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. 2019 Jul 17;93(15):e00616-19. doi: 10.1128/JVI.00616-19

FIG 4.

FIG 4

Confirmation of cDNA origin via recovery of mutant rHAZV. (A) Table outlining the change (underlined) to the cDNA sequence of the HAZV-N ORF and the resulting amino acid sequence. (B) Detection of HAZV-N by Western blotting posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 96 h posttransfection were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected and analyzed by Western blotting for N expression. Recovery of rHAZV containing a HindIII restriction site (rHAZV-G723T) was carried out alongside complete control recovery of rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Schematic showing the location of the inserted HindIII restriction site in the S segment ORF cDNA. UTR, untranslated region. (D) Restriction digest of double-stranded DNA fragments following RNA extraction of rHAZV- and rHAZV-G723T-containing supernatants, first-strand synthesis, and PCR amplification of viral genetic material.