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

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal sequence w as 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 P RV Ea mutant gG(-)/LacZ(+). The resulting recombinant virus expressing M1, designated as gG(-)/LacZ(+), 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 t o study the molecular pathogenesis of PRV.