Qualitative and quantitative analysis of the binding of GII.4 norovirus variants onto human blood group antigens.

Noroviruses (NoVs) are one of the leading causes of gastroenteritis in children and adults. For the last 2 decades, genogroup II genotype 4 (GII.4) NoVs have been circulating worldwide. GII.4 NoVs can be divided into variants, and since 2002 they have circulated in the population before being replaced every 2 or 3 years, which raises questions about the role of their histo-blood group antigen (HBGA) ligands in their evolution. To shed light on these questions, we performed an analysis of the interaction between representative GII.4 variants and HBGAs, and we determined the role of selected amino acids in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the amino acids, directly implicated in interactions with HBGAs. However, the ablation of the threonine residue at position 395 (ΔT395), an epidemiological feature of the post-2002 variants, was not deleterious to the binding of the virus-like particle (VLP) to the H antigen, while binding to A and B antigens was severely hampered. Nevertheless, the ΔT395 VLPs gained the capacity to bind to the Lewis x and sialyl-Lewis x antigens in comparison with the wild-type VLP, demonstrating that amino acid residues outside the HBGA binding site can modify the binding properties of NoVs. We also analyzed the attachment of baculovirus-expressed VLPs from six variants (Bristol, US95/96, Hunter, Yerseke, Den Haag, and Osaka) that were isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs. We showed that the six variants could all attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, Den Haag and Osaka variants additionally bound to carbohydrates present in the saliva of Lewis-positive nonsecretors. The carbohydrate binding profile and the genetic and mutagenesis analysis suggested that GII.4 binding to Lewis x and sialyl-Lewis x antigens might be a by-product of the genetic variation of the amino acids located in the vicinity of the binding site. Analysis of the binding properties for the six variants by surface plasmon resonance showed that only post-2002 variants (i.e., Hunter, Yerseke, Den Haag, and Osaka) presented strong binding to A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post-2002 variants. The combination of increased affinity for ABH antigens and of a newly acquired ability to recognize glycans from Lewis-positive nonsecretors could have contributed to the epidemiological importance of strains such as the Den Haag GII.4 subtype.

Infection by Helicobacter pylori expressing the BabA adhesin is influenced by the secretor phenotype.

Helicobacter pylori (Hp) infects half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. Our aim was to evaluate the significance of secretor and Lewis status in infection and in vitro adherence by Hp expressing BabA adhesin. We enrolled 304 Hp-infected individuals from Northern Portugal. Gastric biopsies, blood and saliva were collected. Polymerase chain reaction (PCR) and immunofluorescence were used to detect BabA+ Hp in gastric biopsies. In vitro adherence by a BabA expressing Hp strain to gastric biopsies was performed. Secretor status was identified by Ulex, a lectin that recognizes secretor-dependent glycan structures in saliva and in gastric mucosa, and by Lewis(a/b) antibodies, and indirectly by identification of an inactivating mutation in the FUT2 gene (G428A). BabA status of infecting Hp was associated with CagA and VacAs1 (p < 0.05), intercellular localization of Hp (p < 0.01) and the presence of intestinal metaplasia (p < 0.05) and degenerative alterations (p < 0.005) in the biopsies. BabA was associated (p < 0.05) with Ulex staining of gastric biopsies and, although not significantly, to absence of homozygosity for FUT2 G428A inactivating polymorphism. In vitro Hp adherence was higher in cases wild-type or heterozygous for FUT2 G428A mutation (p < 0.0001), cases staining for Ulex (p < 0.0001) and a(-)b+ and a(-)b(-) secretor phenotypes (p < 0.001). In conclusion, BabA+ Hp infection/adhesion is secretor-dependent and associated with the severity of gastric lesions.

Detection of antibodies to rabbit haemorrhagic disease virus: an immunoblotting method using virus-coated human erythrocyte membranes.

