Mutational Analysis of Nucleocapsid (N) Protein of Newcastle Disease Virus Using a Minigenome System
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Date
2020-02
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Abstract
The Nuclepcapsid protein (N) protein of Newcastle disease virus (NDV), synonymous with Avian
Avula virus-1(AAv-1) functions primarily to encapsidate the virus genome for transcription, replication and
packaging. In addition to its role in replication, N gene is the major immunogenic and conserved gene of
NDV. Identifying mutation permissive regions in the N protein provides a potential approach for NDV marker
vaccine. In the present study mutations introduced in to the NP gene in the form of epitope replacement
between amino acid positions 446-453 was analysed using an intracellular NDVD58-GFP minigenome assay
for its effect on minigenome RNA synthesis and encapsidation.Site directed mutagenesis was carried out in
pCIneo N plasmid to replace the NDV epitope 446(QFLDLMRAV)453 with a marker epitope TAVSPTTLR.The mutated pCIneo N plasmid was co-transfected with other NDV expression plasmids pCIneo L, pCIneo
P and pNDVD58-GFP minigenome plasmid in BSR/T7 cells. GFP expression was observed 72 hours post
transfection indicating a functional minigenome. Further the presence of encapsidated minigenome RNA in
transfected cell supernatants were also confirmed by Real time PCR. These results indicate that the mutations
did not affect the functionality of the Nucleoprotein and can serve as marker epitopes for NP based ND marker
vaccine.
Description
TNV_20thMVC_PP_Feb-2020_OP48
Keywords
Veterinary Science