Screening Of TiLV Sensitive Cell Lines And Preliminary Study On The Effect Of PIgR On Virus Replication

As the second largest freshwater cultured fish in the world,tilapia has great economic value.But recently years,the Tilapia lake virus disease(Ti LD)caused by Tilapia lake virus(TiLV)had made a great economic loss to the tilapia culture industries.Up to now,the pathogenic mechanism of TiLV has not been clear,and there are no effective control measures.Til V is a highly infectious virus,which can sicken cultured and wild tilapia and cause massive death.It is significant to explore the antiviral immune mechanism of tilapia.Sensitive cell lines are the basic materials and play an important role in the research of viral disease etiology,development of prevention and control products such as diagnostic technology and vaccines.Fish live in the water environment containing kinds of pathogens during their whole life.Their skin and mucosal related tissues and organs constitute the first line of defense against the pathogen invasion.As a transmembrane glycoprotein,poly immunoglobulin receptor(pIgR)is an important factor in the process of mucosal immunity.It can transport poly immunoglobulin(p Ig)from the basolateral to the apical surface of epithelial cells through endocytosis,to protect mucosal tissue from pathogen invasion,and maintain the internal environment stability of mucosal immune system.There are also relevant reports indicate that pIgR may be hijacked by the pathogen to promote their own infection.In order to explore the antiviral immune mechanism of tilapia and develop prevention and control products for TiLV.This study screened the sensitive cell lines to TiLV and studied the biological characteristics of tilapia pIgR and its effect on tilv proliferation.The results are as follows:1.Sensitive cell lines screening for TiLVTilapia brain cells(TIB)and snakehead cells(E-11)were infected with gradient diluted TiLV to determine the optimal concentration for virus inoculation;Then,10kinds of fish cells were infected at optimal inoculation concentration.The cell state was observed every day after experiment infection,and the proliferation of TiLV in various cell lines were quantitatively analyzed by fluorescence quantitative PCR(q PCR).The results showed that the optimal inoculation concentration was 1.0×10~5copies/μL.All of 10 fish cells inoculated with the TiLV produced obvious cytopathic effect(CPE).Fluorescence quantitative detection of TiLV copy number proliferation in each cell showed that Ti B cells derived from tilapia produced the highest virus copies,followed by MSF cell line,CAMK cell line and SS cell line.The viral copy numbers of these four cell lines ranged from 4×10~7 copies/μL to 4.6×10~8 copies/μL.The results of confocal immunofluorescence were consistent with the results of fluorescence quantitative detection.Red fluorescence could be observed in all of 10 cells.TiLV particles with a diameter of 70-110 nm were also observed in cells of three randomly selected fish species using transmission electron microscopy.Researches have shown that Ti B,MSF and CAMK are more sensitive cell lines to TiLV.Among them,Ti B has the strongest proliferation ability and sensitivity to TiLV.2.Study on the molecular characteristics and expression patterns of tilapia pIgRFirstly,the complete open reading frame of tilapia pIgR was amplified by RT-PCR technology,and its structural domain prediction,three-dimensional structure and phylogenetic tree analysis were carried out.Then,RT-q PCR was used to analyze the distribution of pIgR gene in various tissues of healthy tilapia and the changes of pIgR expression in spleen,kidney and skin tissues after infection with TiLV were also analyzed.The results showed that the complete tilapia pIgR ORF sequence obtained by PCR amplification with a length of 1023 bp.The ORF sequence had a signal peptide and two Ig-like domains.The homology modeling results showed that the overall pIgR protein was distorted"L”shape,mainly composed of antiparallel beta sheets and random coils;The phylogenetic tree shows that there are three subgroups of fish,amphibians,birds and mammals;among teleosts,tilapia pIgR cluster with the pIgR of largemouth bass and orange-spot grouper which three fishes all belong to the Perciformes.The expression of pIgR can be detected in all tissues and organs of tested healthy tilapia,and the expression level is higher in the skin,spleen and kidney.After infection with TiLV,pIgR in skin,spleen and kidney of tilapia showed a trend of first increasing and then decreasing.The expression of pIgR in skin reached a peak at 24 h after infection,while the expression of pIgR in spleen and kidney reached peak at 48hours after infection.3.Study on the effect of tilapia pIgR overexpression on TiLV proliferationIn order to explore the effect of OnpIgR on TiLV proliferation,firstly,the pIgR overexpression plasmid was constructed.Then,the constructed recombinant plasmid was transfected into Ti B cells,followed by WB,restriction enzyme digestion and fluorescence observation.The constructed recombinant palsmid was transfected into Ti B cells and then infected with TiLV.The samples were collected at 48h and 72h after infection to detect m RNA and protein levels of TiLV for preliminarily exploring the effect of pIgR on the proliferation of TiLV.The results showed that the enzyme digestion identification showed that the target band appeared at the specific position of the Marker,and the Western Blot results showed that the fusion protein band with the expected size appeared at the position of 55 k Da～70 k Da.After the recombinant plasmid p EGFP-pIgR and empty plasmid p EGFP-N1 were transfected into Ti B cells for 24 h,strong green fluorescence could be seen under the fluorescence microscope.The above results show that the recombinant eukaryotic expression plasmid can normally express the target protein in vitro.Ti B cells,which have over expressed the pIgR,were infected with TiLV.It was found that the relative expression of S8 and S10 m RNA of TiLV increased significantly,and the copy number of tilv also increased significantly.The protein expression of TiLV also increased by using the tilv-s8 monoclonal antibody WB prepared in the laboratory.The results preliminary showed that tilapia pIgR could promote the proliferation of TiLV.Therefore,TiLV infection can promote the expression of pIgR,and the overexpression of pIgR can also promote the proliferation of virus,but the mechanism of their mutual promotion needs to be further studied.