Fig 1.
Schematic representation of the ISA-lation method used to recover infectious viruses from clinical samples.
Viral RNA directly extracted from clinical samples was used to amplify by RT-PCR the entire viral genome in 3 overlapping cDNA fragments. The human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA), were then added respectively at the 5’ and 3’ termini of the first and last fragment as described in the Results/Discussion section. Transfection of the 3 final overlapping DNA fragments into permissive cells enabled the isolation and recovery of infectious viruses after 3 to 9 days.
Table 1.
Summary of the different viruses isolated in this study with the ISA-lation method.
Nature of the sample used for isolation, cell lines used for transfection and passage, relative quantification of the amount of viral RNA in the sample and after two passages by real-time RT-PCR, infectious titres in supernatant media after two passages by TCID50 assay and presence or absence of cytopathic effect (CPE).