PDF(Pubmed)
Abstract：
The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA \"Animal Rule\" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other \"wild type\" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.
摘要：
国防部最近开始努力改进和标准化用于测试治疗剂和疫苗的病毒挑战材料和功效确定策略。这包括在适当情况下以cDNA形式稳定病毒基因组序列，使用人源病毒分离物，以及确定攻击病毒复制的非侵入性策略。最终,希望这些方法能够满足FDA的“动物规则”的许可，当人类数据不太可能可用时，它取代了动物功效数据。为此,我们创建并检查了原型人类感染来源的甲病毒株的cDNA克隆的毒力表型，委内瑞拉(VEEVINH9813),东部（EEEVV105）和西部（WEEVFleming）马脑炎病毒，并创建了荧光和发光报告表达载体，用于评估体内外复制特性。每种病毒的最小传代分离株的序列用于合成全长cDNA克隆以及基于T7转录启动子的细菌繁殖载体。将从cDNA克隆产生的病毒与从cDNA克隆和GenBank序列衍生的其他“野生型”菌株进行比较，以鉴定和消除细胞传代生物储备中积累的推定组织培养物。随后检查小鼠模型中的气雾剂和皮下感染和疾病。在VEEVINH9813生物分离序列中鉴定出增加硫酸乙酰肝素结合的突变,并从cDNA克隆中消除。来自新的人类分离物cDNA克隆的病毒在气溶胶或皮下接种后显示出与现有克隆衍生病毒相似的小鼠毒力。
