Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures

Abstract

A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures. Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful. A low-pH medium (pH 6.8) was also ineffective in virus propagation. Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium. The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation. Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation. Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16. Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13. The infected cells became separated, rounded, and detached, forming holes in the cell monolayer. Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy. Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.