qPCR analysis of the genes related to MHC-I processing pathway in zebrafish following WED - immunization during the first five days. Proteasome activator (PA28), heat shock protein 90 kDa alpha (HSP90α), heat shock protein 4a (HSPa4a), endoplasmin (grp94), TAP binding protein (tapbpl), calreticulin (calr), calnexin (canx) and MHC class I ZE protein (MHC-I) were validated. The relative expression of the above immune-related genes in livers (gray bars) and spleens (white bars) of zebrafish were analyzed by qPCR. Zebrafish were vaccinated via intramuscular injection with WED or PBS. The livers and spleens of 10 fish were taken at 1, 2, 3, 5 d post-vaccination, respectively, and total RNA was extracted and used for qPCR. The mRNA level of each immune-related gene was normalized to that of β-actin. For the gene of each time point, values represent fold change in expression compared to the control treatment, which was set at 1.0. Results are expressed as means ± SD (n = 3). Mock immunized group was subtracted from each group. Independent-sample t-test in the SPSS software (Version 11.5, SPSS Inc.) was used to determine statistical significance of the WED immunized groups relative to mock groups. Significant differences were considered at * p < 0.01.