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

Abstract

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 with 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 with 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.

FIG. 1

Schematic representation of the full-length DNA of pPRKflc2, pPRKflc23 and pPRKflc22. pPRKflc2 is the wild-type full-length DNA copy of CSFV strain C. The amino acid sequence numbering is for the open reading frame of the CSFV strain C (19). The starts of the E^rns and E1 proteins are based on N-terminal sequencing (24), but the carboxy termini of the capsid and E^rns proteins have not yet been determined. N^pro, autoprotease; C, capsid protein; E^rns, E1 and E2 envelope proteins; p7, nonstructural protein p7; 5′, 5′ noncoding region; 3′, 3′ noncoding region.

FIG. 2

Infection of CSFV in SK6 and SK6c26 cells with a multiplicity of infection of 0.05 and detection by immunostaining with MAb b3 against E2. SK6c26 cells infected with Flc22 (a), Flc23 (b), and Flc2 (c) SK6 cells infected with Flc22 (d), Flc23 (e), and Flc2 (f) are shown. Immunostaining was performed 4 days after infection.

FIG. 3

Growth kinetics of the recombinant CSFV Flc22 and Flc23 and the wild-type virus Flc2. Subconfluent monolayers of SK6c26 cells were infected at a multiplicity of infection of 0.1. Viruses were adsorbed for 1.5 h. Virus titers of the total lysates at various time points were determined by end-point dilution on SK6c26 cells.

FIG. 4

RT-PCR of SK6c26 cells infected with viruses Flc22, Flc23, and Flc2 using primers flanking E^rns and E2. −, Negative control (mock-infected SK6c26 cells); M, 200-bp marker.