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

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens

Les infections à influenzavirus de sous-types H5 chez des canards domestiques (étude des possibilités de prévention par la vaccination avec des pseudoparticules virales)

Les influenzavirus aviaires faiblement pathogènes (LPAI) de sous-types H5 et H7 peuvent après mutation devenir hautement pathogènes (HP) et avoir de graves conséquences en santé publique et animale. Les canards ont un rôle essentiel dans la transmission des LPAI, il est donc primordial de contrôler et d empêcher la circulation des LPAI chez cette espèce. Parmi les vaccins recombinants ou inactivés évalués chez le canard, aucune donnée n existe concernant la protection offerte vis-à-vis des LPAI H5. Nous avons généré un baculovirus triple recombinant permettant l expression des protéines HA, NA et M. La protection offerte par un lysat de cellules exprimant les 3 protéines recombinantes a été évaluée chez des canards de Barbarie après infection par le LPAI H5N3 homologue. Une diminution significative de l excrétion virale cloacale et un retard du pic d excrétion virale trachéale ont été observés. Pour optimiser la formation de VLPs, le plasmide triple recombinant a été modifié. Les VLPs présentent à leur surface des protéines HA et NA biologiquement actives et antigéniques. Un baculovirus quadruple recombinant a été généré en ajoutant le gène M2 et la formation de VLPs a été confirmée. Le pouvoir vaccinant des deux types de VLPs devra être évalué.H5 and H7 low pathogenic avian influenza (LPAI) viruses can mutate into highly pathogenic (HP) and lead to serious problems in both animal and public health. Domestic ducks playing a pivotal role in the transmission cycle of H5 LPAI viruses, it s essential to control and prevent the circulation and spread of these viruses in this species. A few inactivated and recombinant vaccines have been assessed in ducks, mainly for preventing HP H5N1 influenzavirus. Nevertheless, H5 LPAI virus duck vaccination is poorly documented. We generated a triple recombinant baculovirus allowing the expression of HA, NA and M proteins. The protection afforded by cell lysate expressing three recombinant proteins was assessed in Muscovy ducks against H5N3 LP virus challenge. A significant decrease of cloacal shedding and a delayed peak of tracheal shedding were observed. To improve the VLPs formation, triple recombinant plasmid was modified. HA and NA were present on the VLPs surface and were biologically active and antigenic. A quadruple recombinant baculovirus was generated by adding the M2 gene, and VLPs formation was confirmed. The protection afforded by the two types of VLPs will have to be evaluated.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Serological evidence for a non-protective RHDV-like virus

The data were recorded during a Rabbit haemorrhagic disease outbreak that occurred in France in 2001 in a wild population of rabbits that we have been monitoring since 2000. These data suggested the existence of non-protective antibodies due to a putative RHDV-like virus. Twenty-one blood and 22 liver samples were taken from the 26 corpses of recently dead rabbits that were found. RHDV was found in all liver samples. A first screening for RHD antibodies, carried out using an ELISA based on the detection of VP60-RHDV antigen, showed that 20 of the rabbits were seropositive. Moreover, we determined antibody titres for 13 of these 20 seropositive samples. All were $\geq$ 1/400. Such titres normally indicate antibody levels sufficient to confer protection to all known RHDV or RHDV-like strains. For 16 samples, we determined whether these rabbits had died of a chronic or an acute form of the disease, by employing monoclonal antibody (Mabs) – based differential ELISA. All had died of an acute form of RHD. Because the antibodies detected by this VP60-ELISA test are known to appear 5–6 days after infection and since acute RHD generally kills the rabbits 2–3 days after infection, we assumed that the detected antibodies must have been present before the exposure to the virus that killed these rabbits. A second detection of antibodies was made with Mabs that are specific for RHDV. The results were negative, showing that the antibodies detected with the VP60 ELISA test were not specific for RHDV. We sequenced a portion of the VP60 gene of viruses isolated in 17 rabbits. All RHDV isolates were very similar to the RHDV strains commonly isolated in France during this period, suggesting that this viral strain was not a putative variant that is not neutralised by antibodies. Therefore we conclude that the detected antibodies were probably due to a RHDV-like virus that induces the production of detectable but non-protective antibodies