Haiku: New paradigm for the reverse genetics of emerging RNA viruses
The entire viral genome, flanked respectively at the 5’ and 3’ untranslated regions (UTRs) by the human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) is schematized at the top of the figure. For each design, called A to E and the classical ISA design (named ‘ISA’), the number of overlapping subgenomic amplicons ranged from three to five (Left panel). These amplicons were amplified by PCR/RT-PCR, purified, pooled and then transfected into permissive cells. Each design was tested with a panel of viruses in mosquito (C6/36, U4.4) and mammalian (SW13, HEK-293) cell lines. After cell transfection and two serial passages, virus replication was demonstrated using a combination of several criteria (CPE, production of viral RNAs and infectious particles in cell supernatant medium). The percentage of success to recover a virus was calculated using the result of four independent experiments of each procedure and is reported (Right panel).