Volkova Eugenia, Frolova Elena, Darwin Justin R, Forrester Naomi L, Weaver Scott C, Frolov Ilya
Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019, USA.
Virology. 2008 Jul 20;377(1):160-9. doi: 10.1016/j.virol.2008.04.020. Epub 2008 May 22.
The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the viral structural proteins, and ultimately viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.
不同甲病毒感染性cDNA的开发为通过广泛修饰病毒基因组来探索其减毒提供了机会，这种方法可能会最小化或排除向野生型致病表型的逆转。此外，此类甲病毒的基因组可经改造以包含仅在脊椎动物而非昆虫来源的细胞中具有功能的RNA元件。在本研究中，我们开发了一种重组委内瑞拉马脑炎病毒（VEEV），它比TC-83毒株的减毒程度更高，并且只能在脊椎动物细胞中复制。这种表型是通过使病毒结构蛋白的翻译以及最终的病毒复制依赖于脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）来实现的。这种重组病毒是有活力的，但需要在非结构蛋白2（nsP2）中发生额外的适应性突变，这会显著提高其复制速率。尽管在培养的脊椎动物细胞中能有效复制，但经基因改造的VEEV在新生小鼠中表现出高度减毒的表型，不过仍能诱导针对VEEV感染的保护性免疫。
