Characterization of a Cytopathogenic Reporter CSFV

Abstract

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

DNAJC14-Independent Replication of the Atypical Porcine Pestivirus

“…Indirect immunofluorescence assays were performed as described earlier ( 74 ). Cells were fixed with 4% paraformaldehyde for 20 min at 4°C, permeabilized with 0.5% (vol/vol) Triton X-100 (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS), stained using the respective MAbs, and visualized using goat anti-mouse IgG conjugated with Cy3 (Dianova, Hamburg, Germany) or goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC; Dianova) in PBS with 0.05% (vol/vol) Tween 20 (PBS-T; Invitrogen, Karlsruhe, Germany).…”

“…Since genomic duplications turned out not to be stable in the context of a normal plasmid (for example, pBR322), a different strategy was chosen to generate duplication mutants. Hence, the APPV clone with a duplication of the NS3-to-NS4B cassette was constructed in a bacterial artificial chromosome (BAC) backbone as described for CSFV ( 74 ). The BAC backbone (pBeloBac11; NEB) was amplified using the oligonucleotides SP6-BACGA_rev and APPV-3′-BACGA_forw and Q5 polymerase (NEB).…”

Immunogenicity of the recombinant adenovirus fusion-expressing E0-E2 gene of the classical swine fever virus

“…Frontiers in Microbiology frontiersin.org pigs to produce neutralizing antibodies against CSFV infection (Xu et al, 2020;Reuscher et al, 2021). The glycoprotein E2 has been proven to be the main structural protein of CSFV and can induce a protective response in pigs (Panyasing et al, 2018).…”