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Generation and characterization of a recombinant Newcastle disease virus expressing the red fluorescent protein for use in co-infection studies

Miller, Patti J
Virology Journal 2012 v.9 pp. 227
DNA-directed RNA polymerase, Newcastle disease virus, chickens, evolution, fluorescent labeling, gene expression, genetic recombination, mixed infection, mutation, reporter genes, viruses
Newcastle disease virus (NDV) is known for its rapid evolutionary dynamics. Mutations introduced by the viral RNA polymerase during replication seem to play a major role in the evolution of the virus, while recombination has only been rarely reported for NDV. The low frequency of recombination in NDV seems to reflect general aspects of the life cycle of negative sense ssRNA viruses and their interactions with the host. Most notably, is the requirement for multiple virus strains to co-infect a same host cell in order to recombine. Here we assessed the ability of two NDV strains (LaSota and B1) to co-infect chicken cells in vitro. We generated a recombinant NDV strain LaSota expressing the red fluorescent protein (RFP) by using reverse genetics. The recombinants rLS-RFP and rB1-GFP were used to co-infect DF1 cells. Cells were either inoculated with both viruses at the same time or at different intervals between primary infection and superinfection. When both viruses were inoculated at the same time point, a 15% co-infection rate was observed, whereas when they were inoculated at intervals of 1, 2, 3, and 24 h, the co-infection rates were 13.6%, 9.3%, 7%, and 2.8%, respectively. These results indicate that although different NDV strains can co-infect host cells in vitro, the superinfection rates are low and yet decrease when the interval between the primary infection and superifection is increased.