Poster (Scientific congresses and symposiums)
Regain of fitness through in vitro replication for a recombinant murine norovirus
de Oliveira-Filho, Edmilson; Ludwig, Louisa; Toffoli, Barbara et al.
2016noro2016, Second International Conference on Noroviruses: “Norovirus and Beyond - Glycans as Drivers in Viral Infection”
 

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Abstract :
[en] INTRODUCTION Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Recombination can create considerable changes in viruses, allowing for complete antigenic shifts, host jumps and fitness and pathogenesis modifications. Mathijs et al. recently isolated a viable recombinant murine norovirus (RecMNV) in vitro after coinfection of two parental MNV strains (MNV1-CW1 and -WU20) in a mouse leukaemic monocyte-macrophage cell line (RAW 264.7). The ensuing RecMNV possessed reduced in vitro fitness compared to its parental strains but has also been shown to have retained in vivo infectivity (Mathijs et al, submitted). The aim of this study was to follow the replicative and genetic adaptations of RecMNV over serial in vitro passages in order to characterise its capability of replicative fitness adaptation. MATERIALS AND METHODS: RecMNV was serially replicated in vitro in monolayers of RAW 264.7 cells over ten passages. Following a first initial infection at an MOI of 0.05, cell layers were consecutively infected with 100 μl neat supernatant of the preceding passage. Two independent lysis plaque assays were performed in triplicate with RecMNV progenies resulting from the first (early) and tenth (late) passage (RecE and RecL). Viral plaque sizes of RecE and RecL were measured with image processing program Image J and statistical analyses of plaque size diameters were subsequently performed. To obtain the complete genome sequences of RecE and RecL, a sequencing strategy was developed in which the MNV genome was divided into seven regions and amplification was performed using overlapping primers. Nucleotide sequences of RecE and RecL were analysed via BioEdit Sequence Editor. Growth curves of RecE and RecL progenies were established for high (10) or low MOI (0.01). RESULTS After ten in vitro passages, viral lysis plaque size diameters had increased significantly. Molecular analysis of RecMNV and both parental strains showed nine nucleotide mutations in the RecMNV genome, comprising three non-silent mutations. In addition, a mutation at position 7245 (A187G) introduced a stop codon, resulting in a 20 AA shorter VP2 in RecMNV (for both RecE and RecL). A comparison of RecE and RecL revealed four non-silent mutations in the NS1-2 and NS7 region of ORF1, two of which were present in the latter region (G1384D and S1393N). DISCUSSION This is the first study in which the fitness of a recombinant NoV strain was evaluated in vitro. Our data provides evidence of viral adaptation to a new environment (here a cell culture system) after a recombination event. Evidence of gain-of-function of RecMNV was demonstrated by differences in growth curves and viral lysis plaque size. In addition, non-silent mutations associated to the gain-of-function/in vitro adaptation were detected. It is noteworthy, that the mutation causing a shorter VP2 in RecE and RecL did not compromise its ability to infect and replicate either in vitro or in vivo (Mathijs et al, submitted). As a perspective we should like to characterise the precise mutation(s) responsible for the fitness regain via infectious clone assay.
Disciplines :
Veterinary medicine & animal health
Author, co-author :
de Oliveira-Filho, Edmilson;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Ludwig, Louisa  ;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Toffoli, Barbara;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Di Felice, Elisabetta;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Mathijs, Elisabeth;  Department of Virology - Molecular Platform, Veterinary & Agrochemical Research Centre, 1180 Brussels, Belgium
Thiry, Etienne ;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Mauroy, Axel ;  Université de Liège > Département des maladies infectieuses et parasitaires (DMI) > Virologie vétérinaire et maladies virales animales
Language :
English
Title :
Regain of fitness through in vitro replication for a recombinant murine norovirus
Publication date :
18 March 2016
Event name :
noro2016, Second International Conference on Noroviruses: “Norovirus and Beyond - Glycans as Drivers in Viral Infection”
Event organizer :
University of Lübeck and the Society for the advancement of Glycosciences
Event place :
Lübeck, Germany
Event date :
17-03-2016 to 19-03-2016
Audience :
International
Funders :
CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico [BR]
Fonds Léon Fredericq [BE]
DADD - German Academic Exchange Service [DE]
SPF Santé - Service Public Fédéral Santé publique. Sécurité de la Chaîne alimentaire et Environnement [BE]
Funding text :
FSE Regione Abruzzo –Università degli Studi di Teramo progetto speciale dottorati di ricerca in Clinica e Terapia d’urgenza Veterinaria XXVII ciclo, anno 2011 (E.D.F.).
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