title: Characterization of myeloid-specific peroxidase, keratin 8, and dual specificity phosphatase 1 as innate immune genes involved in the resistance of crucian carp (Carassius auratus gibelio) to Cyprinid herpesvirus 2 infection.
abstract: Myeloid-specific peroxidase (MPO), keratin 8 (KRT-8), and dual specificity phosphatase 1 (DUSP-1) are believed to play essential roles in innate immunity. Through suppression subtractive hybridization (SSH) analysis, we previously identified MPO, KRT-8, and DUSP-1 as the three genes that were the most significantly upregulated in crucian carp (Carassius auratus gibelio) that survived Cyprinid herpesvirus 2 (CyHV-2) infection. Here, we have further characterized these three genes and their response to pathogen challenge. The open reading frames (ORF) of MPO, KRT-8, and DUSP-1 were cloned by RACE technique and sequenced. The full-length cDNAs of the three genes contained ORFs of 2289, 1575 and 1083 bp respectively. The polypeptides from each ORF were projected to contain 762 (MPO), 524 (KRT-8), and 360 (DUSP-1) amino acids. Phylogenetic analysis showed that the three genes were most closely related to zebrafish. We found that MPO, KRT-8, and DUSP-1 were expressed at low levels in all of the tissues examined in healthy crucian carp. Quantitative real-time RT-PCR analysis indicated that MPO, KRT-8, and DUSP-1 mRNA expression was significantly upregulated within 72 h of CyHV-2 infection compared to mock infected controls. Maximum expression of MPO was detected at 24 hpi (2.71-fold, P < 0.05). While, 12 hpi (3.80-fold, P < 0.01) and 6 hpi (8.70-fold, P < 0.01) were the highest expression time points for KRT-8 and DUSP-1, respectively. In contrast, after Aeromonas hydrophila challenge, the transcripts of these three genes remained unchanged or slightly down-regulated. For the fish survived from viral infection, expression levels of MPO and KRT-8 were 2.72 fold and 2.47 fold higher than those of fish died from acute infection, and similar level of DUSP-1 was observed in samples of survived fish. These data suggested MPO, KRT-8 and DUSP-1 might be involved in the antiviral, but not antibacterial innate immune response in crucian carp. These findings also support the use of MPO and KRT-8 as immunological markers for a response to viral infection in crucian carp.
authors.affiliation: Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China.
authors.forename: Patarida
authors.initials: P
authors.lastname: Podok
authors.affiliation: Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Xu
authors.affiliation: Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xu
authors.affiliation: Key Laboratory of Aquatic Genetic Resources of the Ministry of Agriculture, Shanghai Ocean University, 201306, PR China. Electronic address: lqlv@shou.edu.cn.
authors.forename: Liqun
authors.initials: L
authors.lastname: Lu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism; D005393:Fish Diseases / Q000276:immunology* / Q000821:virology*; D020869:Gene Expression Profiling; D006054:Goldfish; D006566:Herpesviridae Infections / Q000276:immunology / Q000662:veterinary*; D006824:Hybridization, Genetic / Q000235:genetics*; D007113:Immunity, Innate / Q000235:genetics*; D053533:Keratin-8 / Q000235:genetics / Q000378:metabolism; D022423:Myeloid Cells / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D013045:Species Specificity
chemical_list: D018076:DNA, Complementary; D053533:Keratin-8; D009195:Peroxidase; D054637:Dual-Specificity Phosphatases
keywords: Crucian carp Carassius auratus gibelio; Cyprinid herpesvirus 2; Dual specificity phosphatase 1; Keratin 8; Myeloid-specific peroxidase