Reference : Virus-like Particles of Human and Bovine Noroviruses
Scientific congresses and symposiums : Poster
Life sciences : Veterinary medicine & animal health
Virus-like Particles of Human and Bovine Noroviruses
Mauroy, Axel mailto [Université de Liège - ULiège > Département des maladies infectieuses et parasitaires > Virologie vétérinaire et maladies virales animales >]
Scipioni, Alexandra [> >]
Mast, Jan [> >]
Ziant, Dominique [> >]
Ruth, Nadia [> >]
Thiry, Etienne mailto [Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales >]
7th International Congress of European Society of Veterinary Virology
September 24-27th 2006
European Society of Veterinary Virology
[en] 1.Introduction and Objectives
Noroviruses have a positive-sense, single-stranded RNA genome of about 7.5 kb containing three open reading frames (ORFs). ORF1 encodes the non-structural proteins, ORF2 encodes the single capsid protein with a molecular weight of 56 kD and ORF3 encodes a minor structural protein. These viruses are very stable and successful infection can be achieved even with very low infectious dose. Human noroviruses are the major cause of non bacterial, food-borne gastroenteritis worldwide. Contamination is usually oro-fecal (Green et al. 2001). Belonging to the Caliciviridae family, the genus Norovirus also contains viruses infecting animals, in particular bovines. This genus is divided into five genogroups (Ando et al. 2000). The existence of bovine noroviruses raises the question of their possible zoonotic character; the viruses could be transmitted to human by contaminated effluents for example (Scipioni et al., this congress). The study of norovirus is hampered by the lack of a cell culture system. However virus like particles (VLPs) can be produced and they are antigenically and morphologically similar to the wild virus (Jiang et al. 1992). The aim of this work was to obtain VLPs from human and bovine strains. Such VLPs will be used as antigens to produce specific polyclonal and monoclonal antibodies.

2.Material and Methods
The capsid protein genes of a human norovirus strain (H384) and a bovine strain (B309) were amplified by RT PCR from stools collected from human and veterinary diagnostic laboratories. These sequences were analysed by the Basic Local Alignment Search Tool (BLAST) available on the NCBI website. Plasmids containing the ORF2 sequence of two reference human strains, the Norwalk (NV, belonging to the genogroup I) and the Hawaii (HV, belonging to the genogroup II) strains, were kindly received from Jan Vinje (University of North Carolina, USA). The baculovirus protein expression system (Invitrogen®) was used to obtain VLPs. Briefly, cells and supernatants were harvested after freezing/thawing cycles. Cells were pelleted. VLPs were purified from supernatants by ultracentrifugation through a 30% sucrose cushion and then by isopycnic ultracentrifugation in CsCl. Fractions resulting from the gradient were analysed by SDS-PAGE. Protein assembly into VLPs was checked by electron microscopy.

H384 strain ORF2 is closely related to the HuNV/Altenkirchen 140/01/DE strain (99% of identity). Thus, H384 most likely belongs to genogroup II genotype 4 (GII.4, the Lordsdale group). B309 capsid gene is closely related to genogroup III genotype 2 (Newbury2). Electron microscopy confirmed the presence of norovirus-like particles in the supernatant of recombinant baculovirus infected cell culture. By SDS PAGE applied on the fractions resulting of the CsCl gradient, a protein of 56 kD was detected and confirmed the expression of the capsid protein. Therefore, VLPs were obtained for all the 4 strains investigated.

4.Discussion and Conclusions
Currently we dispose of representative surrogates of three norovirus genogroups (GI, GII and GIII). H384 (GII.4) is a representative of the most commonly diagnosed genotype from current outbreaks in many countries (Blanton et al. 2006). These VLPs will therefore serve as antigens to raise polyclonal and monoclonal antibodies. These tools will be used for the diagnosis of the related infections in human and domestic animals.
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