Title: Generation and characterization of a recombinant Newcastle disease virus expressing the red fluorescent protein for use in co-infection studies Authors
Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2012
Publication Date: October 3, 2012
Repository URL: http://handle.nal.usda.gov/10113/57517
Citation: Li, J., Hu, H., Yu, Q., Diel, D.G., Li, D., Miller, P.J. 2012. Generation and characterization of a recombinant Newcastle disease virus expressing the red fluorescent protein for use in co-infection studies. Virology Journal. 9:227. DOI 10.1186/1743-422X-9-227. Interpretive Summary: The genetic make-up of Newcastle disease viruses (NDV) is known to change over time. There are two ways that changes in the genetic structure can occur. The common way occurs when the virus reproduces and errors are made during the replication. The viruses don’t have any way to fix these errors, so they accumulate and are passed to the “offspring” over time. Another, less common way for the genetic structure of NDV to change occurs when two viruses have infected one host cell at the same time and during replication, some of both viruses are replicated in the offspring viruses. This is referred to as recombination. We were able to create a NDV that fluoresces with a red color in ultra violet (UV) light. We infected cells with this red-NDV and also with another NDV that fluoresces green. We then looked at the ability of individual cells to be infected by both viruses at the same time. We found that both viruses were able to infect one cell more often when the two viruses were inoculated together or at closer time points. When we waited 24 hours between infecting with the different viruses, only about 3% of the cells were able to be infected by both viruses. This decreased ability for a cell to be co-infected as time increased between being exposed to the 2nd virus may explain why there is infrequent documention of recombination in NDV.
Technical Abstract: 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.