Effects Of Porcine CD151 Protein And Sialic Acid On The Proliferation Of Porcine Deltacoronavirus On Cells

In recent years,outbreaks of various coronaviruses that infect humans or livestock have brought disasters to the world.The study found that coronavirus has a high mutation rate and frequent cross-species transmission,which makes it very difficult to prevent and control.Up to now,no effective vaccine or antiviral drug against PDCoV has been found.Porcine Deltacoronavirus(PDCoV)is a highly-contact intestinal infectious disease characterized by severe watery diarrhea,dehydration,vomiting and other symptoms in piglets.Its first outbreak was in the United States in 2014,and then spread rapidly to many countries,which brought huge economic losses to the pig industry.Coronavirus need to bind to host cells when infect,therefore,research on coronavirus receptors can help us to understand the mechanism of virus infection and cross-species transmission.It also has great implications for research of new antiviral drugs and vaccines,and the prevention and control of virus infection.In this study,porcine CD151 gene was successfully expressed in a PDCoV non-susceptible cell line 293T with eukaryotic expression vector.By detecting the susceptibility of porcine CD151-expressing 293 cells to PDCoV,it was confirmed that 293T cells which stably expressing CD151 cannot be infected by PDCoV.Pretreat cells with NaIO4 solution and neuraminidase solution,which can remove sugars and specifically destroy sialic acid on the cell surface respectively,then infect pretreated ST/LLC-PK1 cells with PDCoV and samples were tested by RT-qPCR,TCID50 and indirect immunofluorescence,and it was found that the destruction of cell surface sugars and sialic acid can inhibit PDCoV infection.The research results are as follows:1.Study of CD151 overexpression in vitro on PDCoV infection.In this experiment,the CD151 gene was amplified and ligated to the eukaryotic expression vector pEGFP-N1.The recombinant vector pEGFP-N 1-CD 151 was transfected into PDCoV non-susceptible 293T cells,green fluorescent protein was visible by fluorescence microscopy,which proved that the target protein was successfully expressed.Detection of the protein level of transfected cell samples showed that the cells can successfully expressed the CD151 protein.PDCoV was used to infect 293T cells that transiently expressed CD151,and PDCoV infection was detected by PCR.The results showed that 293T transfected and stably expressing CD 151 could not be infected by PDCoV.2.Study of PDCoV proliferation effected by cell-surface sugars.In order to determine the role of cell-surface sugars when PDCoV infect host cells,this test first examined the safe concentration of NaIO4 solution on ST cells and LLC-PK1 cells by MTT assay.After confirmed the safe concentration range,0.2mM,1mM,and 5mM NaIO4 solutions were selected to treat ST cells and LLC-PK1 cells.After 24 hours,the supernatant,intracellular and mixed solutions were collected.Virus content in supernatant was detected by TCID50(the PDCoV content of the samples in the supernatant did not reach the RT-qPCR minimum detection line),and virus contents in cells and mixed samples was detected by RT-qPCR and TCID50.The results showed that as the concentration of NaIO4 solution increased,the PDCoV content also showed an overall downward trend,indicating that as the concentration of NaIO4 solution increased,PDCoV inhibited cell infection.The indirect immunofluorescence method was used to detect the PDCoV infection degree before and after the cells were treated with NaIO4 solution.The results showed that in ST/LLC-PK1 cells,using NaIO4 to destroy cell surface sugars could significantly reduce PDCoV infection of cells.3.Study of PDCoV proliferation effected by cell-surface Sialic Acid.In order to further determine which carbohydrates are involved in the process of PDCoV infection of host cells,this test first explored the safe concentration of neuraminidase solution in ST cells and LLC-PK1 cells by MTT test.After determining the safe concentration range,0.0625U/mL,0.25U/mL,lU/mL and 4U/mL neuraminidase solutions were selected to treat ST cells and LLC-PK1 cells,and the supernatant,intracellular and Mixed liquid were collected respectively.Virus content in supernatant was detected by TCID50(the PDCoV content of the samples in the supernatant did not reach the RT-qPCR minimum detection line),and virus contents in cells and mixed samples was detected by RT-qPCR and TCID50.The results showed that with the increase of neuraminidase concentration,the PDCoV content detected by RT-qPCR and TCID50 showed an overall downward trend,indicating that as the concentration of neuraminidase solution increased,PDCoV infection of cells was suppressed.The indirect immunofluorescence method was used to detect the PDCoV infection degree before and after neuraminidase solution treatment of cells.The results showed that in ST/LLC-PK1 cells,the use of neuraminidase to destroy the sialic acid on the cell surface could significantly reduce PDCoV infection of cells.In summary,this test shows that CD151 cannot mediate PDCoV invasion into host cells;PDCoV may bind to sialic acid on the cell surface during the invasion stage to allow viral particles to adsorb on the host cells.This result provides a basis for the study of PDCoV receptors,and also provides some ideas for the screening of PDCoV antiviral drugs.