Differential gene modulation of pattern-recognition receptor TLR and RIG-I-like and downstream mediators on intestinal mucosa of pigs infected with PEDV non S-INDEL and PEDV S-INDEL strains
VIROLOGY
Authors: Temeeyasen, G.; Sinha, A.; Gimenez-Lirola, L. G.; Zhang, J. Q.; Pineyro, P. E.
Abstract
Porcine epidemic diarrhea virus (PEDV) strains can be divided into non-S-INDEL and S-INDEL strains. PEDV pathogenesis is strain-specific, and studies in neonatal pigs have demonstrated that the PEDV non-S-INDEL strains are more pathogenic than the PEDV S-INDEL strains. RNA viruses, including PEDV, can interact with a large number of pattern recognition receptors (PRRs) in the intestinal mucosa, including toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). We investigated the differential gene modulation of TLRs, RIG-I, and downstream mediators on the intestinal mucosa of neonatal pigs infected with PEDV S-INDEL and non-S-INDEL strains. Ten five-day-old piglets were inoculated orally with 10 ml of 10(4) TCDI50/ml of either PEDV non-S-INDEL or S-INDEL strains. PEDV S-INDEL infection induced pro-inflammatory cytokines through the non-canonical NF-kappa B signaling pathway by activating RIG-I. In contrast, PEDV non-S-INDEL infection suppressed the induction of pro-inflammatory cytokines and type 1 interferon production by down-regulation of TLRs and downstream signaling molecules.
Development of a one-step RT-PCR assay for detection of pancoronaviruses (alpha-, beta-, gamma- and delta-coronaviruses) using newly designed degenerate primers for porcine and avian 'fecal samples
JOURNAL OF VIROLOGICAL METHODS
Authors: Hu, Hui; Jung, Kwonil; Wang, Qiuhong; Saif, Linda J.; Vlasova, Anastasia N.
Abstract
Coronaviruses (CoVs) are critical human and animal pathogens because of their potential to cause severe epidemics of respiratory or enteric diseases. In pigs, the newly emerged porcine deltacoronavirus (PDCoV) and reemerged porcine epidemic diarrhea virus (PEDV) reported in the US and Asia, as well as the discovery of novel CoVs in wild bats or birds, has necessitated development of improved detection and control measures for these CoVs. Because the previous pancoronavirus (panCoV) RT-PCR established in our laboratory in 2007-2011 did not detect deltacoronaviruses (delta-CoVs) in swine fecal and serum samples, our goal was to develop a new panCoV RT-PCR assay to detect known human and animal CoVs, including delta-CoVs. In this study, we designed a new primer set to amplify a 668 bp-region within the RNA-dependent RNA polymerase (RdRP) gene that encodes the most conserved protein domain of alpha-, beta-, gamma-, and delta-CoVs. We established a one-step panCoV RT-PCR assay and standardized the assay conditions. The newly established panCoV RT-PCR assay was demonstrated to have a high sensitivity and specificity. Using a panel of 60 swine biological samples (feces, intestinal contents, and sera) characterized by PEDV, PDCoV and transmissible gastroenteritis virus-specific RT-PCR assays, we demonstrated that sensitivity and specificity of the newly established panCoV RT-PCR assay were 100%. 400 avian fecal (RNA) samples were further tested simultaneously for CoV by the new panCoV RT-PCR and a one-step RT-PCR assay with the delta-Coy nucleocapsid-specific universal primers. Four of 400 avian samples were positive for CoV, three of which were positive for delta-Coy by the conventional RT-PCR. PanCoV RT-PCR fragments for 3 of the 4 CoVs were sequenced. Phylogenetic analysis revealed the presence of one gamma-CoV and two beta-CoV in the sequenced samples. The newly designed panCoV RT-PCR assay should be useful for the detection of currently known CoVs in animal biological samples..