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. 2014 Sep;88(18):10377-91.
doi: 10.1128/JVI.01008-14. Epub 2014 Jul 9.

Genogroup IV and VI canine noroviruses interact with histo-blood group antigens

Sarah Caddy  1 , Adrien Breiman  2 , Jacques le Pendu  2 , Ian Goodfellow  3
Affiliations

Genogroup IV and VI canine noroviruses interact with histo-blood group antigens

Sarah Caddy et al. J Virol. 2014 Sep.

Abstract

Human noroviruses (HuNV) are a significant cause of viral gastroenteritis in humans worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism among human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I (GI), II, and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI to GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV and GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV and to compare it to the binding specificities of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs) and identified α1,2-fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry confirmed that α1,2-fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs, and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into the evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans.

Importance: Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect nonhuman species and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alpha-galactosidase (α-Gal) carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood group antigens. The binding specificity of canine noroviruses is therefore very similar to that of the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses and suggests it may be possible for canine norovirus to infect humans.

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Figures

FIG 1
FIG 1
Binding of CNV VLPs to synthetic oligosaccharides. (A and B) CNV strain C33 VLPs were incubated with a panel of 26 neoglycoconjugates at 37°C (A) and a panel of 5 neoglycoconjugates at 4°C (B) immobilized on immunoplates to determine binding ability. Bound VLPs were detected using an anti-CNV antibody. Synthetic oligosaccharides were conjugated to either HSA or PAA (unlabeled). An asterisk indicates that the oligosaccharide was available for testing attached to both types of conjugate, and the mean OD450 for both is presented. VLP binding to Lewis b was significantly different for each conjugate, and hence, each is shown independently. Schematic structures of the neoglyconjugates recognized by CNV VLPs are presented above the associated bars on the chart. (C) Neoglycoconjugates shown to bind CNV strain C33 were also incubated with two additional CNV strain VLPs (170 and HK) at 37°C. The error bars represent standard errors.
FIG 2
FIG 2
Alignment of GI.1, GII.4, and CNV major capsid protein sequences. Major capsid protein sequences of representative GI.1 (GenBank AAA59229.1) and GII.4 (GenBank ADR78268.1) human noroviruses and the three CNV strains used in this study were aligned using ClustalW. The 8 (GI.1) and 7 (GII.4) residues implicated in HBGA binding from crystallographic studies are shaded. Amino acid residues in the three different CNV strain major capsid proteins that are identical to the key HBGA-binding residues of GI.1 or GII.4 are boxed.
FIG 3
FIG 3
Phenotyping of canine saliva and gastrointestinal samples using ELISA-based assays. (A) Twenty-six canine saliva samples were analyzed to determine the expression of H antigen, A antigen, and Lewis y antigen. Saliva samples 1 to 23 were collected from kenneled dogs, whereas samples D, E, and F were collected from research dogs, from which tissues were also collected. The Fut2 gene was sequenced for 14 dogs, identified by asterisks, in addition to dog B, from which saliva was not available. (B and C) Phenotyping for H antigen (B) and A antigen (C) was also performed for tissue scrapings from the duodenum, jejunum, ileum, cecum, and colon of the six dogs A to F. The error bars represent standard errors.
FIG 4
FIG 4
Immunohistochemical analysis of CNV VLPs binding to canine intestinal tissue sections. VLPs were incubated with tissue sections overnight, and binding was detected using anti-CNV antibody and biotinylated secondary antibody. HBGA expression was determined using anti-A antigen antibody and Ulex lectin. Binding of either VLPs or antibodies/lectin is indicated by the presence of red signal. (A) Binding of CNV strain C33 to jejunal tissue from an A antigen-negative dog. (B) C33 binding to jejunal tissue from an A-positive dog. (C) Binding of C33 to tissue from the pyloric duodenal region of the intestine of an A-positive dog.
FIG 5
FIG 5
CNV VLP binding to canine and human saliva samples. (A) ELISA plates were coated with 26 canine saliva samples with characterized phenotypes (A antigen and Lewis), and the abilities of CNV VLPs (strain C33) to bind were assessed. The samples are ordered according to their CNV VLP binding abilities (low to high). Lewis antigen expression is not shown. (B) Six human saliva samples, each with a different ABO and Lewis phenotype, were used to assess binding of CNV VLPs from the three CNV strains available. (C) CNV strain C33 was next selected as the representative strain to analyze binding to a wider panel of 26 human saliva samples. Le, Lewis antigen.
FIG 6
FIG 6
Binding of CNV and GI.1 VLPs to CHO cells transfected with glycosyltransferases. CHO cells were transfected with rat α1,2-fucosyltransferase B (FTB) cDNA to induce H antigen expression [CHO-FTB (H)] and were cotransfected with FTB and A enzyme to express A antigen (CHO-A) or transfected with FTB and B enzyme to express B antigen (CHO-B). CHO cells transfected with the empty PDR2 vector were used as control cells not expressing HBGAs. Binding of the three CNV strain VLPs (C33, 170, and HK) and GI.1 VLPs to the different cell lines was assessed using FACS. The black lines represent signal in the absence of VLPs but in the presence of the primary and secondary antibodies. The gray lines represent VLP binding. The number above each histogram is the mean fluorescence intensity (MFI) (geometric mean).
FIG 7
FIG 7
Blocking CNV binding to canine samples using synthetic neoglycoconjugates. Neoglycoconjugates were incubated with CNV VLPs for 1 h at 37°C prior to the VLPs being added to either duodenal scrapings in an ELISA-based assay (A) or human HT29 cells in suspension for FACS (B). Synthetic oligosaccharides were conjugated to either HSA or PAA, as indicated beneath each bar. The same conjugates were used in the FACS experiments, where the binding of both CNV strain C33 VLPs and human norovirus GI.1 VLPs was studied. The dashed lines represent signal in the absence of VLPs but in the presence of the primary and secondary antibodies. The black lines represent VLP binding when VLPs were preincubated with PBS only. The gray lines represent the binding of VLPs to cells after they had been preincubated with different synthetic oligosaccharides. The number in gray above each histogram is the MFI (geometric mean) of all cells when VLPs were preincubated with neoglycoconjugate. The second number, in black, for the H type 1 histograms is the MFI of all cells when VLPs were preincubated with PBS only. The error bars represent standard errors.
FIG 8
FIG 8
Enzymatic treatment of canine samples reduces CNV VLP binding. Duodenal scrapings and intestinal tissue sections from an A antigen-positive and an A antigen-negative dog were treated with 1,2-α-fucosidase or a control for 1 h (scrapings) or 18 h (tissue sections) at 37°C. The abilities of CNV VLPs (strain C33) to bind to the scrapings or tissue sections were assessed using an ELISA-based assay (A) and immunohistochemistry (B), respectively. Confirmation that the 1,2-α-fucosidase removed H antigens was achieved by incubating treated tissue sections with Ulex (Anti-H). The error bars represent standard errors.
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