Classical swine fever virus Erns 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 deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of E-rns from this cell line were indistinguishable from those of CSFV E-rns. Two E-rns deletion mutants were constructed, virus Flc23 and 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 protease cleavage sites. Virus Flc22 is not recognized by a panel of E-rns antibodies, due to a deletion of 66 amino acids in E-rns. The E-rns deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by E-rns-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that E-rns from SK6c26 cells was incorporated in the viral particles. 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 structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) E-rns in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.