影响非洲猪瘟病毒对培养细胞感染性的因素分析

Analysis of Factors Affecting the Infectivity of African Swine Fever Virus on Cultured Cells

猪原代肺泡巨噬细胞(PAMs)是非洲猪瘟病毒(ASFV)的主要靶细胞,除宿主单核/巨噬细胞外,ASFV难以在其他体外细胞系中持续生长繁殖。本研究旨在研究影响ASFV在体外感染能力的因素。通过比较ASFV接种易感细胞PAMs和非易感细胞系3D4/21、PK15细胞的全基因组转录谱差异;使用小分子药物改变细胞代谢或周期、物理方法改变细胞黏附;通过荧光定量PCR、Western blot、红细胞吸附检测病毒在3D4/21和PK15细胞系中的增殖情况。结果表明:ASFV接种3D4/21和PK15两种细胞与PAMs细胞的共同差异基因显著富集在细胞黏附、细胞周期和细胞代谢等方面;使用多种调控细胞周期、细胞代谢的小分子药物处理细胞后对ASFV感染PK15和3D4/21细胞的能力无显著影响;通过悬浮培养来调节细胞的黏附后,显著提高ASFV感染PK15和3D4/21细胞的能力,但随着传代次数的增加,病毒的复制能力逐渐下降。综上所述,细胞黏附可能是影响ASFV体外感染能力的重要因素之一,但是改变细胞黏附后并不能维持ASFV的复制能力。本研究为ASFV体外细胞系的建立提供了初步参考。

Porcine primary alveolar macrophages(PAMs)were the main target cells of African swine fever virus(ASFV),and it was difficult for ASFV to grow and multiply continuously in other in vitro cell lines except host monocytes/macrophages.The aim of this study was to investigate the factors affecting ASFV in in vitro passaging cultures.By comparing the genome-wide transcriptional profile differences between ASFV inoculated susceptible cells PAMs and non-susceptible cell lines 3D4/21 and PK15 cells;Using small molecule drugs to alter cell metabolism or cycle,physical methods to alter cell adhesion;detecting virus proliferation in 3D4/21 and PK15 cell lines by fluorescence quantitative PCR,Western blot,red cell adsorption.The results showed that:ASFV inoculation of both 3D4/21 and PK15 cells and PAMs cells were significantly enriched in genes common to both cell adhesion,cell cycle and cell metabolism;The ability of ASFV to infect PK15 and 3D4/21 cells was not significantly affected by treatment of cells with various small molecule drugs that regulate cell cycle and cell metabolism;The ability of ASFV to infect PK15 and 3D4/21 cells was significantly affected by suspension culture to regulate The ability of ASFV to infect PK15 and 3D4/21 cells was significantly improved after cell adhesion was regulated by suspension culture,but the replication ability of the virus gradually decreased with the increase in the number of passages.In conclusion,cell adhesion may be one of the important factors affecting the in vitro infection ability of ASFV,but altering cell adhesion did not maintain the replication ability of ASFV.This study provides a preliminary reference for the establishment of ASFV in vitro cell lines.