Woźniakowski Grzegorz, Mazur-Panasiuk Natalia, Walczak Marek, Juszkiewicz Małgorzata, Frant Maciej, Niemczuk Krzysztof
Department of Swine Diseases, Puławy, Poland.
Director General National Veterinary Research Institute, 24-100 Puławy, Poland.
J Vet Res. 2020 Jun 3;64(2):197-205. doi: 10.2478/jvetres-2020-0039. eCollection 2020 Jun.
African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain.
The host immune response modulation genes , , and were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells.
The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate.
Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the , , and genes might occur with low frequency, resulting in difficulties in separation of various virus populations.
非洲猪瘟（ASF）在一些东欧国家是一个紧迫的经济问题。它还使中国母猪数量减少了50%。控制这种疾病依赖于扑杀感染猪或捕杀野猪以建立卫生区。高效疫苗研发的主要制约因素是病毒逃避宿主免疫反应的机制。本研究旨在使一株非洲猪瘟病毒（ASFV）野毒株适应已建立的细胞系，并构建重组非洲猪瘟病毒株。
使用成簇规律间隔短回文重复序列/半胱天冬酶9（CRISPR/Cas9）诱变系统缺失宿主免疫反应调节基因、和。从波兰分离出一株具有代表性的病毒分离株（Pol18/28298/Out111），并在猪原代肺泡巨噬细胞（PPAM）中进行培养。尝试使该病毒适应几种已建立的细胞系。设计、合成了编码CRISPR/Cas9基因以及与靶序列互补的引导RNA（gRNA）的质粒，并将其转染到感染了ASFV的PPAM细胞中。
重组病毒与亲本病毒分离株相比，显示出相似的复制动力学。
考虑到所开发的CRISPR/Cas9系统的实用性，研究表明、和基因的修饰可能以低频率发生，导致难以分离各种病毒群体。
