Porcine epidemic diarrhea virus is an alphacoronavirus responsible for significant morbidity and mortality in pigs. A key determinant of viral tropism and entry, the PEDV spike protein is a key target for the host antibody response and a good candidate for a protein-based vaccine immunogen. We used electron microscopy to evaluate the PEDV spike structure, as well as pig polyclonal antibody responses to viral infection. The structure of the PEDV spike reveals a configuration similar to that of HuCoV-NL63. Several PEDV protein-protein interfaces are mediated by non-protein components including a glycan at Asn264 and two bound palmitoleic acid molecules. The polyclonal antibody response to PEDV infection shows a dominance of epitopes in the S1 region. This structural and immune characterization provides new insights into coronavirus spike stability determinants and explores the immune landscape of viral spike proteins. : bioRxiv preprint Baculovirus expressed proteins were produced by infecting 1 L of Sf9 at 2´10 6 cells with 10 mL of second amplification baculovirus and incubating for four days. Infected cell supernatants were harvested by centrifugation at 1500´g to remove cells followed by 12,000´g to remove cell debris. Supernatants were filtered through a 0.45 µm filter and then applied to a column of Streptactin agarose (Qiagen, Cat. #30004). The column was washed with 20 column volumes of Tris-buffered saline and then the protein was eluted with same buffer with 2 mM desthiobiotin. The eluted protein was purified by Superose6 Increase 10/30 (GE Life Sciences, Cat. #29091596) gel filtration. Mammalian expressions were done by transient transfection. For both 293F and 293S cells, 1 L of cells at 1´10 6 cells were transfected with complexes composed of 250 µg of plasmid DNA and 750 µg of polyethylenimine (Polysciences, Cat. # 24765-1). For the expression involving kifunensine, three hours after transfection kifunensine (Cayman Chemicals, Cat. #10009437) was added to a final concentration of 5 µM. Expressions were allowed to proceed for five days. Cell supernatants were collected by centrifugation at 1,500 ´ g to remove cells followed by 12,000 ´ g to remove cell debris. Supernatants were filtered through a 0.45 µm filter and then applied to a column of Streptactin superflow. The column was washed with 20 column volumes of Tris-buffered saline and then the protein was eluted with same buffer with 2 mM desthiobiotin. The eluted protein was purified by Superose6 Increase 10/300 gel filtration. Proteins were concentrated for structural studies using ultrafiltration (Sigma-Millipore, AmiconUltra 4 mL, 30K MWCO, Cat. #UFC903024). After 3-day acclimation, pigs in the challenged group were inoculated with a prototype US PEDV isolate US/Iowa/18984/ 2013 (GenBank accession #KF804028) (Hoang et al., 2013), 10 3 plaque-forming units/pig, via oro-gastric gavage as previously described (Madson et al., 2014). Pigs in the control group received virus-free cell culture media. Some of these pigs were inoculated again with the same strain in the identical manner at day 56 post-inoculation (PI).
Output: Structure and immune recognition of the porcine epidemic diarrhea virus spike protein