An attenuated African swine fever virus expressing the E2 glycoprotein of classical swine fever virus protects pigs against challenge of both viruses

Figures

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  Figure 1.. Construction of the recombinant HLJ/18-7GD-E2 expressing the CSFV E2 protein. (a) Schematic diagram of the replacement of EGFP gene at nucleotides 66,695–67,421 of the HLJ/18-7GD genome by p72E2 gene cassette in HLJ/18-7GD-E2. (b) Purity assessment of the recombinant HLJ/18-7GD-E2 virus by use of PCR. PAMs (lane 1); HLJ/18-7GD (lane 2); and HLJ/18-7GD-E2 (lane 3). (c) Western blotting analysis of E2 expression in PAMs. Uninfected PAMs (lane 1), HLJ/18-7GD-infected PAMs (lane 2), HLJ/18-7GD-E2-infected PAMs (lane 3), and purified CSFV E2 protein (lane 4). (d) Red fluorescence (mCherry) was observed in HLJ/18-7GD- or HLJ/18-7GD-E2-infected PAMs. Green fluorescence (EGFP) was just observed in HLJ/18-7GD-infected PAMs. No EGFP fluorescence was observed in PAMs infected with HLJ/18-7GD-E2. Scale bars represent 50 μm.

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  Figure 2.. Characterization of the recombinant HLJ/18-7GD-E2. To determine the stability of E2 gene in HLJ/18-7GD-E2 genome, the virus was passaged in PAMs for 15 times, and E2 genes were amplified from the cell supernatants at different passages by using PCR (a). “M” means DNA marker. 7GD means HLJ/18-7GD virus. E2 protein expression was detected in the recombinant virus-infected PAMs during passages by Western blot (b). The numbers indicate passage numbers of HLJ/18-7GD-E2. “NC” indicates negative control (uninfected PAMs). “P” indicates the recombinant E2 protein as a positive control. To determine growth kinetics of HLJ/18-7GD and HLJ/18-7GD-E2, PAMs were infected with HLJ/18-7GD or HLJ/18-7GD-E2 at an MOI of 0.1. At the indicated timepoints, viral DNA was assessed by using qPCR (c) and live viruses were titrated by using TCID50 titration (d) in cell supernatants. Data were presented as the mean ± standard deviation.

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  Figure 3.. The virulence of the recombinant HLJ/18-7GD-E2 in pigs. (a) To investigate the virulence of HLJ/18-7GD-E2, 10 SPF pigs were intramuscularly inoculated with 106 TCID50 of HLJ/18-7GD-E2, and eight naïve pigs were inoculated with cell culture medium as controls. All pigs were monitored daily for clinical signs and survival for 28 days post-inoculation (dpi). To determine the protection induced by HLJ/18-7GD-E2 against ASFV and CSFV, Groups of five immunized pigs and four control pigs were challenged with 102.5 HAD50 of ASFV HLJ/18 and 105 TCID50 of CSFV Shimen virus on 28 dpi. (b) Body temperatures of the pigs immunized with HLJ/18-7GD-E2 during the 28-day observation period. (c) The survival of the pigs immunized with HLJ/18-7GD-E2 during the 28-day observation period.

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  Figure 4.. Immune responses induced by HLJ/18-7GD-E2 in pigs. To determine the immune responses induced by HLJ/18-7GD-E2, the serum samples were collected from the immunized pigs and control pigs. Anti-ASFV antibodies were detected by using a commercial ELISA against the p30 protein (a), and cellular response of PBMCs from the immunized pigs and control pigs was detected on 25 dpv by using ELISpot assay (b). In addition, anti-CSFV E2 antibodies were detected at the indicated timepoints by using a commercial ELISA kit (c). The horizontal dashed line represents the cut-off value.

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  Figure 5.. Protective efficacy of HLJ/18-7GD-E2 against challenge with virulent ASFV. Groups of five pigs were vaccinated with 106 TCID50 of HLJ/18-7GD-E2 and then intramuscularly challenged with 102.5 HAD50 of HLJ/18 on 28 dpv (a–c). Groups of four unvaccinated pigs were challenged as controls (d–f). The body temperature (a and d) and survival (b and e) were monitored daily, and tissues were collected for viral genome detection by using qPCR (c and f). The dashed black lines indicate the normal temperature (40°C). LN 1, intestinal lymph node; LN 2, inguinal lymph node; LN 3, submaxillary lymph node; LN 4, bronchial lymph node; LN 5, gastrohepatic lymph node; and LN 6, mediastinal lymph node.

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  Figure 6.. Protective efficacy of HLJ/18-7GD-E2 against challenge with lethal CSFV. Groups of five pigs were vaccinated with 106 TCID50 of HLJ/18-7GD-E2 and then intramuscularly challenged with 105 TCID50 of CSFV Shimen virus on 28 days post-vaccination (a–c). Groups of four unvaccinated pigs were challenged as controls (d–f). The body temperature (a and d) and survival (b and e) were monitored daily, and the indicated tissues were collected for viral genome detection by using RT-PCR (c and f). The dashed black lines indicate the normal temperature (40°C).