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

• The genes for MPO, KRT-8, and DUSP-1 from crucian carp were cloned and sequenced.
• MPO, KRT-8 and DUSP-1 were stimulated within 72 h post infection of CyHV-2, but not bacterial.
• Expression levels of MPO and KRT-8 were significantly in resistant infection than susceptible infection.
• MPO and KRT-8 could serve as immune markers representing CyHV-2 resistant infection in crucian carp.

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.