Production of Recombinant African Swine Fever Viruses: Speeding Up the Process
Production of Recombinant African Swine Fever Viruses: Speeding Up the Process
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DOI：
10.3390/v12060615
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发表时间：
2020-06-01
期刊：
影响因子：
4.7
通讯作者：
Dixon, Linda K.
中科院分区：
文献类型：
--
作者：
Rathakrishnan, Anusyah;Moffat, Katy;Dixon, Linda K.
非洲猪瘟(ASF)是猪的一种破坏性疾病，没有疫苗可供控制。非洲猪瘟病毒(ASFV)的基因操作通常是繁琐和耗时的。在这里，我们描述了一种操纵病毒基因组的方法，以在大大缩短的时间内产生基因缺失病毒。该方法将传统的同源重组和荧光激活细胞分选(FACS)相结合，分离纯化表达荧光报告基因的病毒。通过三轮FACS单细胞分离和两轮限制性稀释法，我们从缺失DP148R基因的贝宁97/1ASFV中删除了另外两个基因EP153R和EP402R。通过结合不同的荧光标记，这种方法有可能极大地促进对ASFV基因功能的研究，并开发候选的减毒活疫苗。
African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines.