Establishment of a rescue system for an autonomous Parvovirus mink enteritis virus.

Construction and characterization of a full-length infectious clone (pMEV) of mink enteritis virus are described. Feline kidney cells (F81) were transfected with pMEV containing an engineered BamHI site that served as a genetic marker. The rescued virus was indistinguishable from its parental virus. The availability of a MEV infectious clone will facilitate studies of viral replication and pathogenicity and will permit the elucidation of determinants of the host range of the parvovirus.

Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave Bioreactor microcarrier culture in 1-10 L scale.

A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2g/L Cytodex 1 microcarriers and 2.0 x 10(5)cells/mL was successful. A maximum cell yield of 1.2 x 10(6)cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave Bioreactor could not be determined accurately due to the fast sedimentation of microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus-host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave Bioreactor using a multiple harvest strategy. Maximum virus titres of 10(6.6) to 10(6.8) TCID(50)/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.