Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in all cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.