Minibest viral dna extraction kit ver 5

To determine the changes of LCDV copy numbers in PBLs, the viral genomic DNA (gDNA) in PBLs from LCDV-infected flounder at 1, 3, 6, 12, and 36 hpi was extracted with a MiniBEST Viral DNA Extraction Kit Ver.5.0 (TaKaRa, Kusatsu, Japan) according to the manufacturer’s instruction, and LCDV genome copies in PBLs of flounder were detected by qPCR. The specific primers P1/P2 were designed as described before according to the sequence of the LCDV ORF038 gene: 5′-TCT TGT TCA GCA TTT ACT TCT CGGC-3′, 5′ TCT TCT CCTTA GAT GAT TTC CC-3′ [19 (link)]. The qPCR reaction system contained 10 μL SYBR qPCR Master Mix (Roche, Switzerland), 2 μL primer P1/P2, and 50 ng template DNA, adding sterile distilled water to a final volume of 20 μL. The reaction procedures were as follows: 10 min at 95 °C, then 45 cycles of 10 s at 95 °C, 10 s at 55 °C and 20 s at 72 °C. The LCDV copy numbers were calculated according to the Ct values via the standard curve generated before [19 (link)].

To further identify whether the 27.8R was a functional receptor involved in LCDV infection of PBLs, an antibody-blocking assay using anti-27.8R MAb and isotype control was carried out in PBLs in vitro. The PBLs from healthy flounder were incubated with mouse anti-27.8R MAb at concentrations of 0.12 μg/mL, 1.2 μg/mL, and 12 μg/mL for 2 h, respectively, and the same volume of mouse anti-WSSV MAb 1D5 was used as controls. After three washes with PBS, the PBLs were inoculated with 100 μL LCDV at 4 TCID₅₀/mL. Following three washes again, the cells were collected at 24 h for DNA extraction using MiniBEST Viral DNA Extraction Kit Ver.5.0 (TaKaRa, Japan) for detection of LCDV copy numbers by qPCR as above.