Significant inhibition of re-emerged and emerging swine enteric coronavirus in vitro using the multiple shRNA expression vector

Abstract

A B S T R A C TSwine enteric coronaviruses (SECoVs), including porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine deltacoronavirus (PDCoV) have emerged and been prevalent in pig populations in China for the last several years. However, current traditional inactivated and attenuated PEDV vaccines are of limited efficacy against circulating PEDV variants, and there are no commercial vaccines for prevention of PDCoV and SADS-CoV. RNA interference (RNAi) is a powerful tool in therapeutic applications to inhibit viral replication in vitro. In this study, we developed a small interfering RNA generation system that expressed two different short hairpin RNAs (shRNAs) targeting the M gene of PEDV and SADS-CoV and the N gene of PDCoV, respectively. Our results demonstrated that simultaneous expression of these specific shRNA molecules inhibited expression of PEDV M gene, SADS-CoV M gene, and PDCoV N gene RNA by 99.7%, 99.4%, and 98.8%, respectively, in infected cell cultures. In addition, shRNA molecules significantly restricted the expression of M and N protein, and impaired the replication of PEDV, SADS-CoV, and PDCoV simultaneously. Taken together, this shRNAs expression system not only is proved to be a novel approach for studying functions of various genes synchronously, but also developed to test aspects of a potential therapeutic option for treatment and prevention of multiple SECoV infections.

Identification of a Novel Linear B-Cell Epitope on the Nucleocapsid Protein of Porcine Deltacoronavirus

“…To date, six porcine coronavirus diseases (PEDV, TGEV, PRCV, SADS-CoV, PHEV, PDCoV) have been reported [42,43]. Because they present with similar clinical symptoms, diagnosis is a challenge, a sensitive and discriminate serologic method is clearly needed.…”

A review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin

“…An important phase in the life cycle of the CoV is the human cell entry and fusion derived from proteolytic activity of the relative fusion protein (FP) by respective proteases (Jaimes & Whittaker, 2018). A conventional approach to explore the CoV life cycle is to clone the FP gene into an appropriate vector, transfect the cells, incubate with the related protease, purify the protein and carry out the protein assays (Cong et al, 2019;Li et al, 2019). This approach shows a number of drawbacks: the need of viral genome, expensive synthesizing process, availability of monoclonal antibodies, and time-consuming process.…”

Evaluation of Cross-Protection between G1a- and G2a-Genotype Porcine Epidemic Diarrhea Viruses in Suckling Piglets

“…The highly virulent Chinese G2a PEDV strain, CH/JX/01 (GenBank accession number: KX058031) was isolated in our laboratory, and the G1a PEDV strain CV777 was generously provided by Dr. Zhen Li from Shanghai Academy of Agricultural Sciences. Both CH/JX/01 and CV777 were propagated in Vero-81 cells according to the previously described method [25,26]. Briefly, culture medium was removed from 90% confluent Vero-81 cell monolayers in T-25 culture flasks (Biofil, Guangzhou, China), and the cells were washed twice with sterile phosphate-buffered saline (PBS, 0.01M, pH 7.2, Solarbio, Beijing, China).…”