( Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China )

Abstract

The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNA M1 (EGFP S147/P) and SV40 poly(A) signal seque nce 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, desig n ated as gG^-/LacZ⁺, was isolated and confirmed by plaque purification, PCR, So uthern blot and Western blot. PK-15 cells were infected with the purified recom binant virus at 0.1 pfu/cell and fluorescence emission was monitored at differen t times post-infection (p.i.) using fluorescence microscopy. Fluorescence emiss ion could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximu m 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 abov e 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.