|MARCANO, VALERIE - University Of Georgia|
|CARDENAS-GARCIA, STIVALIS - University Of Georgia|
|GOGAL JR, ROBERT - University Of Georgia|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2017
Publication Date: 9/20/2017
Publication URL: http://handle.nal.usda.gov/10113/5838019
Citation: Marcano, V.C., Cardenas-Garcia, S., Gogal Jr, R.M., Afonso, C.L. 2017. Intracellular fixation buffer inactivates Newcastle Disease Virus in chicken allantoic fluid, macrophages and splenocytes for immune assessment during infection. Journal of Virological Methods. 251:1-6. http://dx.doi.org/10.1016/j.jviromet.2017.09.025.
Interpretive Summary: To improve poultry vaccines in depth studies of the avian immune system need to be done. For this to occur, viruses such as Newcastle Disease Virus need to be properly inactivated in order to prevent their accidental release into the environment. Inactivation of Newcastle disease virus (NDV) has been routinely achieved with several other methods including ultraviolet light and formalin in the past. However, these strategies have not been validated for the dual purpose of characterization of surface molecules in viral-infected chicken immune cells and virus inactivation. We demonstrate here the efficacy of the buffer for the dual purpose.
Technical Abstract: Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, ß-propiolactone, binary ethylenimine, ultraviolet light and formalin, however these strategies have not been validated for cell surface ligand or receptor phenotype in viral-infected chicken immune cells. To study the capacity of fixation buffers to preserve surface markers while inactivating NDV, a primary splenocyte culture was infected with NDV and incubated with a commercial intracellular fixation buffer (ICB), formulated with 4% formaldehyde. Splenocytes were fixed with a 1:2 dilution of inactivation buffer (ICB) in buffer for 45 min at 23°C or 4°C and inactivation of Newcastle Disease virus (NDV) was tested in addition to recognition of antigens by antibodies in fixed and non-fixed splenocytes via flow cytometric analysis. The binding and percentage of splenic CD4 and CD8+ cells were not affected. In addition, NDV titers as high as 10x9.5 and 10x7.6 egg infectious doses in allantoic fluid and macrophages, respectively, were successfully inactivated after 45 min at 23°C and 4°C, confirming the ICB’s effectiveness in inactivating high concentration of NDV. In conclusion, high concentrations of NDV in allantoic fluid, chicken splenocytes, and macrophages can be inactivated using ICB. Additionally, this method did not compromise cell phenotyping of enriched chicken splenocytes.