Construction of PRV BAC and deletion mutants

The PRV BAC was constructed from an isolate of the emergent PRV variant following published methods (Fig. 1) [16, 26]. Briefly, After cotransfection of ~1.5 μg PRV AH02LA strain DNA and ~4 μg pHA2-pUC19-H1-H2 DNA, mini-F recombinant viruses with green fluorescence were purified to obtain a homogeneous population [27]. Next, mini-F containing PRV DNA was isolated and then transferred into E. coli cells DH10B and subsequently into E.coli strain GS1783 cells by electroporation for mutagenesis [23]. Finally, BAC DNA was isolated and RFLPs were identified as described [24] using BamH I, Kpn I, Pst I and Sph I, using the complete genome sequence of PRV ZJ01 strain (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"KM061380.1","term_id":"746719444","term_text":"KM061380.1"}}KM061380.1) as a reference for in silico prediction.

To generate the gE deletion mutant of PRV AH02LA strain, another homologous recombination was performed to recover the gI gene and the undeleted sequences of gE as shown in Fig. 1. Briefly, PCR with primers ΔgE T^V overlap1 F and ΔgE T^V overlap2 R (Table 1) was performed with the PRV AH02LA DNA as template. And then CECs were co-transfected with approximately 5 μg PRV BAC DNA and 2 μg DNA of fragment from the second PCR reaction of overlap PCR with primersΔgE T^V overlap1 F andΔgE T^V overlap2 R by calcium phosphate precipitation. Using illumination at 488 nm, non-fluorescent virus isolates were selected and purified to homogeneity. Product structure was confirmed by sequencing with the same primers and other specific primers from the SEQ-gI/gE series (Table 1).

For generation of the gI/gE repair virus via homologous recombination, CECs were transfected with approximately 5 μg PRV BAC DNA and 2 μg PCR product generated using DNA of parental PRV as template and primers matching PRV Homo 1 F and PRV Homo 2R [16] (Fig. 1). Two to three days after transfection, non-fluorescent plaques (488 nm) were isolated and purified to obtain a homogeneous population. The structure of the recovered viruses was confirmed by PCR and sequencing with primers PRV Homo 1 F, PRV Homo 2R and sequencing primers of SEQ-gI/gE (Table 1).