Composition of CHIKV-Cbn and CHIKV-LR populations grown in mosquito or mammalian cells.

(A) Analysis of the 3'UTR of Caribbean lineage restricted to replicate in mosquito or mammalian cells. RNA was extracted from culture media and used for reverse transcription reaction. The pool of cDNA was amplified by PCR and cloned into pGEM-T Easy vector and individual clones were analyzed. Representative agarose gels for PCR amplification of the 3'UTRs of individual clones recovered from the input in vitro transcribed RNA (upper gel), and from viral populations after five successive passages in C6/36 (middle gel) and BHK (bottom gel) cells. Full-length 3'UTR of CHIKV-Cbn (Cbn) and DNA ladder (M) were used as reference. The sizes of DNA bands in the ladder (in base pairs) are indicated on the right. (B) Pie charts for the frequencies of parental 3'UTR (3'Cbn, grey) and viral variants with novel 3’UTRs, collectively referred to as New 3’UTRs (indicated in orange for C6/36 and in red for BHK cells). Mean percentages from experiments I and II are indicated in white. (C) Schematic representation of the alignment of sequences of CHIKV-Cbn populations grown in C6/36 (left) and BHK (right) cells from two independent experiments (I and II). Deletions within viral variants are indicated with orange and red lines. An 80-nt insertion in Clone 5 from mammalian-grown population is indicated with a yellow line. (D) Titers of infectious particles in the supernatant of mosquito (C6/36, left) and mammalian (BHK, right) cells after the transfection of CHIKV RNAs bearing the 3’UTRs of two viral variants obtained after passaging in mammalian cells. Clone 25 carried the deletion of a DR3 copy and was the most abundant viral variant from experiment II. Clone 15 had a drastic deletion of both DR3 copies. Data were analyzed by two-way ANOVA with Bonferroni´s test using PRISM5 (GraphPad Software). ***, p(E) Analysis of the 3'UTR of La Reunion lineage restricted to replicate in mosquito or mammalian cells. Representative agarose gels for PCR amplification of the 3'UTRs of individual clones recovered from the input in vitro transcribed RNA (left gel), and from viral populations passaged in C6/36 (right upper gel) and BHK (right bottom gel) cells. DNA ladder (M) was used as reference. The sizes of DNA bands in the ladder (in base pairs) are indicated on the right.