Purification of anatid herpesvirus 1 particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation

Abstract

Anatid herpesvirus 1 (AHV-1) infection causes substantial economic losses to the world-wide waterfowl production. However, little is known about the efficient method used to study the purification of AHV-1 and the negative staining morphology of the purified virus particles. This lack of knowledge is one of the important factors that have affected the progress of research studies on AHV-1 molecular virology to such an extent that they are lagging far behind those on other members of the same family Herpesviridae. Therefore, an efficient method for purifying AHV-1 from cell-culture medium has been developed. Abundant AHV-1 particles, whose morphological features match those of herpesvirus, were obtained by using the following procedures: (1) conventional differential centrifugation for removal of debris after cell disruption, (2) tangential-flow ultrafiltration coupled with sucrose density gradient ultracentrifugation for isolation of the virus, and (3) conventional differential ultracentrifugation for virus concentration. The purified AHV-1 particles were subjected to transmission electron microscopy (TEM), infectivity and recovery tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting assay, and agar gel diffusion test (AGDT). The results of examinations revealed that purified AHV-1 particles were free of visible contamination or degradation. The purified AHV-1 particles were biologically active and were successful in initiating infection upon inoculation into susceptible duck embryo fibroblast. The procedures are reliable technically and feasible for purification of large volumes of viruses.