Tight junction proteins are important for the maintenance and repair of the intestinal mucosal barrier. The present study investigated relationships among tight junction protein gene expres- sion, porcine epidemic diarrhea virus (PEDV) infection, and intestinal mucosal morphology in piglets. We compared the expression of six tight junction proteins (ZO-1, ZO-2, Occludin, Claudin-1, Claudin-4, and Claudin-5) between seven-day-old piglets infected with PEDV and normal piglets, as well as in PEDV-infected porcine intestinal epithelial cells (IPEC-J2). We also evaluated differences in mucosal morphology between PEDV-infected and normal piglets. The expression of six tight junction protein genes was lower in PEDV-infected piglets than in the normal animals. The expression of ZO-1, ZO-2, Occludin, and Claudin-4 in the intestine tissue was significantly lower (p<0.05) in PEDV-infected than in normal piglets. The expression of Claudin-5 in the jejunum was significantly lower in PEDV-infected piglets than in the normal animals (p<0.01). The expression of Claudin-1 and Claudin-5 genes in the ileum was signifi- cantly higher in PEDV-infected piglets than in normal piglets (p<0.01). Morphologically, the intestinal mucosa in PEDV-infected piglets exhibited clear pathological changes, including breakage and shedding of intestinal villi. In PEDV-infected IPEC-J2 cells, the mRNA expression of the six tight junction proteins showed a downward trend; in particular, the expression of the Occludin and Claudin-4 genes was significantly lower (p<0.01). These data suggest that the expression of these six tight junction proteins, especially Occludin and Claudin-4, plays an important role in maintaining the integrity of the intestinal mucosal barrier and resistance to PEDV infection in piglets.
To explore the role of Toll-like receptors (TLRs) and interferon (IFN) in the innate immunity against porcine epidemic diarrhea virus (PEDV), we detected the expression of TLR genes in PEDV-infected IPEC-J2 cells by real-time PCR. We also detected the level of interferon α (IFN-α) and interferon γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA). Results showed that IPEC-J2 cells exhibited a clear pathological change after PEDV infection at 24 h. In addition, TLR7, TLR9 and TLR10 expressions were significantly upregulated in PEDV-infected IPEC-J2 cells at 24 h. Interestingly, the expression patterns of TLR2 and TLR4 were consistent at different stages of PEDV infection. The expression level of TLR3 decreased significantly with the increase of infection time, but the expression levels of TLR5 and TLR8 genes at 6 h and 12 h were significantly lower than those in the control group (p<0.01). There were significant correlations among the expression levels of TLR genes (p<0.05). Cytokine detection showed that the secretion level of IFN-α in the PEDV-infected group was significantly higher than that in the control group (p<0.01), and IFN-γ at 6 h and 12 h after PEDV infection was significantly higher than that in control group (p<0.01). Therefore, our results suggest that PEDV infection can induce innate immune responses in intestinal porcine jejunum epithelial cells, leading to changes in the expression of Toll-like receptors, and can regulate the resistance to virus infection by affecting the release levels of downstream cytokines.
The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.
Coronaviruses present a considerable concern for humans and animals. The current world- wide pandemic of SARS-CoV-2 virus showed many gaps in understanding of coronaviruses spread and transmission. Because of lack of effective vaccine against SARS-CoV-2 the only preventive measures are represented by wearing protective masks and gloves thus limiting potential risk of contact with the airborne virus. Inversely, the limited time of protective function of the masks presents another drawback of their use. Therefore, the application of disinfection agent dispersed on the surface of protective masks may enhance their effectivity and safety of their application. The aim of the study was to examine the virucidal efficacy of low-concentra- ted sodium hypochlorite dispersed using ultrasonic humidifier on the surface of surgery masks. The study was conducted using SARS-CoV-2 surrogate virus, namely porcine epidemic diarrhea virus (PEDV) representing a model with similar biophysical properties and genomic structure to human coronaviruses. Five different concentrations of the disinfectant with different content of sodium hypochlorite were selected for the study. A final concentration of 0.228 g/L sodium hypochlorite effectively inactivated the PED virus and may support the biosafety of masks usage.
en
pl
