Three Amino Acid Substitutions in the Spike Protein Enable the Coronavirus Porcine Epidemic Diarrhea Virus To Infect Vero Cells.

Porcine epidemic diarrhea virus (PEDV), a continuously evolving pathogen, causes severe diarrhea in piglets, with high mortality rates. To prevent or mitigate the disease, it is common practice to develop live or inactivated PEDV vaccines based on cell-adapted viral variants. Propagating wild-type PEDV in cultured cells is, however, often challenging due to the lack of knowledge about the requirements for the cell adaptation of PEDV. In the present study, by using the RNA-targeted reverse genetic system for PEDV to apply S protein swapping followed by the rescue of the recombinant viruses, three key amino acid mutations in the S protein, A605E, E633Q, and R891G, were identified, which enable attenuated PEDV strain DR13 (DR13att) to efficiently and productively infect Vero cells, in contrast to the parental DR13 strain (DR13par). The former two key mutations reside inside and in the vicinity of the receptor binding domain (RBD), respectively, while the latter occurs at the N-terminal end of the fusion peptide (FP). Besides the three key mutations, other mutations in the S protein further enhanced the infection efficiency of the recombinant viruses. We hypothesize that the three mutations changed PEDV tropism by altering the S2' cleavage site and the RBD structure. This study provides basic molecular insight into cell adaptation by PEDV, which is also relevant for vaccine design. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a lethal pathogen for newborn piglets, and an efficient vaccine is needed urgently. However, propagating wild-type PEDV in cultured cells for vaccine development is still challenging due to the lack of knowledge about the mechanism of the cell adaptation of PEDV. In this study, we found that three amino acid mutations, A605E, E633Q, and R891G, in the spike protein of the Vero cell-adapted PEDV strain DR13att were critical for its cell adaptation. After analyzing the mutation sites in the spike protein, we hypothesize that the cell adaptation of DR13att was achieved by altering the S2' cleavage site and the RBD structure. This study provides new molecular insight into the mechanism of PEDV culture adaptation and new strategies for PEDV vaccine design.

Antigenicity Alternations of Variant PEDV S Protein Disclosed by Linear B Cell Epitope Mapping.

The spike protein (S) plays a crucial role in porcine epidemic diarrhea virus (PEDV) infection and induces neutralizing antibodies. Mutations of the S protein are supposed to provide the main antigenic shift leading to the antigenic escape of PEDVs. It is therefore a significant question how much accumulation of antigenic shift could lead to the antigenic escape of the variant PEDV. To provide an answer in the study, B cell epitopes (BCEs) on the S protein of the PEDV vaccine strain CV777 (SCV777) and variant strain SD2014 (SSD2014) were mapped using biosynthetic peptides and rabbit anti-PEDV S serum. Seventy-nine and 68 linear BCEs were identified from SCV777 and SSD2014, respectively. While 66.2% of the BCEs of SSD2014 could be recognized by anti-SCV777 serum and 67.1% of SCV777 BCEs could be recognized by anti-SSD2014 serum, more than 40% of the BCEs identified using anti-SCV777 serum on SCV777 could not be recognized by anti-SSD2014 serum and vice versa. The completely shared BCEs took low percentages of 29.4% and 25.3% for SSD2014 and SCV777, respectively. These results indicate a low conservation of antigenicity of the S protein compared to a relatively high amino acid sequence similarity of 92.2% between the two strains. The study provided a BCE shift reference of PEDV antigenic escape and surveillance control.

Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus In Vitro and In Vivo.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the family Coronaviridae that causes severe diarrhea and high mortality in neonatal suckling piglets. Currently, there is no effective medication against this pathogen. Cepharanthine (CEP), tetrandrine (TET), and fangchinoline (FAN) are natural bis-benzylisoquinoline alkaloids with anti-inflammatory, antitumor, and antiviral properties. Here, we first found that CEP, TET, and FAN had anti-PEDV activity with IC50 values of 2.53, 3.50, and 6.69 µM, respectively. The compounds could block all the processes of viral cycles, but early application of the compounds before or during virus infection was advantageous over application at a late stage of virus replication. FAN performed inhibitory function more efficiently through interfering with the virus entry and attachment processes or through attenuating the virus directly. CEP had a more notable effect on virus entry. With the highest SI index of 11.8 among the three compounds, CEP was chosen to carry out animal experiments. CEP in a safe dosage of 11.1 mg/kg of body weight could reduce viral load and pathological change of piglet intestinal tracts caused by PEDV field strain challenge, indicating that CEP efficiently inhibited PEDV infection in vivo. All of these results demonstrated that the compounds of bis-benzylisoquinoline alkaloids could inhibit PEDV proliferation efficiently and had the potential of being developed for PED prevention and treatment.

Accessory protein: a type of protein that cannot be ignored by coronavirus

Coronaviruses (CoVs) are the largest single-stranded positive-stranded RNA viruses in the genome. Most of them can spread across species and infect humans. They are currently one of the pathogens that cause major public health incidents and seriously threaten human health. The viral genome is about 25-31 kb in length, encoding multiple non-structural proteins, structural proteins (S, E, M, N) and accessory proteins. For most coronaviruses, although accessory protein is a non-essential protein for virus replication, it often plays an important role in the pathogenic process of the virus and is an important functional protein for coronaviruses. This type of protein is located at the 3′ end of the viral genome, and the transcription of the mRNA is regulated by the transcription regulating sequence (TRS) located at the start position of the gene, and the codon usage preference of the protein coding sequence also produces protein translation. Significant influence. The accessory protein has the properties of a transmembrane protein and a unique protein transport motif. The latter plays a decisive role in the formation of the transmembrane region, the topological structure of the protein, and the intracellular transport process of the protein, which directly affects the function of the accessory protein. . This article first summarizes the latest classification and genome structure of coronaviruses;then systematically summarizes relevant research progress in terms of the types, functions, protein transport motifs, topological structures, and codon usage preferences of accessory proteins, and then provides an overview of the next step The research direction has been prospected, which provides an important reference for a more comprehensive understanding of the biological characteristics of the coronavirus accessory protein.

Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Enhances Viral Proliferation by Inhibiting Apoptosis of Infected Cells.

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 ORF3 Protein Is Transported through the Exocytic Pathway.

Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

Mechanism of apoptosis regulation induced by coronavirus in infected cells

Coronaviruses are the common pathogenic microorganisms that infect human and animals and cause health hazards. Cell immune responses are induced to fight against coronavirus infection in infected cells. In order to initiate transcription and translation and to assemble the next generation in infected cells, viruses respond to cellular immune response and participate in many cellular activities. When specific receptors such as death receptors are bound by viral proteins, cells initiate apoptotic processes. Some viral proteins play critical roles in promoting or inhibiting apoptosis in the apoptotic process. For example, S protein induces external apoptotic pathway by binding to death receptor in cell membrane, M and S proteins induce internal apoptotic pathway by causing endoplasmic reticulum stress and Ca2+ imbalance. On the other hand, E protein inhibits apoptosis in infected cells. This article reviews the mechanism of pro-apoptotic or anti-apoptotic effects of coronavirus on infected cells. By understanding the different roles of different viral proteins in extrinsic and intrinsic apoptotic pathways, it is expected to provide ideas for artificial intervention in cell regulation for prevention and control of coronavirus infection.