Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.

Dengue virus infection is the most frequent arthropod-borne infection affecting humans in the world. Our understanding of the pathophysiological events leading to mild or severe outcomes of the disease remains limited by the fact that viral target cells in the human body are poorly characterized. One of the most sensitive strategies for detecting cells supporting active replication of this positive-strand RNA virus is the search for the replicative intermediate, an antigenome of negative polarity, by RT-PCR. However, a phenomenon described as 'false priming' of the reverse transcriptase (RT) prevents strand-specific detection. The results of the current study showed that this event corresponds to cDNA synthesis that is independent of any primer addition. This property was general to all RNAs tested and was not associated with small free nucleic acids, such as tRNAs and microRNAs. Rather, it corresponded to initiation of cDNA synthesis from the 3' end of the RNA template, and a model is proposed in which the template RNA snaps back upon itself and creates a transient RNA primer suitable for the RT. Such a property would explain why many assays proposed for detection of a replicative intermediate are not specific, and may help in the development of a molecular biology protocol that could allow replication studies of RNA viruses of human interest, such as dengue virus, hepatitis C virus and enteroviruses.

Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences.

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The 'Recombination Detection Program', bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5' and 3' genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.