Cells and viruses
The Chinese perch brain cell line (CPB)was used for SCRV replication and was grown in Leibovitz’s L-15medium (Gibco, USA) supplemented with 10% fetal calf serum (FBS;HyClone,USA)at 28°C. The SCRV-QY and SCRV-GM strains were obtained from the Pearl River Fishery Research Institute (Guangzhou, China). Largemouth Bass, with a body length of 7±1.0 cm and an average weight of 12.0±0.9g, were obtained from an SCRV-free zone in FOSHAN (Guangdong, China).
Fish pathogenicity experiments
Largemouth Basses (70 in each group) were intraperitoneally (i.p.) injected with 0.1 mL of 50TCID50 SCRV-QY or SCRV-GM; the control group was i.p. injected with 0.1 mL of PBS. The Largemouth Basses were monitored over 50 days for clinical signs.
Detection of viral RNA copy numbers by real-time qRT-PCR
Three largemouth Basses from each group were sampled from the first day to the fourteenth day, then on the twenty-first and twenty-eighth days. The different tissues were collected and were stored at -80°C until use.
The viral RNA copy numbers were detected by quantitative real-time RT-PCR (qRT-PCR).First, RNA was extracted using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer’s protocol. Then, the isolated RNA was reverse transcribed using the First Strand cDNA Synthesis Kit (Takara, Japan). The cDNA was used as a template for qRT-PCR, which was performed using PremixExTaqTM (PerfectRealTime) (Takara, Japan) to assay samples with the following SCRV N gene-specific primers: F, 5'-GACATGTTCTTCTACAGATTCAAC-3' and R, 5’-CAATCCAGCACTCCACTG-3'. The probe was 5'-AGGTTCAAAGACTGTGCAGCTCTGT-3', which was labeled on its 5’endwithFAMandonits3’endwithEclipse. To estimate virus replication, virus-speciﬁc mRNA expression was measured using qRT-PCR and was expressed as the number of RNA copies per mg of tissue. The results were analyzed using Applied Biosystems 7500software.
Detection of innate immune activity by real-time qRT-PCR
Three largemouth Basses from each group were sampled on the first, seventh, fourteenth, twenty-first, twenty-eighth and forty-second days. The different tissues were collected and stored at -80℃ until use.
First, total RNA was extracted using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer’s protocol. The isolated RNA was then reverse transcribed using the TransScript first-Strand cDNA Synthesis SuperMix (Trans, Guangzhou, China). The cDNA was used as a templatefor qRT-PCR performed using TransStart Top Green qPCR SuperMix (Trans, Guangzhou, China) to assay samples with the following primers: GAPDHgene-specific primers: F, 5'-ATCAAGGAAGCGGTGAAGAAGG-3' and R, 5'-CGAAGATGGAGGAGTGGGTGTC-3'; IRF-7gene-specific primers, F, 5'-AGTGAAGGTGGTCCCTCTGA-3' and R, 5'-CGGCAGACCAAAGACAGAGT-3'; IRAK1 gene-specific primers, F, 5'-CGCTGTTAGCCGTTAGCCTG-3' and R, 5'-CGTAGAGAAACCGTCCCCTC-3'; Mxgene-specific primers, F, 5'-GGATTCTGACATCGGGAGCAA-3'; gene-specific primers, R,GTGCAGTAGACTCATGCTGTand Viperin gene-specific primers, F, 5'- CCAAGAGGGGCCTCAAACTT-3' and R, 5'-CTGACACTTGGGAGCTGGAG-3'.GAPDH was used as an internal control. To detect the innate immune activity, the mRNA expression levels of four specific innate immune-related genes were measured using qRT-PCR. The results were analyzed using Applied Biosystems 7500software.
Histopathological examination of liver, heart, spleen, kidney, brain and intestine
Three largemouth Basses from each group were sampled on the first, seventh and fourteenth days. The pieces of liver, heart, spleen, kidney, brain and intestine were fixed with paraformaldehyde and embedded with paraffin. The tissue sections were stained with hematoxylin-eosin (HE). The sections of tissue were then examined by light microscope.
All experimental measurements are expressed as the mean ± SE. The data were analyzed by one-way ANOVA and Student's t test using SPSS 13.0 statistical software.