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Fig. 4 | Biological Procedures Online

Fig. 4

From: Reverse Genetics Assembly of Newcastle Disease Virus Genome Template Using Asis-Sal-Pac BioBrick Strategy

Fig. 4

Schematic representation of the reverse genetics technique for the rescue of recombinant Newcastle Disease Virus. Plasmids expressing the whole anti-genome, and the helper genes (NP, P, and L), constructed using the Asis-Sal-Pac BioBrick strategy, were used to transfect the T7-BHK monolayer cells. The amounts of different plasmids for transfection were adjusted in a way to express genes with different mRNA levels. Reconstituted RNPs in the cytoplasm, with the transcribed antigenome RNA, are used to assemble the new viral genomes and to produce new viral particles, which are released via budding from the infected cells into the supernatant. Forty-eight hours post-infection, the supernatant was harvested and inoculated into 10 days-old embryonated eggs. After 5 days, the allantoic fluid of the inoculated eggs was analyzed for the presence of viral hemagglutinin by HA assay, and for the presence of viral RNA by RT-PCR and Sanger sequencing. –vRNA: anti-sense viral RNA; +vRNA: sense viral RNA; F: fusion protein; HN: hemagglutinin-neuraminidase, M: matrix; L: large polymerase protein; P: phosphoprotein; NP: nucleoprotein

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