Histo-Blood Group Antigens Act as Attachment Factors of Rabbit Hemorrhagic Disease Virus Infection in a Virus Strain-Dependent Manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50–90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level

Norovirus-glycan interactions

Noroviruses (NVs) are among the most common viral pathogens which target the gastrointestinal (GI) tract and cause severe diarrhea, vomiting and episodes of abdominal cramps with fever. Millions of people around the world get infected with NVs annually, of which 200,000 cases are estimated to be fatal. Yet for decades, the failure of propagating the human NVs in a cell-culture model has hampered NV infection research and consequently treatment and vaccine development. Thus, the research has mainly been focused on epidemiology and studies on the interaction of model virus-like particles (VLPs) with potential host receptors or attachment factors to gain understanding of the first steps of the infection in order to successfully pave the way for effective clinical therapy or prophylaxis. Motivated by this theme, the emphasis of the present work is mainly on protein-carbohydrate interactions of host glycans with NV VLPs. About 80 % of NV outbreaks reported to date are caused by GII.4 genocluster of NVs. Therefore, due to its dominating clinical importance GII.4 NV-like particles and capsid protein dimers are used for in vitro and in silico studies included in the thesis work. NVs have been shown to recognize host histo-blood group antigens (HBGAs) as viral receptors or attachment factors. To investigate the molecular details of interactions of GII.4 NVs with a repertoire of fucosylated HBGAs, molecular dynamics studies were initially carried out based on the crystal structure of B-trisaccharide HBGA in complex with VA387 GII.4 norovirus P dimer. The results, which were later confirmed by crystallographic studies, could explain, on an atomic level, the binding characteristics from a mutagenesis study carried out earlier on the same NV strain. Along with the modelling studies theoretical binding energies were also estimated for different HBGAs binding to VA387 P dimers. The atomic details of binding modes revealed how a single fucose binding site could exploit two different binding modes of the same glycan. This was supported by a literature review of the occurrence of similar fucose binding sites and modes observed in nature for fucose binding lectins and antibodies. One of the objectives of the thesis was to understand the dynamics of virus host interactions at the cell surface membrane, as it holds clues to very early steps of virus infection. Therefore, total internal reflection fluorescent microscopy (TIRFM) was developed to study the binding events of glycosphingolipid (GSL)-containing vesicles to single NV like particles bound to a supported lipid bilayer (SLB). The advantage of this single vesicle binding assay is the ability to analyze the attachment-detachment kinetics both in transient and steady state conditions. Therefore, it enabled us, for the first time, to discriminate between compositionally different GSL-containing vesicles based on their detachment activation energy. This relates directly to the binding strength of the virus-vesicle complex thereby providing new insights into the characteristics of binding virus-like particles to various lipid bound glycans. Moreover, the differences in the distribution of detachment energy of activation for different GSL-containing vesicles were also analyzed. Microdomains or clustered patches of GSLs with or without cholesterol are dynamic integral parts of most of the plasma membranes. Their role has been implicated in virus infection of HIV and influenza virus but not in NVs. However for the first time, NVs were shown to recognize galactosylceramide (GalCer) microdomains in supported lipid bilayers. The atomic details of the binding mode of these interactions are, however, still to be clarified. In conclusion, the thesis describes details of viral protein - host carbohydrate interactions at the molecular level, of relevance for understanding virus infection and design of novel anti-viral strategies

Strain-Dependent Norovirus Bioaccumulation in Oysters ▿

Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction

FUT 2 polymorphism and outcome in very-low-birth-weight infants

BACKGROUND: To determine whether the secretor gene fucosyltransferase (FUT)2 polymorphism G428A is predictive for adverse outcomes in a large cohort of very-low-birth weight (VLBW) infants. METHODS: We prospectively enrolled 2,406 VLBW infants from the population-based multicenter cohort of the German Neonatal network cohort (2009-2011). The secretor genotype (rs601338) was assessed from DNA samples extracted from buccal swabs. Primary study outcomes were clinical sepsis, blood-culture confirmed sepsis, intracerebral hemorrhage (ICH), necrotizing enterocolitis (NEC) or focal intestinal perforation requiring surgery, and death. RESULTS: Based on the assumption of a recessive genetic model, AA individuals had a higher incidence of ICH (AA: 19.0% vs. GG/AG: 14.9%, P = 0.04) which was not significant in the additive genetic model (muitivariable logistic regression analysis; allele carriers: 365 cases, 1,685 controls; OR: 1.2; 95% CI: 0.99-1.4; P = 0.06). Other outcomes were not influenced by FUT2 genotype in either genetic model. CONCLUSION: This large-scale multicenter study did not confirm previously reported associations between FUT2 genotype and adverse outcomes in preterm infants