The reaction was developed by the addition of TMB (3,3,5,5-tetramethyl-benzidine, Sigma) and stopped after 10?min incubation with 4?N H2SO4

The reaction was developed by the addition of TMB (3,3,5,5-tetramethyl-benzidine, Sigma) and stopped after 10?min incubation with 4?N H2SO4. block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Ley) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. Conclusion Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation. family and are genetically classified into 6 genogroups (GI-GVI) with a recently proposed genogroup VII [4], although genogroup I (GI) and GII cause most human NoV infections. Despite this diversity over the past two decades most reported NoV outbreaks and epidemics have been caused by NoV GII.4 genotype. Phylogenetic analyses of the GII.4 strains circulating in the last 20?years have shown that this genotype can be divided into distinct variants, which peak and wane over time in a similar pattern to that described for SB290157 trifluoroacetate influenza viruses [5C7]. Several studies have linked NoV susceptibility to histo-blood group antigens (HBGAs), namely with the secretor status associated with the presence of at least one functional allele, and with Lewis antigens (Lea and Leb), determined by the gene [8, 9]. The HBGAs, including the ABO, secretor and Lewis families, are distributed on cell membranes and mucosal epithelia with high polymorphism. HBGAs are synthesized from various disaccharide precursors through sequential additions of monosaccharides with specific linkages catalysed by different glycosyltransferases [10]. The syntheses of the secretor, Lewis and ABO antigens are catalyzed by an -1,2 fucosyltransferase (FUT2), an -1,3 or -1,4 fucosyltransferase (FUT3) and two glycosyltransferases (A and B enzymes), respectively. Homozygote carriers of inactive alleles essentially lack Lea and Leb antigens; such individuals are denoted Lewis-negative and constitute about 5?% of the Caucasian population. Secretor-positive individuals express Leb antigen, while secretor-negative individuals express Lea antigen [11]. Human NoVs are known to recognize HBGAs as attachment factors, with different NoV strains showing different properties regarding the ability to bind to different antigens [8, 10]. The NoV genome is organized in three open reading frames (ORFs). The VP1, encoded by SB290157 trifluoroacetate ORF2, is the major capsid protein, which is further organized into the N-terminal (N), the shell (S), and the protruding (P) domains. The P domain SB290157 trifluoroacetate is divided into two subdomains: P1 and P2 [12]. The P1 subdomain forms the anchoring portion of the P dimer connecting it to the S domain, while the P2 subdomain is exposed on the surface of the capsid protein and is the most variable region of the virus. The main epitopes for immunorecognition and the histo-blood group antigen (HBGA) binding domains reside within this P2 subdomain. The emergence and accumulation of mutations along the P2 subdomain is the main driver of evolution for GII.4 strains, Rabbit Polyclonal to ZNF329 which results in epidemic strains with altered antigenicity and HBGA binding properties [13C16]. It has been reported that NoVs attach to either HBGA expressed on the gastroduodenal epithelial cells of secretor-positive individuals [17, 18]. Human secretor positive saliva and synthetic HBGAs have been used in VLP binding and/or blocking assays in different studies [19C21]. However, it has also been shown that NoV can bind to enterocytes independently of HBGAs [22]. Human NoVs have for long time been elusive to propagation in cell cultures [23, 24], although it has been recently reported that human NoVs can infect B lymphocytes in the presence of HBGA-expressing bacteria [25]. Caco-2 cells, originally derived from a human colonic adenocarcinoma, show morphologic and physiologic markers of differentiation characteristic of the mature small intestine enterocytes, express carbohydrates of the histo-blood group family on their surface, and allow significant attachment of norovirus VLPs mainly when these cells are differentiated [26]. In this study we have assessed the binding properties of VLPs of different variants of NoV GII.4 genotype to Caco-2 cells. Moreover, blockade activity of the VLP binding by porcine gastric mucin (PGM) and monoclonal antibodies (mAbs) against NoV VLPs, anti-Lewis antigens (Lea, Leb, Lex and Ley) or anti-H antigens (H1 and H2) mAbs was investigated in order to better understand the interactions between NoVs and the cellular surface of intestinal cells. Results Distribution of Lewis antigens, secretor status and ABO types among the saliva.

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