Characterization and Expression of Three Immune-Related ORFs from Cyprinid Herpesvirus 3 (CyHV3)
Cyprinid herpesvirus 3 is a recently recognized species in the family Alloherpesviridae (order, Herpesvirales) representing an emerging group of viruses found as the cause of epidemic mortality in cultured and wild populations of common carp and koi. Cyprinid herpesvirus 3 (CyHV3) is a large double stranded DNA virus with a genome of approximately 295 kbp with 156 unique open reading frames (ORFs). A total of 15 core genes of CyHV3 have homologs found in the related ictalurid herpesvirus 1 (IcHV1). Among the newly identified nonconserved ORFs of CyHV3 are three putative immune-related genes that may provide interesting insights into potential immunomodulatory functions as related to the severity of the disease induced by CyHV3. Furthermore, an understanding of these putative gene functions may improve our understanding of host (carp) antiviral immune responses that have yet to be described. The series of studies represents the initial characterization and expression of ORFs 4, 12, and 134 from CyHV3 that share homologies, respectively, with three key immunomodulatory genes, two tumor necrosis factor receptor superfamily members (TNFRSF) TNFRSF-14 and TNFRSF-1b, and the cytokine interleukin-10.
Rapid amplification of cDNA ends and subsequent amplification of mRNA with primers designed to the untranslated regions from CyHV3 infected KF-1 cells produced transcripts that were not spliced for ORFs 4 and 12. The c-terminal portion of ORF 4 and entire cDNA sequence of ORF 12 shared highest amino acid identities to counterpart homologs belonging to the murinae subfamily at 26.4 % for ORF 4, and 30.6 % for ORF 12. The coding sequences for both CyHV3 ORFs aligned with the cysteine rich domains, which are the hallmark of TNFRSF family members. Taqman real-time PCR detected a significant increase in transcription for both ORFs from KF-1 between 6 h and 6 d post CyHV3 infection. Both TNFRSF-like homologs when exposed to cycloheximide and phosphonoformic acid indicated delayed-early transcription kinetics. Polyclonal antibody from genetically immunized mice detected expressed proteins of approximately 38 kDa and 16 kDa for ORFs 4 and 12 preferentially in the cell lysates of 293T and a 38 kDa ORF 4 protein band from cell lysates of infected koi fin (KF-1) cells by western blotting.
ORF 134 is encoded by two exons and shares 20.4 % amino acid identity to koi IL-10. Transcription of this viral homolog is also significantly increased (p < 0.05) from KF-1 cells between 6 h and 6 d post CyHV3 infection. Administration of cycloheximide and phosphonoformic acid to infected KF-1 cells in vitro indicated delayed-early transcription kinetics as assayed by real-time PCR. Successful CyHV3 and koi IL-10 expression in mammalian cell lines as c-terminal 6x HIS tagged constructs was detected by the presence of approximately 21 - 25 kDa proteins for CyHV3 IL-10 and 18 kDa for koi IL-10 in supernatants of transfected cells. Western blotting with rabbit polyclonal antibodies to bacterial expressed c-terminal 6x HIS tagged CyHV3 IL-10 protein detected a prominent 18 kDa viral IL-10 band from the supernatant of CyHV3 infected but not uninfected KF-1 cells. Compared to koi IL-10, mammalian expressed viral IL-10 may have the ability to suppress koi TNF α 1 and 2 mRNA transcription and increase TNF α 3 transcription from LPS exposed head kidney macrophage suspensions. Evaluation of the 5.8S, 18S, and 28S as candidate housekeeping genes in CyHV3 KF-1 infected cells exposed to cycloheximide and phosphonoformic acid was conducted. The 18S gene was chosen as the most suitable for the real-time PCR evaluation of transcription kinetics of CyHV3 ORF 4, 12, and 134 using the comparative delta delta Ct method. The use of the 18S gene for normalization of Ct values obtained from CyHV3 infected KF-1 cells under the conditions of this study should be applicable to examine temporal regulation of transcripts from other CyHV3 ORFs which have yet to be characterized.