The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

The Open Reading Frame 3 (ORF3), an accessory protein of porcine epidemic diarrhea virus (PEDV), has been shown to interact with a myriad of cellular proteins, among which include the IκB kinase β (IKBKB). Here, specific IKBKB domains responsible for ORF3–IKBKB interaction were identified. Dysregulation of NF-κB and Type I interferon (IFN) in the presence of ORF3 was also demonstrated. We showed that while ORF3 was capable of up-regulating IKBKB-meditated NF-κB promoter activity, it surprisingly down-regulated the activation of IKBKB-meditated IFN-β promoter and expression of IFN-β mRNA. When overexpressed, ORF3 could suppress Poly I:C mediated type I IFN production and induction. Finally, we demonstrated that IKBKB- and RIG-I-mediated type I IFN induction by ORF3 resulted in different outcomes. Our study is the first to demonstrate the potential and complex roles of ORF3 in the involvement of aberrant immune signaling as well as in the virus–host interaction.