Expression Of Cyprinid Herpesvirus 3 Encoded Viral Interleukin 10 And Its Regulation On Immune Related Genes

Interleukin-10(IL-10)is a multifunctional cytokine with both immunostimulatory and immunosuppressive effects,but its main role is immunosuppression.Some viruses can evade host immune surveillance and clearance by up-regulating the expression of host IL-10 or encoding the functional orthologs of IL-10,which was termed as viral IL-10(vIL-10),to build a persistent infection.Cyprinid herpesvirus 3(CyHV-3)is one of the few lower vertebrate viruses currently known to encode vIL-10.In this experiment,prokaryotic expression system has been used to obtain the recombinant viral IL-10 protein(rvIL-10)encoded by CyHV-3 vIL-10 gene successfully.To investigate the effect of CyHV-3 vIL-10 on the expression of immune-related genes and the apoptosis of epithelioma papulosum cyprinid(EPC)cells,rvIL-10 was used to stimulate the head kidney-derived macrophages from common carp and EPC cells respectively.And detected the expression of immune related genes and apoptosis related genes using fluorescent quantitative PCR,detected apoptotic rate using Tunel staining and flow cytometry.CyHV-3 vIL-10 consists of 179 amino acids and contains a conserved domain called IL-10 superfamily.CyHV-3 vIL-10 has a signal peptide but not contains a transmembrane region.CyHV-3 vIL-10 contains a potential phosphorylation site and two potential Nglycosylation sites.The secondary structure of CyHV-3 vIL-10 consists of only alpha-helix and random coils.The amino acid sequence of CyHV-3 vIL-10 has lesser similarity and homology with human IL-10,fish IL-10 or vIL-10 s,but its tertiary structure is quite similar,suggesting conservative biological functions between IL-10 s and vIL-10 s.rvIL-10 of about 38 kDa size was obtained by prokaryotic expression system and detected by Westen blot.In order to investigate the effect of rvIL-10 on the expression of immune-related genes,this expreiment used rvIL-10 to stimulate macrophages from common carp head kidney,and detected the expression of immune related genes by realtime PCR.The results showed that CyHV-3 vIL-10 alone could not significantly affect expression of interleukin-1beta(IL-1?),tumor necrosis factor ?1(TNF-?1),interferongama1(IFN-?1),CXC chemokine a(CXCa),induced nitric oxide synthase(iNOS),interleukin-12(IL-12),IL-10,major histocompatibility complex I(MHC I),interleukin-8(IL-8)and interleukin-6(IL-6)genes on macrophages,but 10 ?g/mL rvIL-10 could significantly promote the expression of IL-10 gene.However,rvIL-10 stimulated on head kidney-derived macrophages in combination with lipopolysaccharide(LPS)showed that CyHV-3 vIL-10 inhibited LPS-induced expression of IL-1?,TNF-?1,iNOS,CXCa,IL-12 and MHC I genes,and the inhibitory effect on TNF-?1 and iNOS genes was significant,but CyHV-3 vIL-10 could not affect the expression of IL-10,IL-8 and IL-6 genes induced by LPS.It is interesting that the regulation of IFN-?1 gene expression by CyHV-3 vIL-10 was double,0.1 ?g/mL rvIL-10 could significantly inhibit the expression of IFN-?1 gene induced by LPS,while 10 ?g/mL rvIL-10 could significantly promote the expression of IFN-?1 gene induced by LPS.In order to study the antagonism of rvIL-10 on apoptosis of EPC cells,the LPS was used to stimulate the EPC cells pre-incubated with rvIL-10,and detected apoptosis rate and expression of apoptosis-related genes by Tunel staining,flow cytometry and real-time PCR.rvIL-10 significantly inhibited apoptosis induced by LPS stimulation because of decrease in apoptotic positive cells and the apoptotic rate.CyHV-3 vIL-10 also inhibited LPS-induced expression of Bax,caspase-9 and caspase-3 genes,and significantly promoted LPS-induced expression of anti-apoptotic Bcl-2 gene,but did not effecte the expression of the Apaf-1 gene.In this experiment,the CyHV-3 vIL-10 gene was cloned and the purified rvIL-10 protein was obtained successfully.It is confirmed that CyHV-3 vIL-10 could inhibit the expression of LPS-induced inflammatory genes,and antagonize the apoptosis of EPC cells.This experiment provides a theoretical and experimental basis for studying CyHV-3 escape of immune surveillance of host.