Construction and growth properties of recombinant pseudorabies virus expressing modified enhanced green fluorescent protein

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence was amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of PRV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/M1(+), was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p. i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p. i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24-36 h p. i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG(-)/M1(+) in tissue cultures were further examined and the titer of gG(-)/M1(+) was similar to that of PRV Ea wild strain and the parental strain gG(-)/LacZ(+). The above results indicated that the recombinant virus expressing the modified EGFP can be used as an in vivo marker to monitor replication and spread, as well as to study the molecular pathogenesis of PRV.