Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B

Abstract

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE(-) was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE(-) vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE(-)/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.

Fig 1

White blood cell and platelet counts in pigs inoculated with the GPE⁻, GPE⁻/P-11, or ALD/A76 strain. Groups of 5 pigs were inoculated with the indicated viruses, and blood was collected on days 0, 3, 5, 7, 9, 11, and 14 p.i. The white blood cell (WBC) (A) and platelet (PLT) (B) counts were determined for each time point and are shown as mean values, with error bars representing the standard deviations. The statistical significance of the differences between the cell counts from the pigs inoculated with GPE⁻ virus and the pigs inoculated with the other viruses was calculated with the t test. a, P < 0.05 between GPE⁻ and GPE⁻/P-11; b, P < 0.05 between GPE⁻ and ALD/A76. One pig died on day 11 p.i. (†). All remaining pigs were euthanized on day 14 p.i.

Fig 2

Schematic representation of the cDNA clones of the GPE⁻/P-11 and mutant vGPE⁻ viruses. (A) Three amino acid substitutions, T830A, V2475A, and A2563V, were found in the GPE⁻/P-11 virus. (B) Seven recombinant viruses with all possible combinations of amino acid substitutions were generated by site-directed mutagenesis of the GPE⁻ backbone. The white and gray boxes indicate the nonstructural and structural proteins, respectively.

Fig 3

White blood cell and platelet counts in pigs inoculated with the vGPE⁻ parent and mutant viruses. Groups of 5 pigs were inoculated with the indicated viruses, and blood was collected on days 0, 3, 5, 7, 9, 11, and 14 p.i. The WBC and PLT counts were determined for each time point and are shown as mean values, with error bars representing the standard deviations. The statistical significance of the differences between the cell counts from the pigs inoculated with vGPE⁻ virus and the pigs infected with the other viruses was calculated with the t test. *, P < 0.05. In each graph, the mean WBC and PLT counts of the pigs inoculated with the vGPE⁻ parent are shown for comparison purposes. The pigs were euthanized on day 14 p.i.

Fig 4

Growth kinetics of the vGPE⁻ parent and mutant viruses and of the ALD/A76 strain in swine cells and guinea pig cells. Swine kidney cell lines (SK-L cells) and the primary guinea pig kidney (GPK) cells were inoculated at a multiplicity of infection (MOI) of 0.001 with the parent and mutant viruses as indicated. The SK-L and GPK cells were incubated at 40°C in the presence of 5% CO₂ and at 30°C, respectively. The supernatants were collected at the indicated times. The titers were determined in SK-L cells and expressed as 50% tissue culture infective doses (TCID₅₀) per ml. In each graph, the titers of the vGPE⁻ and ALD/A76 strains are included for comparison purposes. *, virus titer was below detection the limit of 10^1.8 TCID₅₀/ml.

Fig 5

Focus formation by vGPE⁻, mutant viruses, and ALD/A76 in SK-L cell cultures. SK-L cells were inoculated at an MOI of 0.001 with the parent and mutant viruses as indicated. The cell monolayers were overlaid with MEM containing 1.0% Bacto agar and incubated at 37°C in the presence of 5% CO₂. After 72 h of incubation, the cells were fixed with acetone and viral NS3 was detected by immunofluorescence using MAb 46/1. The number of infected cells per focus was counted for 30 independent foci (n = 30). Error bars represent the standard deviations, and the significance of the differences was calculated with the t test. *, P < 0.05.

Fig 6

Effect of amino acid substitutions in NS4B on RNA replication efficiency. In order to assess the effect of the amino acid substitutions in NS4B on the viral RNA replication efficiency, confluent SK-L cell monolayers were inoculated at an MOI of 1.5 with the parent and mutant viruses carrying the indicated amino acid changes in NS4B. Parallel cultures of infected cells were incubated at 37°C in the presence of 5% CO₂ for 2 h, 7 h, and 12 h. Total RNA was extracted from the infected cells at the indicated times. The relative RNA copy numbers compared with the copy number at 2 h postinoculation are shown. Each bar represents the mean value from 3 independent experiments, and the error bars show the standard deviations. Pairwise comparisons of the values were carried out using the t test. *, P < 0.05.

Fig 7

Effect of amino acid substitutions in NS4B on the viral RNA replication efficiency. (A) A GPE⁻-derived bicistronic replicon consisting of the 5′ and 3′ UTRs flanking a chimeric N^pro-firefly luciferase gene separated from the NS3 to NS5B genes by the IRES of EMCV was constructed. Single amino acid substitutions at positions 2475 and 2563 were introduced by site-directed mutagenesis as indicated. (B) In order to analyze the effects of the amino acid substitutions on the viral RNA replication efficiency, 10^6.8 CPK cells were transfected with 1 μg of each replicon RNA and with 100 ng of pRL-TK (Int⁻) reporter plasmid for constitutive Renilla luciferase expression. After 6 h, 12 h, and 24 h of incubation at 37°C in the presence of 5% CO₂, the firefly and Renilla luciferase activities expressed in relative light units were assayed in cell lysates. The firefly luciferase activity was normalized with the corresponding Renilla luciferase activity from the same lysate. Each bar represents the mean value from 3 independent experiments, with error bars showing the standard deviations. The values obtained with each construct were compared pairwise using the t test. *, P < 0.05.