Hyun-Jin Shin

The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus

Abstract The spike protein of Porcine epidemic diarrhea virus is the main surface glycoprotein involved in virus attachment and entry and therefore is the target of neutralizing antibodies. Here, the immunogenicity of a novel antigenic domain found on the carboxy-terminal of the spike protein characterized by the peptide motif GPRLQPY, was evaluated. A synthetic peptide whose linear sequence is identical to the 24a.a. carboxy-terminal portion of the spike protein (S-CT24) elicited a strong antibody response in BALB/c mice that had specific reactivity against the S-CT24 and PEDV. These antibodies were shown to have a specific affinity to the GPRLQPY motif, as demonstrated by non-reactivity with a peptide that lacks this motif. In addition, antiS-CT24 antibodies exhibited neutralizing activities against KPEDV-9 in focus reduction neutralization tests suggesting that the GPRLQPY motif induces neutralizing antibodies against PEDV.

Phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (PEDV) neutralizing epitopes.

Abstract Seven-mer phage random peptide libraries were panned against 2C10, a monoclonal antibody that showed neutralizing activities against PEDV. Recombinant M13 phages displaying the peptides SHRLP(Y/Q)(P/V) or GPRPVTH on the g3p minor coat protein showed strong binding affinity with 2C10 (70% and 30% of recovered phages, respectively) after multiple panning. Sequence analysis suggested that these peptides are similar with 1368GPRLQPY1374 found at the carboxy-terminal of the S protein. In neutralization inhibition assays, the two peptide motifs and a 24-mer synthetic peptide corresponding to the C-terminal endodomain of PEDV S protein were observed to compete for the antigen binding site of 2C10, as demonstrated by the loss or reduction of neutralizing activity of the monoclonal antibody. This new finding suggests that the newly discovered peptide motifs mimic a neutralizing epitope PEDV.

Receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion

Porcine epidemic diarrhea virus (PEDV) infection in Vero cells is facilitated by trypsin through an undefined mechanism. The present study describes the mode of action of trypsin in enhancing PEDV infection in Vero cells during different stage of the virus life cycle. During the viral entry stage, trypsin increased the penetration of Vero-cell-attached PEDV by approximately twofold. However, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon PEDV infection. Trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. Furthermore, we also show that the PEDV spike (S) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. These findings indicate that trypsin affects only cell-attached PEDV and increases infectivity and syncytium formation in PEDV-infected Vero cells by cleavage of the PEDV S protein. These findings strongly suggest that the PEDV S protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion.

Porcine epidemic diarrhea virus infects and replicates in porcine alveolar macrophages

Abstract Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine epidemic diarrhea; consequently, the small intestine was believed to be its only target organ. In this study, we found that PEDV infected not only the small intestines, but also the respiratory tract. Infection and replication of PEDV in the respiratory tract from naturally PEDV-infected piglets were examined by reverse transcription polymerase chain reaction, immunohistochemistry, and virus re-isolation. Our observations were confirmed by experimental inoculation, and we found that PEDV infection in the respiratory tract was specifically associated with alveolar macrophages in the lung. Vero cell-adapted PEDV was able to replicate in both primary alveolar macrophages and continuous porcine alveolar macrophage cells. Sequencing analysis of the spike (S) glycoprotein revealed that mutations in S might be a potential determinant of auxiliary targets for PEDV. The discovery that PEDV infects and replicates in alveolar macrophages provides new insights into its pathogenesis.

Antioxidative and Antiviral Properties of Flowering Cherry Fruits (Prunus serrulata L. var. spontanea)

The phenolic compounds of many fruits have been known to be efficient cellular protective antioxidants. In this study, antioxidative and antiviral properties of flowering cherry cultivars (Prunus yedoensis, Prunus sargentii, Prunus lannesiana, and Prunus cerasus) in Korea were investigated. The antioxidant property was assayed for specific activities including 2,2-diphenyl-1-picrylhydrazyl (DPPH) hydroxy radical scavenging activity, reducing power capacity, and superoxide dismutase (SOD) like activity. In addition, antiviral activity was determined by inhibition studies on the infection cycle of porcine epidemic diarrhea virus (PEDV), measured as minimum concentration of cherry extracts that inhibited 50% of cytopathic effect (CPE) on PEDV. Our results show that the four varieties of cherries contain substantially high antioxidants and antiviral activities. In particular, P. cerasus contains higher antioxidants and antiviral activities as well as polyphenolic content than other varieties. Our data indicate that Korean native cherry cultivars could be beneficial supplements of dietary antioxidants and natural antiviral agents.

Clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into Vero cells

Abstract Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. Similar to other coronaviruses, PEDV spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. However, the entry mechanism of PEDV is not studied. Here, we determined the entry mechanism of PEDV into Vero cells. Our data confirmed that PEDV entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. The internalized PEDV was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. In addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to PEDV. Our data revealed that PEDV entry followed clathrin-mediated endocytosis and was dependent on a low pH and serine proteolysis for successful entry into cells.

Evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study

Background: Porcine epidemic diarrhea virus (PEDV) is an infectious, highly contagious virus, and is an etiological agent of acute entero-pathogenic diarrhea in swine. Objectives: Evaluation of the antibody response of two types of PEDV vaccines is to be carried out. Animals and methods: Sows were vaccinated with either live or killed commercial PEDV SM98 (GenBank: GU937797.1) vaccines. Four different groups of sows with five sows in each group were used in this study: the unvaccinated negative control group, the killed virus vaccination group with killed virus boosting (K/K), the live virus vaccinated group with live virus boosting (L/L), and the combination group vaccinated with live virus and subsequently boosted with killed vaccine (L/K). Sows were vaccinated intramuscularly twice at four and two weeks prior to farrowing with 2ml/head vaccine dose. Antibody titers in sow and piglet serum one week after farrowing and that in colostrum were compared by enzyme-linked immunosorbent assay (ELISA) and serum neutralization test. Results: Vaccination with K/K vaccine induced the highest level of IgG and IgA in sow serum, colostrum, and especially in piglet serum, with the lowest levels found in the L/L group. The major neutralizing activity was also found in the K/K group, particularly in colostrum, with piglets bearing higher neutralizing activity compared to sow sera. Among recombinant spike S1, S2, S3, and nucleocapsid N protein of PEDV, S3 protein presented the highest antibody level in the K/K group. Conclusion: Killed PEDV SM98 vaccine induced higher antibody levels. Clinical importance: This study clearly confirms that killed vaccine has induced higher antibody levels and may contribute to the design of future research and vaccine programs.

Application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus.

Abstract A focus formation assay (FFA) for detection and titration of porcine epidemic diarrhea virus (PEDV) in a micro-culture system using Vero cells and PAP staining technique was evaluated. A linear correlation between the virus dilution and virus titer determined by FFA was observed between the range of 10 and 30 foci per well. Comparative analysis between FFA and plaque assay showed no significant difference in estimating the titer of cell adapted PEDV. However, the culture time required for detecting the virus was considerably shorter for FFA. In addition, FFA had higher sensitivity for detecting field isolates of PEDV as well as positive identification of the virus with the antibody specific reaction. A broader range of dilutions and number of replicates may be used for titration. A FFA may be applied as an alternative method for detection and titration of PEDV.

Development of transgenic mouse model expressing porcine aminopeptidase N and its susceptibility to porcine epidemic diarrhea virus.

Abstract Porcine coronavirus infections have known as they are specific to pigs with predominantly enteric or respiratory diseases. No laboratory animal model is yet been developed in porcine coronaviruses study. Here, we report that development of a transgenic mouse model expressing porcine APN which is susceptible to porcine coronavirus infection. The porcine APN transgene was constructed by fusing with mouse proximal APN promoter at 5′ terminus and bovine growth hormone polyadenylation site at its 3′ terminus. After screen on pubs from the microinjected mice, we confirmed two transgenic lines expressing porcine APN in various organs. We confirmed the susceptibility to porcine epidemic diarrhea virus, one of the porcine coronaviruses. These transgenic mice will be an important tool for research into the porcine coronaviruses.

Comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs

Background: Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen of swine. Objective: Serum neutralization (SN) and enzyme-linked immunosorbent assay (ELISA) test results as well as the utility of spike proteins S1, S2, and S3 and entire nucleocapsid protein were compared. Animals and methods: Serum samples from 400 pigs vaccinated against PEDV strain SM98P were collected from 78 farms in Korea. SN test and ELISA were performed to confirm the presence of antibodies. For prokaryotic expression of partial fragments of spike protein the size and location of S1, S2, and S3, and full nucleocapsid protein, polymerase chain reaction was performed using specific primers. Results: Comparison of these results demonstrated that there was a correlation between the SN and ELISA results. Sera with higher neutralizing activity also had higher IgG titer. The antibody profiling data presented the correlation of neutralizing activity with the level of spike protein antibody. In particular, the S3 region may have an important role in neutralizing activity. Conclusions: We confirmed that the carboxy-terminal region that includes the endodomain of the S protein induced stronger neutralizing activity than the region that includes the ectodomain. Clinical relevance: The region of the S protein may have a stronger neutralizing KPEDV-9 epitope and could be useful for the evaluation of future PEDV vaccine efficacy.