Quantification of endogenous viral polymerase, 3D(pol), in preparations of Mengo and encephalomyocarditis viruses.
Measurement of an antigenic response to the aphthovirus infection-associated antigen (VIA), the viral RNA polymerase 3D(pol), is frequently used as a discriminating assay for the extent of viral replication in animals. In practice, animals seropositive for VIA are assumed to have been exposed to live virus, although in fact it is suspected that endogenous 3D(pol) in commercial inactivated vaccines may occasionally stimulate analogous responses and result in false-positive tests for virus exposure. Cardiovirus infections in mice produce similar anti-VIA antibodies, and in view of recently developed attenuated Mengo vaccines and live Mengo vectors, these VIA responses are also under investigation as potential correlates of vaccine efficacy. We have purified recombinant Mengo 3D(pol), developed monoclonal antibodies to the protein, and used these reagents in highly sensitive Western blot assays to quantify the levels of endogenous 3D(pol) in Mengo and encephalomyocarditis virus (EMCV) preparations. The presence of 3D(pol) was detected at all stages of standard vaccine purification procedures, including materials purified by CsCl. Clarified suspensions of Mengo- or encephalomyocarditis virus-infected HeLa cells were found to contain very high quantities of 3D(pol), averaging approximately 1.2-1.5 micrograms of protein/micrograms of virus. Pelleting through 30% sucrose or purification by CsCl removed much of this material, but even these samples retained approximately 0.2-0.4 ng of 3D(pol)/micrograms virus. These ratios represent approximately 1 3D(pol) molecule/20 virus particles in the most highly purified materials and probably indicate that 3D(pol) is a contaminant on the particle surface rather than an intrinsically packaged molecule. In clarified cell lysates, which are commonly used as vaccine inocula, the protein to virus ratio was approximately 210:1, a level that could represent serious contamination problems for future VIA detection if such inocula are used without further purification.
Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin, 53706, USA. vfrolov@biochem.wisc.edu
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D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003588:Cytopathogenic Effect, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D008574:Mengovirus / Q000201:enzymology*; D051379:Mice; D012324:RNA-Dependent RNA Polymerase; D014776:Virus Cultivation
C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases