Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny

Réarrangements génomiques et génétique inverse des rotavirus

Les rotavirus (RV) sont les principaux agents des gastro-entérites aigües du nourrisson. Leur génome est composé de 11 segments d ARN bicaténaire. Le réarrangement génomique est l un des mécanismes de la variabilité génétique des RV. Cet événement rare consiste en une duplication partielle de la séquence d un segment suite à une recombinaison intra-moléculaire. Les conséquences des réarrangements génomiques sur la capacité réplicative de RV à génome réarrangé n ont été que peu étudiées. Nous avons montré que les segments réarrangés 7 et/ou 11 ne confèrent pas d avantage réplicatif aux RV humains par rapport à leur homologue sauvage. Paradoxalement, les segments réarrangés sont ségrégés préférentiellement dans la descendance virale lors d expériences de compétition entre un RV à génome réarrangé et un RV sauvage. L ensemble de ces résultats suggère que les segments réarrangés 7 et/ou 11 des RV humains sont encapsidés préférentiellement à leur homologue sauvage lors de la réplication virale. Nous avons ensuite développé un système de génétique inverse pour les RV basé sur l utilisation de l encapsidation préférentielle des segments réarrangés. Ce système qui utilise comme exogène un segment réarrangé et comme virus auxiliaire un RV bovin a permis l obtention des RV recombinants ayant intégré un segment 7 réarrangé modifié in vitro, notamment un RV recombinant qui exprime une protéine NSP3 modifiée d un poids moléculaire correspondant au double de celui de la protéine sauvage. Enfin, des expériences complémentaires ont été initiées afin de généraliser le système à d autres segments réarrangés ou à d autres virus auxiliairesPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

ETUDE D'UN ROTAVIRUS HUMAIN POSSEDANT UN GENE 7 REARRANGE CODANT UNE PROTEINE NSP3 MODIFIEE

PARIS-BIUM (751062103) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Etude de l'immunité innée anti-rotavirus chez l'homme et vaccination rectale chez la souris fondées sur l'utilisation de pseudo-particules virales

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

DIAGNOSTIC DES INFECTIONS RESPIRATOIRES A CHLAMYDIA PNEUMONIAE ET MYCOPLASMA PNEUMONIAE PAR PCR DUPLEX (DES BIOL. MED.)

PARIS6-Bibl. St Antoine CHU (751122104) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Diversité génétique des erythrovirus humains

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The effect of trypsin on the growth in vitro of adenoviruses present in faeces.

International audienc

Rearrangements of rotavirus genomic segment 11 are generated during acute infection of immunocompetent children and do not occur at random.

International audienceGroup A rotaviruses are the main cause of viral gastroenteritis in infants. The viral genome consists of 11 double-stranded RNA (dsRNA) segments. Dysfunction of the viral RNA polymerase can lead to gene rearrangements, which most often consist of partial sequence duplication of a dsRNA segment. Gene rearrangements have been detected in vivo during chronic infection in immunodeficient children or in vitro during passages at a high multiplicity of infection in cell culture, suggesting that these replication conditions lead to selective advantages favoring the recovery of viruses with rearranged genes. During acute rotavirus infection, the replication level is high, but the occurrence of rearrangement events has never been reported. By the use of a reverse transcription-PCR assay specifically designed to detect small numbers of copies of rearranged forms of segment 11 in a high background of its standard counterpart, we detected 12 rearrangement events among 161 cases (7.5%) of acute rotavirus infection in immunocompetent children. Strikingly, in all but one case, rearrangement took place at the same location within the short direct repeat AUGU sequence. For the unique case with a different rearrangement pattern, the rearrangement occurred within the direct repeat ACAAGUC that was specific for this isolate. In conclusion, we report the occurrence of segment 11 rearrangements during acute rotavirus infection in immunocompetent children. We show that under such conditions of infection, the viral RNA polymerase generates rearrangements which occur not at random but within direct repeats which might constitute hot spots for RNA recombination