Development of multily attenuated Sendai virus vaccine by means of reverse genetics

(1)决定小鼠致病性的仙台病毒基因的鉴定：通过对仙台病毒弱毒变异株(MVC11)和高致病性野生型病毒(M1)的比较，发现C蛋白第170位由Phe到Ser的单点突变消除了仙台病毒对小鼠的毒力。通过反向遗传学方法回收的一些C蛋白缺失的重组病毒(C‘、C、Y1和Y2)的检测，证实了C蛋白与致病性有关。(2)C蛋白突变的注意机制1)干扰素敏感性：当MVC11感染干扰素α/β受体基因敲除小鼠(A129)时，小鼠的体重仅略有下降。结果表明，在A129中，MVC11的减弱程度与具有IFNR的普通小鼠相同，这表明干扰素敏感性与MVC11的减弱无关。2)感染细胞的死亡效应：C蛋白突变的减毒病毒，如MVC11，可诱导坏死和凋亡。感染细胞的死亡导致后代病毒生产的中断，并导致病毒致病性的减弱。另一方面，像M1这样的致病病毒在感染后没有表现出明显的细胞病变效应，并在很长一段时间内持续释放后代病毒。在caspase抑制剂的存在下，MVC11感染的细胞迅速死亡。(3)重组仙台病毒的生长：重组仙台病毒C基因被MVC11基因替换后，从弱毒株(Z)的cDNA中获得的重组病毒不能有效生长。基于这一观察结果，我们正试图在MVC11的F和V基因中引入突变，目的是建立多重减毒病毒。

(1) Identification of Sendai virus genes which determine mouse pathogenicity.Comparing an attenuated mutant virus (MVC11) with a highly pathogenic wild-type virus (M1), we showed the single point mutation from Phe to Ser at the 170th position of the C protein abolished the virulence of Sendai virus against mice. Involvement of the C protein in pathogenicity was confirmed by examining some recombinant viruses recovered by means of reverse genetics which were lacking one or two of the set of the C proteins (C', C, Y1 and Y2).(2) Mechanism of attention of the c protein mutant.1) Interferon sensitivity: When MVC11 was infected to interferon α/β receptor (IFNR)-knock-out mice (A129), mice exhibited only slight decrease of body weight. The result demonstrated that MVC11 was attenuated in A129 to the same degree as in ordinary mice possessing IFNR, suggesting that interferon sensitivity does not relate to attenuation of MVC11.2) Effect of death of infected cells: Attenuated viruses with mutations in the C protein like MVC11 induced necrosis as well as apoptosis. Death of the infected cells caused interruption of progeny virus production afterwards, and resulted in attenuation of viral pathogenicity. On the other hand, pathogenic viruses like M1 did not demonstrate significant cytopathic effect and released progeny virus continuously for a long time after infection. In the presence of caspase inhibitor which suppressed apoptosis but not necrosis, MVC11-infected cells died rapidly. These results suggested that necrosis plays an important role in causing death to attenuated virus-infected cells.(3) Growth of recombinant Sendai virus: Recombinant virus of Sendai virus obtained from cDNA of the attenuated laboratory strain (Z), the C gene of which was substituted by that of MVC11 did not grow efficiently. Based on this observation, we are trying to introduce mutations into the F and V genes of MVC11 with the aim to establish multiply attenuated virus.