The C-strain of the classical swine fever virus (CSFV) is considered the gold standard vaccine for the control of CSF. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA). Consequently, its use can impose severe trade restrictions. The immunodominant and evolutionarily conserved A-domain of the E2 structural glycoprotein is an important target in CSFV-specific ELISAs. With the ultimate aim to render the C-strain suitable as a DIVA vaccine, mutations were introduced that were expected to dampen the immunogenicity of the A-domain. In the first of two approaches, the feasibility of shielding the A-domain by N-linked glycans was evaluated, whereas in the second approach C-strain mutants were created with targeted deletions in the A-domain. Analysis of the antibody responses elicited in rabbits suggested that shielding of the A-domain by an N-linked glycan had a minor effect on the immune response against the A-domain, whereas a targeted deletion of only a single amino acid severely dampened this response. C-strain mutants with larger deletions were highly debilitated and incapable of sustained growth in vitro. By providing the viruses with the opportunity to increase their fitness by mutation, a mutant was rescued that found a way to compensate for the imposed fitness cost. Most of the identified mutations occurred in several independently evolved viruses, demonstrating parallel evolution. By virtue of this compensatory evolution, a well replicating and genetically stable C-strain mutant was produced that can be serologically differentiated from wildtype CSFV. The findings provide the molecular basis for the development of a novel, genetically stable, live attenuated CSF DIVA vaccine.
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