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Updated: Feb 21, 2026
Rescue of Recombinant Newcastle Disease Virus from cDNA
Published on: October 11, 2013
Intracellular fixation buffer inactivates Newcastle disease virus in chicken allantoic fluid, macrophages and
Valerie Marcano1, Stivalis Cardenas-Garcia2, Robert M Gogal3
1Department of Veterinary Pathology, College of Veterinary Medicine, The University of Georgia, 501, D. W. Brooks Dr., Athens, GA 30601, USA; Southeast Poultry Research Laboratory, 934 College Station Rd., Athens, GA, 30605, USA.
Abstract:
Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, β-propiolactone, binary ethylenimine, ultraviolet light and formalin. However, these strategies have not been tested 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 ICB in phosphate buffered saline (PBS) for 45min at 23°C or 4°C and inactivation of 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 109.5 and 107.6 EID50 in allantoic fluid (AF) and macrophages, respectively, were successfully inactivated after 45min at 23°C and 4°C, confirming the ICB's effectiveness in inactivating high concentrations of NDV. In conclusion, high concentrations of NDV in AF, chicken splenocytes, and macrophages can be inactivated using ICB. Additionally, this method did not compromise cell phenotyping of enriched chicken splenocytes.
Insights
Intracellular Fixation Buffer (ICB) effectively inactivates Newcastle disease virus (NDV) in chicken immune cells without altering cell surface marker expression. This method preserves cell phenotyping for research applications.
Area of Science:
- Veterinary Virology
- Immunology
- Cell Biology
Background:
- Newcastle disease virus (NDV) inactivation is crucial for research.
- Existing inactivation methods may affect cell surface markers.
- The impact of fixation buffers on NDV-infected chicken immune cells requires investigation.
Purpose of the Study:
- To evaluate the efficacy of an intracellular fixation buffer (ICB) in inactivating NDV.
- To determine if ICB preserves cell surface marker phenotype in infected chicken immune cells.
- To assess the compatibility of ICB with flow cytometric analysis.
Main Methods:
- NDV-infected chicken splenocytes were treated with ICB (4% formaldehyde).
- Splenocytes were incubated with ICB for 45 minutes at 23°C or 4°C.
- NDV inactivation was confirmed by viral titration.
- Cell surface marker expression (CD4+, CD8+) was analyzed by flow cytometry.
Main Results:
- ICB successfully inactivated high titers of NDV in allantoic fluid and macrophages.
- Treatment with ICB did not affect the binding or percentage of splenic CD4+ and CD8+ cells.
- Cell phenotyping of enriched chicken splenocytes remained unaffected.
Conclusions:
- Intracellular Fixation Buffer (ICB) is effective for inactivating high concentrations of NDV.
- ICB preserves cell surface marker integrity in chicken immune cells.
- This method allows for simultaneous NDV inactivation and cell phenotyping.
