REPLICATION,VIRULENCE & IMMUNOGENICITY IN RECOMBINANT RESPIRATORY SYNCYTIAL VIRUS

人呼吸道合胞病毒（RSV）是世界范围内儿科呼吸道疾病的最重要的病毒因子，并造成巨大的发病率和死亡率。获得许可的疫苗仍有待开发。疫苗开发的障碍包括病毒在细胞培养中生长不良，实验动物感染的半容许性质，以及难以在免疫原性（取决于合理的病毒复制水平）和减毒（取决于病毒复制水平降低）之间实现适当的平衡。我们最近开发了一种通过胞内共表达编码完整RSV复制中间体RNA（反基因组）和N、P、L和M2-1蛋白的cDNA来生产感染性重组RSV的方法，所述蛋白一起构成完全胜任RNA合成的核衣壳。这为基础分子和发病机制研究提供了重要工具，也为微调候选疫苗病毒的减毒水平提供了方法。RSV编码10种mRNA，编码11种蛋白质（M2 mRNA含有两个重叠的ORF，编码两种不同的蛋白质，M2-1和M2-2）。我们研究了单个RSV基因是否可以？被打晕了（缺失的）而不消除病毒在细胞培养物中生长的能力。迄今为止，已经单独敲除了四种RSV基因，即NS 1、NS 2、SH和G，而没有丧失感染性。NS 2基因的缺失在体外和体内是高度减毒的，并且代表了用于疫苗目的的非常有用的突变。NS 1和SH敲除病毒是中度减毒的，并且也是包含在减毒活疫苗中的候选者。G基因敲除的病毒在某些细胞中生长良好，但在其他细胞中则不然，这意味着它使用的是一种分布具有细胞特异性的替代受体。恢复G敲除病毒的能力表明，G对于感染性病毒的形成或传播不是必需的，这一发现对病毒组装和受体使用具有重要意义。最后，我们以前表明RSV可以接受和表达额外的外源基因。在这里，我们表达了某些细胞因子的重组RSV作为一种可能的方法，以提高免疫反应的减毒活疫苗。- 病毒，疫苗，减毒活疫苗，儿科，感染性疾病，呼吸道疾病，重组DNA

Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory tract disease worldwide and is responsible for a huge burden of morbidity and significant mortality. A licensed vaccine remains to be developed. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of the infection in experimental animals, and the difficulty of achieving an appropriate balance between immunogenicity (which depends on reasonable levels of virus replication) and attenuation (which depends on reduced levels of virus replication). We recently developed a method for producing infectious recombinant RSV by the intracellular coexpression of cDNAs encoding a complete RSV replicative intermediate RNA (antigenome) and the N, P, L and M2-1 proteins, which together constitute a nucleocapsid that is fully competent for RNA synthesis. This provides an important tool for basic molecular and pathogenesis studies as well as a method for fine- tuning the level of attenuation of candidate vaccine viruses. RSV encodes ten mRNAs encoding eleven proteins (the M2 mRNA contains two overlapping ORFs encoding two separate proteins, M2-1 and M2-2). We investigated whether individual RSV genes could be ?knocked out? (deleted) without ablating the ability of the virus to grow in cell culture. To date, four RSV genes have been individually knocked out without loss of infectivity, namely NS1, NS2, SH, and G. Deletion of the NS2 gene is highly attenuating in vitro and in vivo and represents a very useful mutation for vaccine purposes. The NS1 and SH knockout virus are moderately attenuating, and also are candidates for inclusion in a live-attenuated vaccine. The G knockout virus grows well in certain cells but not others, implying that it is using an alternative receptor whose distribution is cell-specific. The ability to recover a G knockout virus shows that G is not essential for the formation or transmission of infectious virus, findings which have important implications for virus assembly and receptor usage. Finally, we previously showed that RSV can accept and express an added foreign gene. Here, we have expressed certain cytokines in recombinant RSV as a possible method to improve the immune response to a live-attenuated vaccine. - Virus, vaccine, live-attenuated viral vaccine, pediatrics, infectious disease, respiratory tract disease, recombinant DNA