Dataset Information

Characterization of influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus.

ABSTRACT: The coronavirus spike protein and the influenza virus hemagglutinin are class I viral membrane fusion proteins. While the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. Whether they are functionally interchangeable is currently unknown. In this study, we constructed scIAV-S, a single-cycle influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (PEDV), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. Treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. Moreover, the infectivity of scIAV-S was associated with the degree of cell adaptation of PEDV-S. Intriguingly, scIAV-S lacking functional neuraminidase (NA) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of PEDV. Together, scIAV-S offers a robust platform for the investigation of the entry mechanism of PEDV or, possibly, of other coronaviruses.

SUBMITTER: Wanitchang A

PROVIDER: S-EPMC7087185 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows.MethodsAffinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge.Results and conclusionOf three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.

Project description:BackgroundPorcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied.MethodsTo accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies.ResultsFive antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666-789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 (722SSTFNSTREL731) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure.ConclusionsA mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722SSTFNSTREL731) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.

Dataset Information

Characterization of influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets