Screening And Identification Of Envelope Proteins Encoded By ORF136 And ORF25 From Cyprinid Herpesvirus 3

Cyprinid herpesvirus 3(Cy HV-3),also known as Koi herpesvirus(KHV),is the etiological agent of a highly contagious and fatal disease termed koi herpesvirus disease(KHVD)that afflicting both the common carp and koi carp Cyprinus carpio L populations.Since it was first reported in 1997,the disease caused by Cy HV-3 has spread and prevailed in Asia,Europe,North America,Africa and other counties or regions worldwide,resulting in significant economic losses to the ornamental koi and carp cultural industries.Cy HV-3 is an enveloped herpesvirus with large double-stranded DNA,which currently classified into the genus Cyprinivirus within the family Alloherpesviridae of the order Herpesvirales.Viral envelope proteins play a key role in virus entry mediated by recognized receptor of host cells,but it is less well understood about the study on Cy HV-3 envelope proteins.In this study,proteomic analyses of purified Cy HV-3 virions and envelope fractions for screening putative viral envelope proteins were carried out and followed by localization study of envelope proteins ORF136 and ORF25.As results confirmed,ORF136 and ORF25 were envelope proteins of Cy HV-3,which may lay the foundation for further functional research.Following are three parts of this study:1.Propagation and purification of Cy HV-3: common carp brain cell line(CCB)was infected with Cy HV-3-GZ1301 and Cy HV-3-E,respectively.Virus suspensions were collected when apparent cytopathic effect was observed.Then,the virus suspensions were purified by 20% to 66% sucrose density gradient in combination with ultracentrifugation.As result shown under transmission electron microscope(TEM),the virions with high purity and typical characteristic in diameter 150 to 200 nm in line with previous report were obtained.Moreover,protein bands were apparently shown by using coomassie brilliant blue staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Envelope supernatants and nucleocapsid components were separated by using 1% Triton X-100.This effective protocol of extraction of vial envelope fraction and nuclocapsid were confirmed by transmission electron microscopy,SDS-PAGE and Western blotting analysis.2.Proteomic analysis of Cy HV-3: proteins from purified Cy HV-3 virions and envelope fractions were cut into slices after SDS-PAGE and coomassie brilliant blue staining.Gel slices were digested with tyrisin subsequently followed by analysis through liquid chromatography tandem mass spectrometry(LC-MS/MS)and searching by MASCOT with uniprot databases.As result shown,a total of 46 viral proteins including 14 envelope proteins,4 capsid proteins,2 tegument proteins and 26 unknown proteins were identified in purified virions of two distinct Cy HV-3 isolates,four of which(ORF82,ORF88,RF121 and ORF139)were newly detected except for those previously reported.Moreover,a total of 16 proteins were detected in envelope fractions.To sum up,at least 46 structural proteins including 22 envelope proteins,4 nuclocapid proteins,2 tegument proteins and 18 unclassified proteins were present in purified Cy HV-3 virions.In addition,a search for host proteins associated with Cy HV-3 indicated a total of 22 proteins mainly involved in stress response,signal transductions and metabolism were present based on complete genome datas of common carp Cyprinus carpio.3.Identification of ORF136 and ORF25: ORF136 and ORF25 protein were further identified based on previous study for screening of envelope proteins.First of all,the predicted antigenic determinant based on amino acid sequence of protein encoded by ORF136 gene was amplified by PCR.Then,the recombinant prokaryotic expression vector was constructed by ligating the cloned fragment to vector p ET-32a(+),used for transformation of E.coli.Rosetta(DE3)and expression protein induced by IPTG.Furthermore,the polyclonal antibody from the rabbits immunized by purified recombinant proteins were prepared and analyzed by Western blotting and indirect immunofluorescence.In addition,ORF136 was amplified by PCR and connected with eukaryotic expression vector p VAX1 to construct recombinant eukaryotic expression vector p VAX1-ORF136.Then,ORF136 protein was validated by Western blotting,immunofluorescence by confocal laser scan microscopy and immunoelectron microscopy using polyclone antibody against ORF136.As results of Western blotting shown,specific band was detected in purified virions and envelope fractions with the expected molecular weight of 15 k Da,indicating ORF136 localized in viral envelope.As results shown,the green fluorescence of ORF136 protein particularly accumulated in the cytoplasm of CCB cells infected with Cy HV-3 and transected with p VAX1-ORF136 by confocal laser scan microscopy,indicating ORF136 protein mainly localized in the cytoplasm.Finally,the envelope localization of ORF136 was further confirmed by immunoelectron microscopy.Similarly,ORF25 protein was validated by using Western blotting and immunofluorescence assay.As results of Western blotting shown,specific band was detected in purified virions and envelope fractions,indicating ORF25 localized in viral envelope,though the molecular weight of 100~130 k Da was not consistent with expected the molecular weight of 65 k Da.As result of immunofluorescence assay with confocal laser scan microscopy shown,the green fluorescence was detected in the cytoplasm of CCB cells infected with Cy HV-3 and transected with pc DNA3-ORF25,indicating ORF25 mainly localized in the cytoplasm.In this study,proteomic analysis of purified Cy HV-3 virions and envelope fractions were performed.Envelope proteins ORF136 and ORF25 were confirmed by Western blotting analysis,immunofluorescence assay and/or immunoelectron microscopy,which may lay the foundation for further functional study.