COMPARATIVE STUDIES ON WILD-TYPE AND ATTENUATED STRAINS OF PSEUDORABLES VIRUS

Abstract

Comparative studies of wild-type and passaged strains of pseudorabies virus (PRV) were studied in Vero cells. Morphological differences were compared using electron microscopy. The progression of cytopathic effects (CPE) observed in light microscopy was also correlated by electron microscopy. Polyacrylamide gel electrophoresis (PAGE) technique was used to analyse the virus-specified protein profiles. The antigenicity of the structural and non-structural virus proteins were also studied using Western blotting. Initially the effect of serial passaging at the sub-optimal temperature of was studied. Two sets of passaged virus strains from the TC114.0p37 and TC442.14p37 wild-type virus were used in this study. Comparison was also made with the two vaccine strains PRVAC and Duvaxyn. Cytopathic effects (CPE) were taken as markers for virulence in the in vitro study in Vero cells. The CPE of the wild-type virus infections was expressed in the form of cell rounding as well as syncytial formation. Both the vaccine strains and the passaged virus strains expressed only cell rounding. Although serial passaging at 30°C did alter the form of expression of the CPE in the passaged virus infections, it was not an effective means for attenuation. This was illustrated by the reversion of some of the passaged strains when the infected cells were incubated at 37°C. Generally the cytopathic effects observed by light microscopy and the ultrastructural changes seen with electron microscopy correlated well for most of the wild-type and attenuated virus strains studied. Whenever CPE was not observed no progeny virus particles or nucleocapsids were seen in the thin sections. A high proportion of the nucleocapsids from attenuated virus infections did not possess the dense cores. Even when the cores were present, they were of varying densities. It was also observed that there were more than one mode of envelopment of the nucleocapsids. The most common means was through the nuclear membrane but envelopment by the cytoplasmic membranes was also evident. The consistent structural and non-structural virus-specified proteins expressed by most of the virus strains studied were GP130, GP125, Pll5, GP98, P82, GP74, P65, GP58, P45 and P41. Most of these virus-specified proteins were found to be antigenic by Western blot analysis. This technique seemed to be more sensitive in detecting low accumulations of viral proteins as some of the viral proteins which were not visible in the protein profiles showed reaction bands in the Western blots. The GP98 seemed to be more evident in the wild-type strains compared to the attenuated strains. All the wild-type and passaged virus strains investigated did express the GP130 glycoprotein. This glycoprotein was reported to be absent in an avirulent vaccine strain of PRV. With purified extracellular and intracellular viruses, the antigenic proteins commonly observed were GP130, GP125, PllS, GP98 and GP62. It could be concluded that glycoproteins GP130, GP125 and GP98 are essential gene products as they were found in the infected cells as well as in the structural components of the virus.