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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #322798
Title: Reverse genetics of Newcastle disease virus

Author
item CARDENAS-GARCIA, STIVALIS - University Of Georgia
item Afonso, Claudio

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 1/1/2016
Publication Date: 5/16/2017
Publication URL: https://handle.nal.usda.gov/10113/5763048
Citation: Cardenas-Garcia, S., Afonso, C.L. 2017. Reverse genetics of Newcastle disease virus. Methods in Molecular Biology. In: Perez, D., editor. Methods in Molecular Biology. Volume 1602. New York, NY: Humana Press. p. 141-158. doi:10.1007/978-1-4939-6964-7_10.

Interpretive Summary: Virulent Newcastle disease virus can cause significant disease in non-vaccinated and in poorly vaccinated poultry. Therefore, there is strong interest in developing new more effective vaccines as well as in understanding the mechanisms of disease and pathogenesis. The technique of reverse genetics allows the modification of the viral genome to develop better vaccines and to study the mechanisms of pathogenesis. Here we describe in detail, the materials and the methods involved in cloning the Newcastle disease virus complete genome, as well as methods to perform site-directed mutagenesis, to attenuate the virulence of the viruses and methods of gene replacement to produce more effective vaccines. This review will help researchers to develop better vaccines against Newcastle disease and to study the virulence of the virus, which may facilitate control of the disease.

Technical Abstract: Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among other studies. A system based on infecting cells with highly attenuated Vaccinia virus Ankara strain expressing the T7 ribonucleic acid (RNA) polymerase from bacteriophage T7, with co-transfection of three helper plasmids and a full-length copy deoxyribonucleic acid (cDNA) plasmid, was successfully developed to rescue genetically modified Newcastle disease viruses in 1999. In the present chapter, the materials and the methods involved in cloning the Newcastle disease virus complete genome, site-directed mutagenesis, and gene replacement techniques, are described in detail.