Classical swine fever virus E-rns deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines

An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein E -rns of classical swine fever virus (CSFV) was used to rescue CSFV E-rns de letion mutants based on the infectious copy of CSFV strain C. The biochemic al properties of E-rns from this cell line were indistinguishable from thos e of CSFV E-rns. Two E-rns deletion mutants were constructed, virus Flc23 a nd virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of E-rns (deletion of 215 amino acids) to retain the original protea se cleavage sites. Virus Flc22 is not recognized by a panel of E-rns antibo dies, due to a deletion of 66 amino acids in E-rns. The E-rns deletion muta nts Flc22 and Flc23 could be rescued in vitro only on the complementing SK6 c26 cells. These rescued viruses could infect and replicate in SK6 cells bu t did not yield infectious virus. Virus neutralization by E-rns-specific an tibodies was similar for the wild-type virus and the recombinant viruses, i ndicating that E-rns from SK6c26 cells was incorporated in the viral partic les. Pigs vaccinated,vith Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA,vith a pestiviral str uctural protein and opens new ways to develop nontransmissible modified liv e pestivirus vaccines. In addition, the absence of (the antigenic part of) E-rns in the recombinant viral particles can be used to differentiate betwe en infected and vaccinated animals.