Location: Foreign Animal Disease ResearchTitle: Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis Author
|Pacheco Tobin, Juan|
|Rieder, Aida - Elizabeth|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/2012
Publication Date: 12/10/2012
Publication URL: http://handle.nal.usda.gov/10113/56416
Citation: Lawrence, P., Uddowla, S., Pacheco Tobin, J., Kotecha, A., Fry, E., Rieder, A.E. 2012. Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1 G-H loop as a new tool for studying FMDV pathogenesis. Virology. 136(1):150-161. Interpretive Summary: In this study, we designed, produced, and characterized a Foot-and-Mouth Disease Virus (FMDV) containing a marker protein tag (FLAG tag) embedded in an immunogenic region of the virus structure. The FLAG-tagged virus replicated to similar titers as the parental progenitor virus (A24 Cruzeiro) in cell culture and the mutant FMDV was detected with antibodies specific to the FLAG-tag. Moreover, anti FLAG antibodies could effectively neutralize the mutant virus. When inoculated in cattle, they developed disease similar to the original parental virus, but the animals failed to generate antibodies that react with FLAG peptide. Virus particles could be screened with anti-FLAG antibodies making this variant virus a useful tool in the study of the mechanisms of disease for FMDV.
Technical Abstract: In this study, we generated a recombinant foot-and-mouth disease virus (FMDV) particle derived from A24 Cruzeiro with a FLAG tag (DYKDDDDK) substitution in the hypervariable antigenic site of the G-H loop of the VP1 capsid protein in an effort to expand the immunogenicity of the virus particle and to develop a new recombinant viral vector for vaccine studies. The properties of the wild-type (WT) and FLAG-tagged A24 Cruzeiro virus (A24-FLAG) were similar with respect to their growth curves, achieved titers, and susceptible cell lines. The tractable A24-FLAG was detectable by both Western blot and immunocytochemistry using a polyclonal rabbit antibody against FLAG. Moreover, the same polyclonal anti-FLAG successfully neutralized A24-FLAG, but not its WT counterpart. Cattle experimentally infected with the epitope-tagged virus produced anti-FMDV neutralizing antibodies, but failed to generate antibodies reactive with FLAG-tagged proteins by Western blot and ELISA. The lack of sero-conversion to the FLAG tag was investigated by molecular modeling, which revealed that the flexibility of the G-H loop might render the FLAG tag a cryptic site, inaccessible to the host immune system. The A24-FLAG virus will assist in the design and implementation of marker virus vaccines as well as the study of the FMDV life cycle.