2.6 Sequencing and phylogenetic analysis of gE and gC genes
The complete gE and gC genes of PRV were amplified from viral DNA extracted from PRV isolates using two specific primers gE-F/R and gC-F/R designed in our laboratory (Zhao et al., 2020). Then the genes were ligated with the vector pMD18-T (Takara), and separately introduced toEscherichia coli DH-5a cells (Takara). The positive recombinant plasmids carrying gE and gC genes were sent to Sangon Biotech Shanghai Co., Ltd for DNA sequencing and all sequencing reactions were performed in duplicate.
Phylogenetic trees were constructed based on the gE and gC gene sequences of PRV isolates and reference strains in GenBank using MEGA software, version 7.0 (www.megasoftware.net) by the neighbor-joining method with 1,000 bootstrap replicates (Kumar et al., 2016). Evolutionary distances were computed by the pairwise distance method with the maximum composite likelihood model. Sequences of PRV strains listed in Table 1 retrieved from NCBI were used as references.