Assay for porcine circovirus production
View Patent ↗The present invention provides methods for the determination of the viral titer of a culture of host animal host cells infected with a circovirus. The FACS-based methods of the invention may include determining the viability of the host cells in a cell culture medium supernatant and of those cells that remain adherent to a solid support. Detecting and measuring the percentage of cells that expressed the viral antigens ORF1 and ORF2 may determine the viral load of the cultured host cells. The yield of the virus may be established by the detection and measurement of both antigens in supernatant cells, for example 5 to 7 days from when the host cells are transferred to a serum free medium. The methods of the invention may yield rapid quantitative data. This allows the repeated in-process monitoring of the viral production throughout the incubation period, and ready selection of the most appropriate harvesting point.
1. A method for the production of circovirus, comprising:
seeding a host cell culture with a seed culture of a circovirus;
incubating the host cell culture in the absence of fetal calf serum;
monitoring (i) viability of the host cells using flow cytometry, and (ii) ORF1 and ORF2 expression by adherent and non-adherent host cells using a fluorescent antibody cell sorting (FACS)-based method; and
harvesting the circovirus from the host cell culture when ORF2 expression is approximately the same in the non-adherent cells and adherent cells of the host cell culture.
2. The method according to claim 1 , wherein the circovirus is PCV2.
3. The method according to claim 1 , wherein the host cell is PK-15.