Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs

Abstract

Porcine epidemic diarrhea (PED) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. Consequently, it causes significant economic losses to the pork industry. There are limited studies on the innate immune responses to PED virus (PEDV) in pigs. The aims of our study were to investigate differences in innate immune responses to PEDV infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. Weaned 26-day-old pigs (n=20) and 9-day-old nursing pigs (n=20) were assigned to PEDV inoculated or uninoculated control groups. The pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (PIDs) 1 and 5 to assay immune responses. Blood samples were collected at PIDs 1, 3 and 5. The natural killer (NK) cell frequencies, NK cell activities (lysis of target K562 tumor cells in vitro), CD3+CD4+ T cell and CD3+CD8+ T cell frequencies were measured in blood and ileum at PIDs 1 and 5. The PEDV infected suckling pigs showed severe diarrhea and vomiting at PID 1, whereas the PEDV infected weaned pigs showed milder clinical signs starting at PID 3. PEDV infected suckling pigs had significantly higher diarrhea scores, earlier fecal PEDV RNA shedding and significantly higher viremia (viral RNA in serum) compared to weaned pigs. There was no mortality in either infected suckling or infected weaned pigs. The control pigs not inoculated with PEDV did not show any clinical signs and no detectable fecal or serum PEDV RNA. Strikingly, PEDV infected suckling pigs had significantly lower NK cell frequencies, undetectable NK cell activity and lower IFNγ producing NK cells in blood and ileum compared to PEDV infected weaned pigs. Pro-inflammatory cytokine profiles of PEDV infected suckling pigs differed from those of PEDV infected weaned pigs and coincided with onset of fecal PEDV RNA shedding and serum PEDV RNA titers. The infected suckling pigs have higher and earlier increases in serum IFNα, but lower serum IL-8 and TNFα levels compared to infected weaned pigs. CD3+CD4+ T cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in CD3+CD8+ T cell frequencies. In conclusion, the observations of impaired lytic activity and IFN-γ production by NK cells in suckling pigs coincided with the increased severity of PEDV infection in the suckling pigs compared with the weaned pigs.

Comparative Proteome Analysis of Porcine Jejunum Tissues in Response to a Virulent Strain of Porcine Epidemic Diarrhea Virus and Its Attenuated Strain

“…In addition, Cao et al have proven in vitro that PDEV could inhibit the production of IFN-β in its infected porcine intestinal epithelial cells [28]. However, recently, Thavamathi et al have verified that suckling pigs infected with PEDV have a higher and earlier increase in serum IFNα [29], which may explain the upregulation of those ISG proteins in the present study. It is also known that IFN regulates the production of ISGs by activating the JAK-STAT signaling pathway [30].…”

Susceptibility of porcine IPEC-J2 intestinal epithelial cells to infection with porcine deltacoronavirus (PDCoV) and serum cytokine responses of gnotobiotic pigs to acute infection with IPEC-J2 cell culture-passaged PDCoV

“…The levels of IFNα, IL-6, IL-12, IL-22, and TNFα were quantitated by ELISA in the cell culture supernatants (for IFNα, IL-6, and TNFα) or the serum samples collected from the two PDCoV-inoculated Gn pigs 1-2 and the four mock-inoculated Gn pigs 3-6 at PID 1, as described previously (Annamalai et al, 2015;Azevedo et al, 2006;Chattha et al, 2013). Briefly, Nunc Maxisorp 96-well plates were coated with antiporcine IL-6 (0.75 μg/ml, goat polyclonal antibody), anti-porcine IL-12 (0.75 μg/ml, goat polyclonal antibody), anti-porcine IFNα (2.5 μg/ml, clone K9) (R&D systems, Minneapolis, MN), anti-porcine IL-22 (2.0 μg/ ml, rabbit polyclonal antibody), and anti-porcine TNFα (1.5 μg/ml, goat polyclonal antibody) (Kingfisher biotech, Saint Paul, MN) overnight at 4 ℃ or 37 ℃ (for IFN-α only).…”

“…The porcine IFN-α detection antibody was biotinylated using a commercial kit, as described previously (Chattha et al, 2013). Plates were developed and cytokine concentrations were calculated, as previously described (Annamalai et al, 2015;Azevedo et al, 2006). The samples were tested in duplicate, and cytokine levels were expressed as the mean values.…”

Porcine epidemic diarrhea virus: current insights

“…106 Type I IFN plays an important role in antiviral immune response; however, several recent reports showed that PEDV has the ability to suppress the IFN antiviral response for 45,[106][107][108][109] These in vitro studies have identified eleven viral proteins responsible for this suppression, which include nsp1, nsp3, nsp5, nsp7, nsp14, nsp15, nsp16, ORF3, E, M, and N. The molecular mechanisms of PEDV-inhibition Type I IFN production include: 1) papain-like proteinase 2 of PEDV antagonizing the IFN response by deubiquitinating retinoic acidinducible gene-I and stimulator of IFN genes; 107 2) PEDV N protein suppressing the IFN regulatory factor 3 (IRF3) and nuclear factor kappa-B activities and antagonizing the IFN-β production by disrupting the interaction between IRF3 and TANK-binding kinase 1; 45 3) PEDV infection in intestinal epithelial cells inhibiting dsRNA-mediated IFN-β induction by interfering with IRF3 activity associated with retinoic acid-inducible gene-I-mediated signaling pathway; 108 4) PEDV nsp1 interrupting the enhanceosome assembly of IRF3 and cAMP response element binding protein by degrading CREB-binding protein via the proteasomedependent pathway; 106 and 5) PEDV-encoded 3C-like protease (nsp5)-mediated proteolytic cleavage of NEMO directly being involved in inhibition of IFN-β transcription. 109 A report by Annamalai et al 110 showed that the innate immune responses to PEDV infection vary with the age of host. PEDV-infected suckling pigs had significantly lower natural killer (NK) cell frequencies, undetectable NK cell activity, and lower IFN-γ-producing NK cells in blood and ileum compared to PEDV-infected weaned pigs.…”