The virus of rabbit haemorrhagic disease (RHDV) was purified from infected rabbit liver homogenate by using its property to bind to human red blood cells. Lysates from virus coated cells contained a 60 kDa protein identified as the major viral protein. Immunoblots prepared with that preparation were proved to be useful for immunochemical analysis since the 60 kDa component was intensively stained by subsequent incubation with rabbit sera from infected rabbits and with a secondary labelled antibody. The sera from 114 rabbits were analysed with this test and the data were compared with those obtained by using the haemagglutination inhibition test (HIT). Among the 114 field sera tested by Western blot, 86 contained antibodies to the 60 kDa RHDV antigen whereas only 76 showed positive reaction by HIT. The sensitivity and the specificity of the Western blot were 0.85 and 0.45, respectively, with a concordance between the two techniques of 0.72. Additionally, the European brown hare syndrome virus antibodies reacted with the 60 kDa RHDV protein on immunoblots.

Partial characterization of the human erythrocyte receptor for rabbit haemorrhagic disease virus.

An important, well known property of the rabbit haemorrhagic disease virus is its ability to agglutinate human red blood cells. Accordingly, red cells from human adult donors were agglutinated despite their blood group ABO status, and treatments with proteases or glycosidases did not prevent agglutination. However, we discovered that the cells from human umbilical cords or foetuses were not agglutinated. In order to identify the viral receptor on human erythrocytes, glycolipids and glycoproteins from adult red cells were separated and tested for their potency in inhibiting agglutination. The bulk of the biological activity was associated with the highly glycosylated glycolipids (polyglycosylceramides), whereas a lower but significant activity was also associated with neutral glycolipids. No activity was found in the lipid-free sialoglycoprotein fractions. All these data strongly suggest that the RHDV receptor on human red cells corresponds to a development antigen which is not expressed on foetal cells and is mainly carried by glycolipids. Faint activity was also found in membranes from sheep red cells, suggesting that a similar glycolipid component is carried by these animal cells.

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family.

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.

Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

Evidence of cross-protection within Leptospira interrogans in an experimental model.

Killed whole-cell preparations were used as bacterins against leptospirosis. As this type of protection is considered to be serogroup-specific, several serogroups were added to the usual vaccines, and the most pathogenic serovar was chosen for each group. Different leptospire extracts were evaluated for their protective capacity against acute lethal leptospirosis in gerbils (Meriones unguiculatus). Total extracts induced complete protection against homologous challenges and partial protection against heterologous challenges. LPS fractions protected against homologous but not heterologous challenges, whereas protein extract induced significant protection against both types of challenge. Thus, cross-protection within L. interrogans was related to the protein extract.

Role of the coypu (Myocastor coypus) in the epidemiology of leptospirosis in domestic animals and humans in France.

The coypu (Myocastor coypus), a rodent whose natural habitat is stagnant freshwater, has become a widespread pest in France within the last decade. This study investigated the prevalence of seropositivity and the renal carriage of leptospires in coypus in order to evaluate their role in terms of the risk of infection by Leptospira interrogans in domestic animals and humans. The study involved the application of serological and bacteriological methods to identify leptospires infection and/or carriage in 738 coypus trapped from 1996 to 1999 in six areas of France. Seroprevalence in samples ranged from 16.5 to 66%, and three field strains were isolated (two L. interrogans Icterohaemorrhagiae and one L. interrogans Sejroe). This first report on the isolation of leptospires from coypus in France emphasises the role of this animal in the epidemiology of leptospirosis.

Early stages of rabbit haemorrhagic disease virus infection monitored by polymerase chain reaction.

In order to define more accurately the initial events that take place during rabbit haemorrhagic disease virus (RHDV) infection, different organs of experimentally infected rabbits were analysed for the presence of the virus and correlated with histopathological observations. A total of 24 rabbits were intranasally inoculated with a viral suspension, and tissue samples were taken from the liver, spleen, kidney, lung, thymus, lymph node and tonsil at different intervals post-inoculation (2, 4, 6, 12, 18, 24, 30, 36, 48, 50, 51, 70 and 72 h). Histopathological observations revealed the presence of the first significant lesions at 30 h post-inoculation (p.i.) in the liver. Using an ELISA and a haemagglutination test (HAT), the virus was detected in the liver at 36 h p.i. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the RHDV RNA was present as early as 18 h p.i. in the liver and spleen, whereas thymus, kidney, tonsil and lymph node were found to be positive after more than 36 h p.i. The lungs presented a variable positivity between 0 and 36 h p.i., but remained positive after this time.