Newcastle disease virus (NDV), belonging to the species avian orthoavulavirus 1, genus Orthoavulavirus, and family Paramyxoviridae, is responsible for Newcastle disease in poultry and other avian species. It has shown significant potential as an oncolytic virus and as a vector for vaccine delivery. NDV from infected biological serum is usually isolated or purified using density gradient ultracentrifugation. However, it has many disadvantages, including the fact that it is time consuming and can process only a limited quantity of sample at one time. In our study, native agarose gel electrophoresis and dynamic light scattering (DLS) analysis showed that NDV carried a net negative surface charge. Thus, we purified the virus using a HiTrap Q Sepharose Fast Flow anion exchange column with salt elution. Hemagglutination assay and plaque assay showed that the procedure yielded high‐purity NDV particles with a recovery of more than 80%, and the process was fast and simple. The purity of the virus was confirmed using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot analysis. The hydrodynamic volume and ‘dry state’ diameter of the purified NDV were analyzed using dynamic light scattering and transmission electron microscopy and were to be in the range of 200–300 nm. The viruses did not exhibit any deviation from their known physical properties. The genome of the virus was also detected by amplifying a 423‐bp region using reverse transcription‐polymerase chain reaction. Our study confirmed that NDV could be effectively purified using an anion exchange column. In addition, the procedure could be easily upscaled or downscaled based on the experimental requirements.

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