Quantification of Endogenous Viral Polymerase, 3Dpol, in Preparations of Mengo and Encephalomyocarditis Viruses

Abstract

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.

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

“…The membranes were rinsed (3×, TBS) and treated with horseradish peroxidase-conjugated secondary antibody (in MT), then rinsed (3×, TBS) again before the bands were visualized by chemiluminescence (ECL kit, Amersham Bioscience). Murine monoclonals (5A12; 1:2000 dilution) against Mengo 2A (Aminev, Amineva, and Palmenberg, 2003a) and Mengo 3D po (8D10) as well as murine polyclonal against the Mengo capsid (Frolov, Duque, and Palmenberg, 1999) have been described. Polyclonal antibodies against tubulin, GFP, eIF3n, B23 (1:1000, Santa Cruz Biotech), S6, eIF4E, 4E-BP1, eIF2α and phosphospecific antibodies against S6, S6K, eIF4E and Mnk1 (1:1000, Cell Signaling Technology) were commercial, as were appropriate secondary antibodies (1:8000) and stains for WGA (rhodamine or fluorescein) and DAPI (Sigma).…”

Expression of Foot-and-Mouth Disease Virus Non-Structural Protein, 3D in Insect Cells and its Application in Detection of Anti-FMDV Antibodies

“…At 60 dpv, only 2 sera remained positive, and at 120 dpv, none of the vaccinated cattle had antibodies detectable by the 3D-ELISA. Although some mature virions contain RNA polymerase [18], it is not demonstrated whether endogenous or exogenous contamination of virions with 3D is responsible for the induction of 3D antibodies [9].…”

“…The amount of antibodies to 3D, and to other NSP, that is detected in sera from vaccinated animals depends on the immunogenicity of the NSP concerned, the amount that is present in the vaccine, the number of vaccinations that the animal has received and the time since last vaccination [15]. This would be a possible explanation for the differences among vaccines in the 3D antibody response in vaccinated cattle, which is influenced by the methodology of antigen production, inclusion of purification steps, the antigen concentration, or the presence of adjuvants [4,9,14] (Alonso et al 1988). For epidemiological studies, sera sampling is usually carried out 4-6 months after the last vaccination in cattle.…”

Coxsackievirus B3 induction of NFAT: Requirement for myocarditis susceptibility

“…The H3 variant of CVB3 was made from an infectious cDNA clone as described previously (Knowlton et al, 1996) and was purified by centrifugation through a sucrose cushion (Frolov, Duque, and Palmenberg, 1999). Virus was grown in HeLa cells until at least 70% of the cell were detached.…”