SK6 Cells Research Articles

Chimeric classical swine fever viruses containing envelope protein E RNS or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete E RNS gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which E RNS was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both E RNS and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of E RNS or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or E RNS antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV.

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns).

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.

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) 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.

Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types.

The effect of specific pseudorabies virus (PRV) antibodies on the enlargement of plaques produced by PRV were studied in monolayers of different cell types. The plaque size was used as parameter for the efficacy of the cell-associated spread (CAS) of PRV. First, the effect of anti-PRV hyperimmune serum on the plaque growth was examined in monolayers of the continuous cell lines ST, SK-6 and MDCK and monolayers of the primary cultures of porcine fibroblasts, endothelial cells and endometrial cells. A tenfold increase in the serum concentration did reduce the plaque size with 50% in both SK-cells and fibroblasts and with 40, 28 and 16% in MDCK, endothelial and endometrial cells, respectively. In ST cells, no change in size was observed with increasing antibody concentrations. Secondly, the effects of monoclonal antibodies (mAbs) directed against PRV glycoproteins gB, gC, gD and gE and polyclonal antibodies against gC were evaluated in SK-6 cells. MAbs against gB, gD and gE were able to reduce the CAS with a cumulative effect between mAbs against gD and either mAbs against gB or mAbs against gE. Monoclonal and polyclonal antibodies against gC did not change the plaque size.

Involvement of membrane-bound viral glycoproteins in adhesion of pseudorabies virus-infected cells.

Cell-associated spread of pseudorabies virus (PrV) plays an important role in the pathogenesis of the disease. Besides the already known direct cell-to-cell spread of the virus in monolayers, adhesion and subsequent fusion of suspended PrV infected cells to monolayers of uninfected cells are thought to occur. To study the adhesion of PrV-infected cells, an in vitro model was developed in SK-6 cells. Specific adhesion of PrV-infected cells to an uninfected monolayer started 5 h after infection of the cells and reached a maximum 6 h later. A correlation was found between the surface expression of PrV glycoproteins on the infected cells and the adhesion of these cells. PrV hyperimmune serum completely inhibited binding of the infected cells. To investigate which PrV envelope glycoproteins were responsible for the cell adhesion, the infected cells were incubated with antisera against glycoproteins gII, gIII, and gp50. Antiserum against either gII or gIII inhibited cell adhesion, and antisera against gII and gIII together had a cooperative effect. Antiserum against gp50 had no effect on binding when used alone but enhanced the inhibition induced by gII and gIII antisera. Heparin and neomycin inhibited adhesion, showing that the receptor for adhesion was a heparinlike substance. SK-6 cells infected with a gIII deletion mutant of PrV exhibited a much lower adhesion. This binding was heparin and neomycin independent and was not blocked by anti-gII serum. Nevertheless, it was completely inhibited with PrV hyperimmune serum and with anti-gp50 serum. This finding demonstrates that the ligand for adhesion of gIII(-)-infected cells is glycoprotein gp50. These results strongly suggest that the mechanism for adhesion of a PrV-infected cell to an uninfected monolayer is similar to the mechanism of adsorption and penetration of a PrV virion to a host cell.

Hog cholera virus: Identification and characterization of the viral RNA and the virus-specific RNA synthesized in infected swine kidney cells

Virus-specific RNA in swine kidney cells infected with hog cholera virus and the RNA of gradient-purified virions were identified and characterized. The time curve for virus replication in SK-6 cells was established. The logarithmic phase of virus growth was between 4 and 12 h after virus adsorption. Virus production was maximum at 20 h after virus adsorption. Analysis of cytoplasmic RNA in a neutral agarose gel showed that the time curve for synthesis of a high molecular weight RNA in infected cells closely paralleled that for virus growth. Electrophoresis in an agarose-formaldehyde gel of [ 3H]uridine-labeled RNA, synthesized with actinomycin-D, showed that this high molecular weight RNA species was approximately 12 kilobases (kb) in length. RNA, extracted from virions purified in a glycerol-tartrate gradient, co-migrated in a neutral agarose gel with the 12 kb RNA species; moreover, it hybridized specifically with a cDNA clone prepared against the intracellular 12 kb RNA. These results confirm the virus-specific nature of the 12 kb RNA. Since no prominent subgenomic virus-specific RNA was identified in infected cells, an RNA species of this size may also act as a messenger RNA.