Most variation we observed in the VP1 sequences of recent GII.17 strains mapped in or near these epitopes, which may lead to herd immune escape or alter human susceptibility to GII.17 NoV, thus partially explaining the prevalence of this novel variant (Chen et al., 2015). To identify amino acids regulating antigenic differences over time, six GII.17 D mutants reacted with four GII.17 wild-type strains immunized serum. cross-reactivity and cross-protection of their respective antisera using the expression of norovirus P particles in a flexible hinge (Vinj, 2015). P domain name is further divided into the central P1 subdomain and a highly variable P2 subdomain, with the latter, which is essential for virion binding with potential neutralizing antibodies and histo-blood group antigens (HBGAs; Tan et al., 2003). Structural changes of the capsid may lead to the possible emergence of a new phenotype (Mikael et al., 2003). In 2014/2015, the sudden outbreaks of GII.17 Kawasaki variant have raised widespread concern (Xue et al., 2016b; da Silva Ribeiro de Andrade et al., 2018). GII.17 genotype was grouped into four different clusters (clusters ACD) based on the maximum likelihood analysis (Xue et al., 2016c). Strains in clusters A ranged from 1978 to 2015 and in cluster B emerged from 2005 to 2009. Strains detected after 2012 were divided into cluster C and cluster D (Sang and Yang, 2018). Distances of strains in cluster A ranged from 2.1 to 2 2.6% indicated the stability of the gene over a long period of time. However, the distances between cluster B and cluster A were 7.0C8.0%. The distances between recent strains (cluster C and D) to earlier strains (cluster A and B) increased by 10.0C15.0%, indicating that the recently emerged GII.17 strains were new variants (Chen et al., 2015). GII.17 genotype evolved at a rate of 1 1.6810?3 (95% HPD: 0.7910?3 C 2.6510?3) nucleotide substitutions/site/year (Sang and Yang, 2018). The lack of cell culture systems or small animal models for human diseases severely hinders the development of vaccines and therapeutic agents. Moreover, these viruses are highly heterogeneous and undergoing antigenic variation in response to human immunity, further complicating our understanding of the complex immune interactions that regulate susceptibility and disease (Debbink et al., 2012b). Recent studies have shown that HBGAs are the attachment factors of human norovirus, which are critical in determining NoV CA-074 Methyl Ester host susceptibility and epidemiology (Van Trang et al., 2014; Nordgren and Svensson, 2019). In addition, the blockade assay has been used as a surrogate neutralization Cd55 assay, and pig gastric mucin (PGM) type III is used as a substrate (Harrington et al., 2002; Lindesmith et al., CA-074 Methyl Ester 2011, 2012). NoV specific antibodies that block the binding of P particles to HBGAs are considered as the best correlate of protection against NoV. Around the capsid surface, amino acids surrounding the binding pocket evolve rapidly, altering HBGA and antibody binding affinity (Debbink et al., 2012a). On one hand, these viruses may evolve to bind with different HBGAs, which indicate that individuals resistant to one pandemic strain may be susceptible to the other. On the other hand, minor sequence and structural differences allow these viruses to escape pre-existing herd immunity, ultimately leading to CA-074 Methyl Ester the emergence of new strains, which are competent to infect the same individuals who had been infected before (Donaldson et al., 2008; Physique 1). While the epidemiology and genetic analyses of new GII.17 variants were reported (Xue et al., 2016a,c), and further study to probe the emergence and its high prevalence is usually significant. Extensive antigenic relationships among NoV GII.17 variants are still unknown and the complex relationship between host protective immunity and virus antigenic heterogeneity are the primary obstacles to vaccine development. In this study, we systematically investigated the antigenic and ligand-binding characteristics of the four time-ordered GII. 17 strains and found potentially critical amino acids that contribute to antigenic variation. Open in a separate window Physique 1 Evolution mechanism of norovirus based on capsid protein variation. Materials and Methods Ethical Statement All the animal protocols performed in this study were approved by the Guangdong Institute of Microbiology Laboratory Animal Ethics Committee (permission number GT-IACUC202004271). Expression and Purification of P Particles in prokaryotic expression system (Tan and Jiang, 2005). A cysteine-containing peptide was linked to the N or C terminus of the P domains to promote P-particle formation by forming intermolecular disulfide bridges (Tan and Jiang, 2005). Briefly, the sequences encoding the P domains of NoV GII.17 A-D were amplified by PCR and cloned into the expression vector pGEX-4T-1. After sequence confirmation by sequencing, the bacterial cultures were expressed in BL21 (DE3) with an induction of 0.5 mM isopropyl–D-thiogalactopyranoside (IPTG) at 20C for 20 h. Purification of P protein-glutathione.