Rearrangements of Rotavirus Genomic Segment 11 Are Generated during Acute Infection of Immunocompetent Children and Do Not Occur at Random

Abstract

Group 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.

Frequent rearrangement may explain the structural heterogeneity in the 11th genome segment of lapine rotaviruses — Short communication

“…However, the ability of strains with rearranged genome segment 11 to overgrow other viral strains in vitro has been related to point mutations affecting the phosphorylation pattern of the NSP5, rather than to the length of the rearranged genome segment (Chnaiderman et al, 1998). In addition, a more recent survey on the incidence of rearrangement among viral progenies in immunocompetent children found no evidence for the abilty of strains with rearranged genome segment 11 to overgrow strains with normal genome during acute rotavirus infection (Schnepf et al, 2008). These data, together with the relative rarity of rearranged genome segments in viable wild type rotavirus strains in a variety of host species, indicate that field rotavirus strains, in general, do not systematically exploit this possibility, raising questions on the real evolutionary significance of rearrangement.…”

Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

“…RNA genomic profiles were determined by PAGE of 14% polyacrylamide gels for 16 h at 200 V at room temperature, followed by ethidium bromide staining. The RT-PCR assay for specific detection of rearranged segments 7 in the viral progeny was based on a strategy that we previously described for specific detection of segment 11 rearrangements (36). Primers used for RT-PCR detection of rearranged segments 7 are given in Table 1.…”

“…Fulllength cDNAs for the 7R and 7R⌬ segments were obtained from dsRNAs extracted from the M1 and M3 viruses, respectively. The dsRNAs were reverse transcribed as previously described (36), and PCR amplifications were performed using 5 l of cDNA in a 50-l reaction mixture containing 1ϫ Pfx buffer, 1ϫ Pfx enhancer, 1 mM MgSO 4 , a 0.3 mM concentration of each deoxynucleoside triphosphate (dNTP), a 0.3 M concentration of each primer, and 2.5 U of Pfx DNA polymerase (Invitrogen). Amplification was achieved with a model 9700 Perkin Elmer thermocycler.…”

“…Gene rearrangements were first reported for RV isolated from immunodeficient children with chronic infection (11,26) and can be obtained experimentally by serial passages at a high multiplicity of infection (MOI) in cell culture (10,14,21). We recently showed that rearrangements also occur during acute RV infection (36). Gene rearrangement usually consists of a partial head-to-tail duplication of a segment sequence.…”

Diversity of the G3 genes of human rotaviruses in isolates from Spain from 2004 to 2006: cross-species transmission and inter-genotype recombination generates alleles

“…Four G3 sequences were found to be formed by this event, where the receptor sequences were G3 genotypes and the donors were G1 and G2. This evolutionary mechanism has been extensively described for human genes (Martínez-Laso et al, 2004, probably with variations, and also for RV genes (Cao et al, 2008;Martella et al, 2007; Phan et al, 2007a, b;Parra et al, 2004; Desselberger, 1996;Chnaiderman et al, 1998; Kojima et al, 2000;Patton et al, 2001;Alam et al, 2008;Schnepf et al, 2008).G3-D86273 and G3-D86277. These sequences were generated by the combination of sequence G3-D86282 as a receptor (lineage I) and a fragment from aa 116 to 231 from the G2 genotype as a donor.…”