Fig 1.
Clinical course and viral load in blood after inoculation with the moderately virulent ASFV strain Estonia14.
(A) Body temperature, (B) clinical score, and (C) ASFV genome loads in blood of pigs (n = 10) upon inoculation with ASFV Estonia14. Black symbols show one individual (#94) that developed severe clinical symptoms and succumbed to infection 9 dpi. The dotted lines indicate thresholds for fever (A) and the humane endpoint (B). Lines in panel C represent medians, symbols in all panels represent individual animals.
Fig 2.
Clinical course and viral load in blood after challenge infection with the highly virulent ASFV strain Armenia08.
(A) Body temperature and (B) clinical score of previously Estonia14-inoculated (n = 9, blue) and naïve pigs (n = 10, red) after challenge with ASFV Armenia08. (C) ASFV genome copy numbers in blood of challenged animals. Genome copy numbers were calculated per 1 ml blood. Mind the different scales in (C) for previously inoculated and naïve animals. Numbers indicate ear tags. The dotted lines indicate thresholds for fever (A) and the humane endpoint (B). N.d., Not detected.
Fig 3.
Viral load at necropsy in various tissues after challenge infection with the highly virulent ASFV strain Armenia08.
ASFV genome copy numbers after challenge with ASFV Armenia08 in organs of pigs previously infected with Estonia14 (n = 9, blue, 28 dpc) or naïve animals (n = 10, red, 7-8 dpc). Genome copy numbers were calculated per 100 mg tissue. Symbols represent individual values. Black symbols show animal #94, that died 9 dpi. ghLN, gastrohepatic lymph node; maLN, mandibular lymph node; BM, bone marrow, N.d., Not detected.
Table 1.
Virus isolation results in organs of pigs previously infected with Estonia14 (EST + ARM) or naïve animals (ARM).
Fig 4.
Humoral responses in pigs after inoculation with the moderately virulent ASFV Estonia14 and challenge infection with the highly virulent ASFV Armenia08.
Commercially available competitive ELISAs were used to assess kinetics of antibodies against (A, B) ASFV-p32 and (C, D) ASFV-p72 in sera of (A, C) pigs inoculated with ASFV Estonia14 and then challenged with ASFV Armenia08 (blue, n = 10) or (B, D) naïve pigs (red, n = 10). Grey areas between dotted lines indicate assay threshold, values above were considered positive. ASFV-specific antibodies and their isotypes were assessed by ELISA. Kinetics of (E, F) IgM and (G, H) IgG in sera of (E, G) pigs after inoculation with ASFV Estonia14 and challenge with ASFV Armenia08 (blue, n = 10) and (F, H) naïve pigs after challenge with ASFV Armenia08 (red, n = 10). Dotted lines indicate cut-off values. Animal #94 that succumbed to Estonia14 inoculation is shown in black. Animals that were still positive in qPCR at the end of the trial (28 days after challenge) are shown as bright red squares. Lines represent means, symbols individual values. Mixed-effects analysis (EST + ARM) or repeated-measures ANOVA (ARM) with Holm-Šidák’s correction for multiple comparisons. Compact letter display shows statistically significant differences, with different letters indicating p < 0.05.
Fig 5.
Activation of the complement system after ASFV infection.
Detection of activated complement components (A) C3a and (B) C5a in plasma after inoculation with ASFV Estonia14 and challenge with ASFV Armenia08 (n = 9, blue), or after infection in control animals (dark red, n = 10). The respective reference days (day 0 before infection/challenge) are displayed in grey. Lines represent means, symbols show individual values. Bright red squares among EST + ARM animals indicate individuals where live virus was isolated 28 dpc. Two-way-ANOVA with Holm-Šidák’s correction for multiple comparisons between the timepoints and the respective controls. (C) Correlation analysis with data from 28 dpc in EST + ARM animals (left and middle) and EST + ARM vs. ARM animals 7 dpc (right). N.d., not detected.
Fig 6.
In vitro investigation of complement system responses against ASFV.
Detection of activated complement components (A) C3a and (B) C5a in vitro. Untreated serum of the indicated times after Estonia14 inoculation (plasma of 154 dpi contains ASFV-specific IgG) was mixed with PBS (grey), EDTA (green), or EGTA/MgCl2 (MgEGTA, orange) and then incubated with 106 HAD50/ml ASFV Armenia08 (n = 7-9). Complement activation was assessed by C3a- and C5a-specific ELISAs. (C) Infection rates of monocytes 48 h after infection with Estonia14 (blue) or Armenia08 (red) particles that were pre-incubated with either untreated (white bars) or heat-inactivated (grey bars) plasma from naïve animals (n = 6, in triplicates) for 90 min at 37 °C. One-way-ANOVA with Holm-Šidák’s correction for multiple comparisons. Compact letter display shows statistically significant differences, with different letters indicating p < 0.05. EST, ASFV Estonia14; ARM, ASFV Armenia08.