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.