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Background The mucus layer provides the first defense that keeps the epithelium free from microorganisms. However, the effect of the small intestinal mucus layer on pathogen invasion is still poorly understood, especially for swine enteric coronavirus. To better understand virus‒mucus layer‒intestinal epithelium interactions, here, we developed a porcine intestinal organoid mucus‒monolayer model under air‒liquid interface (ALI) conditions. Results We successfully established a differentiated intestinal organoid monolayer model comprising various differentiated epithelial cell types and a mucus layer under ALI conditions. Mass spectrometry analysis revealed that the mucus derived from the ALI monolayer shared a similar composition to that of the native small intestinal mucus. Importantly, our results demonstrated that the ALI monolayer exhibited lower infectivity of both TGEV and PEDV than did the submerged monolayer. To further confirm the impact of ALI mucus on coronavirus infection, mucus was collected from the ALI monolayer culture system and incubated with the viruses. These results indicated that ALI mucus treatment effectively reduced the infectivity of TGEV and PEDV. Additionally, Mucin 2 (Muc2), a major component of native small intestinal mucus, was found to be abundant in the mucus derived from the ALI monolayer, as determined by mass spectrometry analysis. Our study confirmed the potent antiviral activity of Muc2 against TGEV and PEDV infection. Considering the sialylation of Muc2 and the known sialic acid-binding activity of coronavirus, further investigations revealed that the sialic acid residues of Muc2 play a potential role in inhibiting coronavirus infection. Conclusions We established the porcine intestinal organoid mucus monolayer as a novel and valuable model for confirming the pivotal role of the small intestinal mucus layer in combating pathogen invasion. In addition, our findings highlight the significance of sialic acid modification of Muc2 in blocking coronavirus infections. This discovery opens promising avenues for the development of tailor-made drugs aimed at preventing porcine enteric coronavirus invasion.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteric coronavirus, induces severe watery diarrhea and mortality in suckling piglets. The spike (S) protein, a critical mediator of viral entry, undergoes extensive N-linked glycosylation. To elucidate the functional significance of these post-translational modifications, we employed a reverse genetics system to generate 19 recombinant PEDV strains with single-site mutations at predicted N-glycosylation sites. In vitro experiments revealed that mutations at residues N118, N216, N726, N1232, and N1249 significantly attenuated viral replication and reduced plaque size. Our data demonstrated that these mutations impaired viral attachment and internalization. Importantly, in vivo pathogenicity assays in piglets indicated that the N1232Q and N1249Q mutants presented minimal faecal viral shedding and no clinical symptoms, suggesting their potential as live attenuated vaccine candidates. These findings underscore the critical role of S protein glycosylation in PEDV infectivity and virulence, providing a molecular basis for rational vaccine design.

The genomes of coronaviruses carry accessory genes known to be associated with viral virulence. The single accessory gene of porcine epidemic diarrhea virus (PEDV), ORF3, is dispensable for virus replication in vitro, while viral mutants carrying ORF3 truncations exhibit an attenuated phenotype of which the underlying mechanism is unknown. Here, we studied the effect of ORF3 deletion on the proliferation of PEDV in Vero cells. To this end, four recombinant porcine epidemic diarrhea viruses (PEDVs) were rescued using targeted RNA recombination, three carrying the full-length ORF3 gene from different PEDV strains, and one from which the ORF3 gene had been deleted entirely. Our results showed that PEDVs with intact or naturally truncated ORF3 replicated to significantly higher titers than PEDV without an ORF3. Further characterization revealed that the extent of apoptosis induced by PEDV infection was significantly lower with the viruses carrying an intact or C-terminally truncated ORF3 than with the virus lacking ORF3, indicating that the ORF3 protein as well as its truncated form interfered with the apoptosis process. Collectively, we conclude that PEDV ORF3 protein promotes virus proliferation by inhibiting cell apoptosis caused by virus infection. Our findings provide important insight into the role of ORF3 protein in the pathogenicity of PEDV.

