMPV gene expression and replication. During viral RNA synthesis, both hMPV and aMPV produce capped, methylated, and polyadenylated mRNAs. Capping and methylation of viral mRNA are essential for mRNA stability, efficient translation, and gene expression. The large (L) polymerase protein contains all the enzymatic activities for mRNA processing, including capping, cap methylation, and polyadenylation. Sequence alignments between representative Non-segmented Negative-strand virus (NNS) RNA virus identified six regions of conservation in the L protein (CRI-CRVI) separated by regions of lower sequence homology. conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2’-O (R-2’-O) methylatransferases, and the two methylase activities share a binding site for the methyl donor S-adenosyl methionine(SAM).In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strainscarrying mutations in SAM binding site in the CR-VI of L protein. The vitro trans methylation assay showed that these recombinantat aMPV mutants are specifically defective in ribose 2’-O (R-2-O1), but not in guanine-N-7 (G-N-7) methylation. Besides, the further two-step trans-methylation assay suggested that the G-N-7 methylation of aMPV facilitates R-2-O methylation. These MTase-defective recombinan aMPV mutants showed delayed CPE and formed smaller plaques in cell culture compared to the wildtype aMPV. Therefore, recombinan aMPV mutants were highly attenuated since the ribose 2’-O methylation was abolished.To determine whether recombinant aMPVs are attenuated in animals and if it can be used for live vaccine candidates, two-week-old SPF turkeys were were inoculated oculonasally with 5*10 5 pfu of raMPV-G1696A, raMPV-G1700A, raMPV-N1701A or raMPV-D1755A. The results showed that MTase-defective raMPVs were attenuated in viral replication in the upper and lower respiratory tracts of turkey. Importantly, turkeys immunized with raMPV-G1696A, raMPV-G1700A triggered a high level of neutralizing antibody and were protected from challenge with aMPV Colorado wild type strain or aMPV Minnesota wild type strain.Collectively, our results indicate that (ⅰ) aMPV lacking 2’-O methylation is highly attenuated in vitro and in vivo, and (ⅱ) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV, and perhapsother paramyxoviruses.更多还原