Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo
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RESEARCH
Open Access
Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo Chao Ye1, Jing Chen1, Tao Wang1, Jingjing Xu1, Hao Zheng1,2, Jiqiang Wu1, Guoxin Li1,2, Zhiqing Yu1, Wu Tong1,2, Xuefei Cheng1, Shasha Zhou1 and Guangzhi Tong1,2*
Abstract Background: The alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV. Methods: A novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR. Results: Gibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff. Conclusions: CRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein. Keywords: CRISPR/Cas9, Gibson assembly, Pseudorabies virus, UL41, Vhs activity
Background Pseudorabies virus (PRV) is a member of the alphaherpesviridae subfamily [1]. It is the etiologic agent of Aujeszky’s disease, which causes severe nervous system disorders in newborn piglets and reproductive failure in sows [2]. The combined DIVA (differentiating infected from vaccinated animals) vaccination strategy, has * Correspondence: [email protected] 1 Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai 200241, People’s Republic of China 2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, China
allowed for PRV to be eradicated or well controlled in many countries, mostly in Europe, the Un
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