Porcine epidemic diarrhea virus (PEDV) has caused significant losses in the pork industry, but the mechanism of PEDV infection is still unclear. On the basis of our RNA-Seq data and due to the potential role of sialic acid as a coreceptor, we investigated the function of sialic acid-binding Ig-like lectin 15 (Siglec-15) to determine its role as a receptor in PEDV infection. We found that Siglec-15 enhances PEDV infection by promoting viral adsorption to host cells. Coimmunoprecipitation and immunofluorescence assays revealed that Siglec-15 binds to the S1 subunit and M protein of PEDV. PEDV infectivity was significantly reduced in Siglec-15 knockout mice. In addition, we developed a monoclonal antibody targeting Siglec-15 that can effectively inhibit PEDV infection both in vitro and in vivo. Overall, our study suggests that Siglec-15 may be a receptor for PEDV infection, which is important for related mechanistic studies and reveals a novel target for anti-PEDV therapeutic development.

Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Aminopeptidase N (APN) plays multiple roles in various physiological processes, with its function as a viral receptor in several coronaviruses being one of the most prominent. However, the role of porcine APN (pAPN) in porcine epidemic diarrhea virus (PEDV) has remained controversial. Single-virus tracking enables a more comprehensive dynamic dissection of pAPN utilization during virus entry. In this study, a comparative analysis of pAPN usage by PEDV, transmissible gastroenteritis virus (TGEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) provides more precise and quantitative insights into pAPN’s specific role in PEDV entry. Here, we used molecular docking and surface plasmon resonance (SPR) to demonstrate that pAPN binds to PEDV, with lower affinity than to TGEV. However, pAPN facilitates PEDV replication through internalization only in susceptible cells, not in non-susceptible cells. Using single-virus tracking, we observed that pAPN triggers PEDV internalization via clathrin- and caveolae-mediated endocytosis, resembling a receptor-mediated process. pAPN participates in 35% of PEDV internalization events, but mediates 80% of TGEV internalization, with pAPN-mediated PEDV internalization occurring approximately 60 s slower than TGEV. The dynamic differences in the internalization of PEDV and TGEV mediated by pAPN primarily arise during the binding stage prior to the initiation of accelerated directional movement, whereas their durations of movement are comparable. Additionally, we found that the internalization dynamics of porcine deltacoronavirus (PDCoV), which also uses pAPN as a receptor, are similar to those of TGEV. These findings resolve the controversy surrounding pAPN’s role in PEDV entry, and highlight the dynamic differences in PEDV, TGEV, PDCoV, and SADS-CoV internalization via pAPN at single-virus level, providing a novel theoretical basis for the potential receptor evaluation from kinetic perspective, which could significantly contribute to the development of strategies against future PEDV outbreaks.

Background Porcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV). Results Overall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province. Conclusions Our findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

Surfactin has antiviral activity against various enveloped viruses by inhibiting viral membrane fusion. However, the potential utility of surfactin as an antiviral drug is limited by its cytotoxicity. In this study, 10 surfactin analogues were obtained by chemical synthesis and evaluated to determine their anti-PEDV activities, hemolytic activities, and critical micelle concentrations. The main goal of our study was to develop a safer drug; a surfactin analogue with high anti-PEDV activity and low hemolytic activity. Compared with surfactin, one of the analogues we developed, SLP5, has lower hemolytic activity, with the same antiviral activity. The selectivity index of SLP5 is 52, while the SI for surfactin is 4, in other words, the safe and effective concentration range of SLP5 is 12 times greater than that of surfactin. Like surfactin, SLP5 has a direct antiviral effect on PEDV. Structurally, SLP5 is a linear lipopeptide with three carboxyl groups. Surfactin derivatives similar to SLP5 could be obtained by lactone bond hydrolyzation of surfactin, as well as total synthesis.

Porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. Although the fecal–oral route is generally accepted, an increased number of reports indicate that airborne transmission may contribute to PEDV outbreak. Here, we show that PEDV could cause typical diarrhea in piglets through a nasal spray. Firstly, PEDV can develop a transient nasal epithelium infection. Subsequently, PEDV-carrying dendritic cells (DCs) allow the virus to be transferred to CD3 + T cells via the virological synapse. Finally, virus-loaded CD3 + T cells reach the intestine through the blood circulation, leading to intestinal infection via cell-to-cell contact. Our study provides evidence for airborne transmission of a gastrointestinal infected coronavirus and illustrates the mechanism of its transport from the entry site to the pathogenic site. Outbreaks of porcine epidemic diarrhea virus (PEDV) have seriously affected pig farms around the world. Here, Li et al. show that PEDV can cause disease in piglets when inoculated by nasal spray, and provide insights into the cellular mechanisms underlying PEDV dissemination within the host.