title: Rescue of a recombinant Newcastle disease virus strain R2B expressing green fluorescent protein.
abstract: Newcastle disease virus (NDV), strain R2B is a mesogenic vaccine strain used for booster vaccination in chickens against Newcastle disease in India and many south East Asian countries. A full-length cDNA clone of the virus was generated by ligating eight overlapping fragments generated by reverse transcription polymerase chain reaction having unique restriction enzyme sites within them. This full-length cDNA clone was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme sequences. Defined genetic markers were introduced into the NDV genome to differentiate the rescued virus from the parent virus. A gene cassette containing the reporter gene, green fluorescent protein flanked by NDV gene-start and gene-end signals was generated by PCR and introduced into the full-length clone of NDV between the P and M genes. Recombinant NDV encoding the GFP gene was rescued having precise termini when transfected into permissive Vero cells along with support plasmids harbouring the nucleoprotein, phosphoprotein and polymerase genes. The recombinant virus had similar growth kinetics as that of the parent virus with a moderate reduction in the virulence. The generation of reverse genetics system for NDV strain R2B will help in the development of multivalent vaccines against viral diseases of livestock and poultry.
authors.affiliation: Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, UP, 243 122, India. madhansohini@gmail.com.
authors.forename: Madhan Mohan
authors.identifier: http://orcid.org/0000-0003-0477-6829
authors.initials: MM
authors.lastname: Chellappa
authors.affiliation: Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, UP, 243 122, India.
authors.forename: Sohini
authors.initials: S
authors.lastname: Dey
authors.affiliation: Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, UP, 243 122, India.
authors.forename: Satish
authors.initials: S
authors.lastname: Gaikwad
authors.affiliation: Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, UP, 243 122, India.
authors.forename: Dinesh C
authors.initials: DC
authors.lastname: Pathak
authors.affiliation: Institute of Marine and Environmental Technology, University of Maryland, Baltimore County, Baltimore, MD, USA.
authors.forename: Vikram N
authors.initials: VN
authors.lastname: Vakharia
mesh_terms: D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002645:Chickens; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics*; D003698:Hepatitis Delta Virus / Q000235:genetics; D009521:Newcastle Disease / Q000517:prevention & control*; D009522:Newcastle disease virus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods; D014162:Transfection; D014614:Vaccines, Synthetic / Q000235:genetics*; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; C424862:Newcastle disease virus vaccine MTH-68-H; D016337:RNA, Catalytic; D014614:Vaccines, Synthetic; D014765:Viral Vaccines; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins
keywords: Genetic markers; Green fluorescent protein; Newcastle disease virus; R2B; Reverse genetics