Study on inactivation of porcine epidemic diarrhoea virus, porcine sapelovirus 1 and adenovirus in the production and storage of laboratory spray-dried porcine plasma
Hulst, M.M. ; Heres, L. ; Hakze-van der Honing, R.W. ; Pelser, M. ; Fox, M. ; Poel, W.H.M. van der - \ 2019
Journal of Applied Microbiology 126 (2019)6. - ISSN 1364-5072 - p. 1931 - 1943.
adenovirus - feed-safety - porcine epidemic diarrhoea virus - porcine sapelo virus 1 - spray-dried porcine plasma - thermal and physical inactivation
Aim: Evaluation of the thermal and physical conditions for inactivation of adenovirus (AdV), porcine sapelovirus 1 (PSV1) and the economically important viruses porcine epidemic diarrhoea virus (PEDV) and porcine circovirus 2 (PCV2) in the production of spray-dried porcine plasma (SDPP). Methods and Results: Citrate-treated porcine plasma of pH 7·5, 9·8 and 10·2 (8·5% dry-matter) was spiked with PEDV, PSV1, PCV2 and AdV and incubated at 3°C for maximum 24 h, and at 44 or 48°C for maximum 10 min (Experiment 1). Spiked citrate-treated concentrated plasma of pH 7·5 and 9·8 (24% dry-matter) was spray dried in a laboratory scale apparatus (Experiment 2). Aliquots of SDPP were stored over a period of 0–10 weeks at 11 and 20°C (Experiment 3). Reverse transcription(RT)-quantitative PCR detected no notable reduction in viral genomes in treated plasma and SDPP samples. No infectious PSV1 was re-isolated from plasma and SDPP samples in cell culture. At pH 10·2 and 3°C, infectivity of PEDV in plasma was reduced with a reduction factor of 4·2 log 10 (LRF) at 10 h contact time, whereas heating to 44°C for at least 1 min at alkali pH was needed to achieve a LRF of 4·2 for AdV. Spray drying at an outlet temperature of 80°C reduced AdV infectivity effectively (LRF = 5·2) and PEDV infectivity for 95% (LRF = 1·4). After storage at 20°C for 2 weeks no infectious PEDV was re-isolated from SDPP anymore (LRF ≥4·0). Due to growth of antibiotic-resistant bacteria from plasma in cell cultures used for PCV2 isolation, no data regarding inactivation of PCV2 were obtained. Conclusions: Five percent of PEDV stayed infectious after our spray drying conditions. Spray drying in combination with storage for ≥2 weeks at 20°C eliminated infectivity of PEDV effectively. Significance and Impact of the Study: The conditions for inactivation of virus in plasma and SDPP determined are important for producers to inactivate PEDV during production of SDPP.
Characterization of apoptosis in PER.C6® batch and perfusion cultures
Mercier, S.M. ; Diepenbroek, B. ; Martens, D.E. ; Wijffels, R.H. ; Streefland, M. - \ 2015
Biotechnology and Bioengineering 112 (2015)3. - ISSN 0006-3592 - p. 569 - 578.
hamster ovary cells - high-level expression - flow-cytometry - line per.c6 - death - adenovirus - dna - vaccine - gene - manufacture
Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6® batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6® cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line.
Selective gene transfer in vitro to tumor cells via recombinant Newcastle disease virus
Bian, H. ; Fournier, P. ; Moormann, R.J.M. ; Peeters, B.P.H. ; Schirrmacher, V. - \ 2005
Cancer Gene Therapy 12 (2005). - ISSN 0929-1903 - p. 295 - 303.
single-chain antibody - foreign gene - fusion protein - rna viruses - cloned cdna - expression - adenovirus - carcinoma - antigen - vaccine
We developed a novel strategy to target recombinant Newcastle disease virus (NDV) to tumor cells for gene therapy. Modifying the virus with a bispecific fusion protein allowed virus receptor-independent tumor cell binding and gene transfer. The targeting molecule HN-IL-2 contains an scFv antibody cloned from a neutralizing hemagglutinin-neuraminidase (HN)-specific hybridoma linked to the human cytokine IL-2. A recombinant NDV expressing the enhanced green fluorescent protein (NDFL-EGFP) was applied to show the expression of foreign genes in virus-infected tumor cells. At 24 hours after infection with the modified virus (NDFL-EGFP/HN-IL-2), FACS analysis and fluorescence microscopy revealed neutralization of natural infection in IL-2 receptor-negative Jurkat leukemia cells, but targeted expression of EGFP in IL-2 receptor-positive human leukemia-derived MT-2 cells. The targeted gene delivery of NDFL-EGFP/HN-IL-2 in MT-2 cells was blocked by the target ligand human IL-2. Selective virus entry to IL-2 receptor bearing tumor cells was also observed in a mixture of Jurkat and MT-2 cell lines. These results demonstrate that a recombinant NDV carrying a foreign gene can be successfully targeted to a specific tumor through a bispecific protein, which thereby increases the selectivity of gene transfer.