Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDV AVCT12 . Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDV AVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDV AVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.

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.

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Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARSCoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell cultureadapted PEDV AVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation.IMPORTANCE Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.KEYWORDS porcine epidemic diarrhea virus, nucleocapsid, 3C-like protease, cell adaptation P orcine epidemic diarrhea virus (PEDV) infection results in up to 100% mortality in neonatal piglets and has caused substantial economic damage to the swine industry worldwide. Since the end of 2010, outbreaks in many countries in Asia have caused severe economic losses (1). In 2013 to 2014, a particularly large epidemic swept through all of North America, killing more than 7 million pigs (2, 3). Presently, PEDV has evolved into two distinct clades. Classical PEDV strains are CV777-like strains, while

Sequence analysis of the nucleoprotein (NP) of swine-origin influenza virus H1N1 (S-OIV) reveals a number of atypical characteristics including an early start codon and a highly conserved, non-aromatic residue at position 313. Using an in vitro viral polymerase reconstitution assay, we found that the polymerase complex containing the NP of S-OIV (NP(S-OIV)) yielded substantially lower activity than those assayed with NP derived from other influenza virus strains. Moreover, alteration of the early start codon or introduction of an aromatic residue at position 313 (V313Y) did not increase but instead exacerbated the poor polymerase activity. Interestingly, when NP(S-OIV) was allowed to compete with that of a mouse-adapted influenza virus (A/PR/8/34) to form progeny virions, only progeny bearing NP(S-OIV) were produced, despite the low polymerase activity associated with NP(S-OIV). Our results indicated that NP(S-OIV) requires both the early start codon and the V313 residue for its optimal function. These characteristics are required for a strong compatibility between the S-OIV polymerase subunits and its indigenous NP over that of other strains, which might explain why productive reassortment between S-OIV and seasonal influenza viruses has yet to occur in nature.

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Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication.

Influenza virus nonstructural protein-1 (NS1) is abundantly expressed in influenza virus infected cells. NS1 is well recognized for counteracting host antiviral activities and regulating host and viral protein expression. When used as a plasmid component in DNA transfection, NS1 was shown to significantly increase expression levels of a cotransfected gene of different plasmid. Our previous studies demonstrated that addition of an NS1 plasmid increased the expression levels of influenza virus secreted neuraminidase (sNA) gene in 293T cells. In this study, we improved the utilization of NS1 as an enhancer for transient protein expression by generating pFluNS1 plasmid to contain two expression cassettes; one encoding an NS1 gene and another encoding a gene of interest. pFluNS1 is expected to codeliver the NS1 gene into the same cells receiving the gene of interest. The plasmid is therefore designed to induce higher protein expression levels than a cotransfection of an NS1 plasmid and a plasmid containing a gene of interest. To test the efficiency of pFluNS1, influenza virus sNA and non-viral DsRed genes were cloned into pFluNS1. The expression of these genes from pFluNS1 was then compared to the expression from a cotransfection of an NS1 plasmid and an expression plasmid coding for sNA or DsRed. We found that gene expression from pFluNS1 reached equal or higher levels to those derived from the cotransfection. Because the expression from pFluNS1 needs only one plasmid, a lesser amount of transfection reagent was required. Thus, the use of pFluNS1 provides a transfection approach that reduces the cost of protein expression without compromising high levels of protein expression. Together, these data suggest that pFluNS1 can serve as a novel alternative for an efficient transient protein expression in mammalian cells.