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Q: 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 m 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 g of plasmid DNA and 750 g 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 M. 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 m 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).
A:
Structure and immune recognition of the porcine epidemic diarrhea virus spike protein