Attenuation of porcine deltacoronavirus disease severity by porcine reproductive and respiratory syndrome virus coinfection in a weaning pig model

Figure 1.. Clinical assessment of inoculated pigs. The body temperatures, average respiratory and diarrhea clinical scores and ADWG of inoculated pigs were shown as means ± standard deviations (error bars), the animal amount n = 6 at 0 to 8 DPI and n = 3 at 9 to 21 DPI. The asterisk indicates a significant difference between labeled groups (*p < 0.05; **p < 0.01; ***p < 0.001). (a) The rectal temperature of pigs was daily recorded and shown. (b) Respiratory related clinical signs including cough, fever, anorexia, and sneeze, were scored from 0 to 20, and the mean values of the labeled period were calculated. (c) The diarrhea was daily scored and the mean values of each group at 5 to 9 DPI were individually shown. (d) The ADWG was calculated at 7, 14 and 21 DPI

Figure 2.. Viral shedding and viremia. The viral RNA shedding kinetics of PDCoV in fecal swab (a) and PRRSV in nasal swab (b) were tested by RT-qPCR and the titers of PRRSV viremia (c) were test by TCID50 assay, at 7, 14 and 21 DPI. Data were shown as means ± standard deviations (error bars), the animal amount n = 6 at 0 to 8 DPI and n = 3 at 9 to 21 DPI. The asterisk indicates a significant difference between PDCoV single-infection and coinfection groups (*p < 0.05; **p < 0.01; ***p < 0.001)

Figure 3.. Gross and microscopic lesions and IHC examination of inoculated pigs. 3 pigs from each group were randomly selected for necropsy at 8 DPI, for observing the gross and microscopic lesions and virus distribution. (a) Shown are the representative pictures of intestines and lungs gross lesions. (b) Shown are the representative pictures of intestines (up panel) and lungs (down panel) microscopic pathological lesions. Intestines and lung sections were stained with hematoxylin and eosin (H & E). (c) PDCoV and (d) PRRSV distribution in intestines (up panel) and lungs (down panel) were shown in IHC examination, stained by mAbs against their N protein, respectively. Red arrows indicate the positive signals (stained in brown) in virus-infected cells. (e) The scores of gross and microscopic lesions, PDCoV and PRRSV distribution in both intestines and lungs were shown. Data were shown as means ± standard deviations (error bars) (n = 3). The asterisk indicates a significant difference between labeled groups (*p < 0.05; **p < 0.01; ***p < 0.001)

Figure 4.. Detecting TNF-α, IL-1, and IL-6 in intestines and supernatants of PRRSV infected PAMs. Shown are titers of cytokines TNF-α, IL-1, and IL-6 in intestines (a) at 8 DPI and in the supernatants of PRRSV infected PAMs at 24 hpi (b), detected by ELISA kits. Data were shown as means ± standard deviations (error bars). The asterisk indicates a significant difference between labeled groups (*p < 0.05; **p < 0.01; ***p < 0.001)

Figure 5.. The inhibiting effect of PRRSV-infected PAMs supernatants and TNF-α, IL-1, and IL-6 on PDCoV proliferation in IPEC-J2 cells. (a) The titers of PDCoV in the IPEC-J2 cells treated with PRRSV-infected PAMs supernatants, TNF-α, IL-1, and IL-6, respectively. (b) The expression levels of N protein in the PDCoV-infected IPEC-J2 cells treated with different concentrations of TNF-α, IL-1, and IL-6 were measured by WB. (c), (d), and (e) The titers of PDCoV in the IPEC-J2 cells treated with PRRSV-infected PAMs supernatants together with TNF-α, IL-1, and IL-6 inhibitors, respectively. (*p<0.05; **p<0.01; ***p<0.001)

Figure 6.. Determine the inhibition stages of PDCoV proliferation by TNF-α, IL-1, and IL-6 mixtures. Cells were harvested at 2 hpi and 12 hpi, respectively, for RT-qPCR and WB analysis, after treating with TNF-α, IL-1, and IL-6 mixtures. The results showed that there was no difference at the attachment/entry stage and significant difference appeared at the replication stage, suggesting that TNF-α, IL-1, and IL-6 mixtures inhibited PDCoV proliferation at the replication stage