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

(1)仙台病毒决定小鼠致病性的基因鉴定。将MVC11减毒突变病毒与高致病性野生型病毒M1进行比较，发现C蛋白第170位从Phe到Ser的单点突变消除了仙台病毒对小鼠的毒力。通过检测一些通过反向遗传获得的缺乏一组或两组C蛋白（C′，C， Y1和Y2）的重组病毒，证实了C蛋白参与致病性。(2) c蛋白突变体的注意机制。1)干扰素敏感性：干扰素α/β受体（IFNR）敲除小鼠（A129）感染MVC11后，小鼠体重仅轻微下降。结果表明，MVC11在A129中的减弱程度与具有IFNR的普通小鼠相同，提示干扰素敏感性与MVC11的减弱无关。11.2)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产生中断，导致病毒致病性减弱。另一方面，M1等致病性病毒在感染后未表现出明显的细胞病变作用，并在较长时间内持续释放子代病毒。caspase抑制剂抑制mvc11细胞凋亡，但不抑制坏死，mvc11感染细胞迅速死亡。这些结果表明，坏死在导致病毒感染的减毒细胞死亡中起重要作用。(3)重组仙台病毒的生长：从实验室减毒株(Z)的cDNA中获得仙台病毒重组病毒，其C基因被MVC11取代，不能有效生长。基于这一观察结果，我们正试图将突变引入MVC11的F和V基因，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.