Molecular Characterization And Effection On White Spot Syndrome Virus Infection Of Toll And Caspase From Red Claw Crayfish Cherax Quadricarinatus

With the development of shrimp farming,infection diseases break out frequently caused by various pathogens.Among these different pathogens,white spot syndrome virus(WSSV)has become a major hinder for aquaculture as lacking of efficient pharmaceutical therapy or prevention to date.Therefore,it is crucial to elucidate the WSSV infection mechanism and find an efficient method to control WSSV disease.Different with vertebrate crustacean only have the innate immunity and lack of adaptive immunity.The innate immunity contains humoral and cellular immune response.The study of defensing against WSSV could start with innate immune response.Therefore,in this study,the two immune related factor involved in humoral and cellular immune response were screened and the investigation of an association with WSSV was performed,which was depended on the Hpt cells cultures and a transcriptome library of haematopoietic tissue(Hpt)cells from the red claw crayfish Cherax quadricarinatus post WSSV infection in our lab.In this study,firstly,a full-length cDNA sequence of Toll receptor(named as CqToll)and open reading frame of(ORF)sequence of caspase(named as CqCaspase)were identified from C.quadricarinatus,and the analysis of their domain were carried out.The predicted structure of CqToll protein was composed of three domains,including an extracellular domain of 19 leucine-rich repeats residues,a highly conversed intracellular domain and transmembrane domain.And CqCaspase possessed a CASc domain including two subunits p20 and p10.Next,tissue distribution analysis revealed that CqToll was expressed widely in various tissues determined from red claw crayfish with highest expression in haemocyte.Similarly,CqCaspase also detected in all tissues from C.quadricarinatus and has a highest expression in Hpt.Finally,to determine the relationship between the CqToll or CqCaspase and WSSV,RNA interference experiment and recombinant protein delivery experiment were performed in Hpt cells,respectively.It was shown that significant lower expression of the anti-lipopolysacchride factor(CqALF),an antiviral antimicrobial peptide(AMP)in crustaceans,but not of CqCrustin was observed after gene silencing of CqToll in crayfish Hpt cell cultures,indicating that the CqALF was likely to be positively regulated via Toll pathway in red claw crayfish.Furthermore,the transcription of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly enhanced in Hpt cells if silenced with CqToll,suggesting that the increase of WSSV replication was likely to be caused by the lower expression of the CqALF resulted from the loss-of-function of CqToll.And,the mRNA expression of IE and VP28 were reduced in Hpt cells when recombinant CqCaspase protein was delivered into Hpt cells,which implied that WSSV replication was inhibited by recombinant CqCaspase protein.Above all,we used the Hpt cells as the primary cells to investigate whether CqToll or CqCaspase has effect on WSSV infection in mRNA level.And we speculated that CqToll might participates in antiviral response against WSSV via induction of CqALF.And CqCaspase might inhibit the WSSV replication via promote apoptotic activity,but the mechanism need further study.Our study enriched the research of WSSV infection and provided the novel thoughts about prevent measure of WSSV infection.