Generation of recombinant viruses

The genomic DNA of PRV-GD2013 was extracted and purified using commercially available kits (AXYGEN, LOT#: AP-MIN-BF-VNA-250, USA) according to the manufacturer’s instructions. The pBLE-gI-EGFP-gE transfer plasmid and the PRV-GD2013 genomic DNA were co-transfected into BHK-21 (2 × 10⁵ cells/dish) cells using the Lipofectamine® 2000 transfection reagent (Invitrogen), according to the manufacturer’s instructions. After cytopathic effects (CPEs) were observed, the transfected culture was harvested. After two to three freeze-thaw cycles, monolayers of BHK-21 cells were inoculated with 100 μl of lysate per well and covered with 1% low-melting agarose. Recombinant viruses with green fluorescent plaques were screened under fluorescent microscopy. After several rounds of plaque purification, the recombinant virus stably expressing EGFP was screened and hereafter referred to as PRV-GD2013-ΔgI/gE-EGFP. Similarly, PRV-GD2013-ΔgI/gE-EGFP genomic DNA and pBLE-gI-gE were co-transfected into BHK-21 cells, in the same way, to generate a recombinant virus without EGFP expression, hereafter referred to as PRV-GD2013-△gI/gE (Fig. ​(Fig.1).1). Gene recombination was identified by PCR (using the primers were listed in Table ​Table3,3, gI-F/gI-R, gE-F/gE-R, gIF/gER) and DNA sequencing.