ORF3 protein promotes the proliferation of porcine epidemic diarrhea virus on Vero cells

Abstract:

[Background] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is an acute intestinal infectious disease of pigs that is characterized by diarrhea, vomiting, and dehydration. Its massive outbreak has caused huge losses to the pig industry since 2010. Due to the poor understanding of the immune mechanism and invasion mechanism of PEDV, there is still no effective prevention and treatment about PED. [Objective] The main aim of this study is to explore the effects of ORF3 protein on the proliferation of PEDV in vitro. [Methods] Recombinant PEDV DR13 (attenuated) strains with orf3 genes from different origins or without orf3 gene were rescued by using reverse genetics operating system based on targeted RNA recombination. Then the Vero cells were infected with the acquired recombinant PEDV strains at a multiplicity of infection (MOI) of 0.1. The viral titers (TCID50) at 8, 16, 24, 32, 40, 48 hours post infection (hpi) were measured and the growth curve of each recombinant PEDV was drawn. The intact cells at 25 and 36 hpi were counted by using an automatic cell counting analyzer. Meanwhile, the cell viability were measured at 12, 24, 36, 48 hpi using Cell Counting Kit-8. [Results] Successful rescue of recombinant PEDV strains with or without orf3 was verified by using RT-PCR and observing the cytopathic effect (CPE). The results of immunohistochemistry assay indicated that orf3 gene of recombinant PEDV was expressed in Vero cells. The viral titers of recombinant PEDV strains with orf3 gene were significantly higher than that without orf3 gene. Furthermore, the statistical analysis results using SPSS showed that the numbers of intact cells left in the flasks infected with the recombinant PEDV strain with orf3 gene at both 25 and 36 hpi were significantly higher than that infected with the strain without orf3 gene and viability of the cells infected by recombinant PEDV strain with orf3 gene were significantly higher at 24, 36 and 48 hpi than that infected with the strain without orf3 gene. [Conclusion] PEDV ORF3 protein promotes the proliferation of PEDV on Vero cells, the mechanism of which might be the time delay of death of infected cells by the ORF3 protein. These results provided information on the function of PEDV orf3 gene and were helpful in understanding the replication mechanisms of PEDV.

Key words:orf3, Porcine epidemic diarrhea virus, Proliferation in vitro