The VP1 homology model allowed us predict the S domain name (67C229) and P1-1 (229C280), P2 (281C447), and P1-2 (448C567) subdomains. had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we decided an intermediate-resolution (8.5-?) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that this P domain name dimers were raised slightly (5 ?) above the S domain name. The VP1 homology model allowed us predict the S domain name (67C229) and P1-1 (229C280), P2 (281C447), and P1-2 (448C567) subdomains. Our results suggested that this raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes. IMPORTANCE We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-? resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that this P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions. INTRODUCTION Human sapovirus (genus family. Other genera in the family include cells were infected with the baculovirus at 26C and harvested at 6 days postinfection. The VLPs secreted into the cell medium were separated from the cells by low-speed centrifugation and then concentrated by ultracentrifugation at 30,000 rpm at 4C for 2 h (Beckman SW-32 Balapiravir (R1626) rotor). The VLPs were purified by CsCl equilibrium gradient ultracentrifugation (Beckman SW-55 rotor) at 45,000 rpm at 15C for 18 h. The morphology of the purified VLPs was verified using electron microscopy (EM). Samples were negatively stained with 2% uranyl acetate and Balapiravir (R1626) examined with an electron microscope (JEOL; JEM-1220) operated at 80 kV. Immunoreactivities of the chimeric and parental VLPs. In order to examine the cross-reactivities among the chimeric and parental VLPs, we performed an antigen enzyme-linked immunosorbent assay (ELISA) with polyclonal rabbit antiserum raised against the parental Mc114 and Yokote VLPs as previously described (13, 14). Microtiter plates (Maxisorp, Denmark) were coated with 100 l (2 g/ml) of VLPs in phosphate-buffered saline (PBS) at pH 7.4. Wells were washed with PBS and then blocked with skim milk. After washing, 100 l of serially diluted antiserum was added to each well. The wells were washed, and then secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma) was added to wells. After washing, 100 l of substrate em o /em -phenylenediamine and H2O2 was added to wells for 30 min. The reaction was stopped with the addition of 50 l of 3 N HCl, and the absorbance was measured at an optical density of 492 nm (OD492). All experiments were performed in triplicate. The final OD492 MAP3K3 was calculated as the samplemean minus the PBSmean (i.e., 0.05). A cutoff limit was set at an OD492 of 0.15, which was Balapiravir (R1626) 3 times the value for the negative control (PBS). Cryo-EM structure of Yokote/Mc114 VLPs. We decided the cryo-EM structure of the Yokote/Mc114 VLPs in order to define regions that may contain immunoreactive epitopes and to identify the S and P domains. The Yokote/Mc114 VLPs were embedded in vitreous ice and examined at 100 K with a cryo-electron microscope (JEM-2200FFC; JEOL, Japan),.