Biochemical Engineering Journal 90 (2014) 131–139 Contents lists available at ScienceDirect Biochemical Engineering Journal jo ur nal home page: www.elsevier.com/locate/bej Regular Article Process intensification for an enhanced replication of a newly adapted RM-65 sheep pox virus strain in Vero cells grown in stirred bioreactor Khaled Trabelsi, Samy Majoul, Samia Rourou, Héla Kallel ∗ Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Viral Vaccines Research and Development Unit, Institut Pasteur de Tunis, 13, place Pasteur, BP74, 1002 Tunis Belvédère, Tunisia a r t i c l e i n f o Article history: Received 31 March 2014 Received in revised form 28 May 2014 Accepted 1 June 2014 Available online 6 June 2014 Keywords: Animal cell culture Bioprocess design Production kinetics Optimization Sheep pox vaccine Vero cells a b s t r a c t Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 10 5.875 TCID 50 (median tissue culture infective dose) ml -1 . To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supple- mented with 5 mM glucose, 5 mM fructose and 25 mM sucrose. Spinner culture confirms these data; virus titer was 10 7.375 TCID 50 ml -1 . In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3 g l -1 Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 10 7.275 TCID 50 ml -1 . This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM + 2% FCS. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Sheep occupy an important place in the livestock industry and contribute significantly to the world economy. Their populations are threatened by a number of health hazards among them sheep pox [1]. This disease is an OIE notifiable [2] and transboundary dis- ease of sheep and goats [3]. The etiological agent, sheep pox virus belongs to the genus Capripoxvirus in the subfamily Chordopoxviri- nae of the Poxviridae family. Sheep pox disease is economically important in the enzootic regions of Africa, the Middle East, Central Asia and the Indian subcontinent [4–7]. The disease is characterized by pyrexia, gen- eralized skin lesions, internal pox lesions, and lymphoadenopathy. Following an incubation period of 4–8 days, there is an increase in body temperature, heart rate and respiration, followed by the formation of macules in the skin [3,4,8]. These ultimately develop into pox lesions, which can affect over 50% of the skin surface. These lesions contain high viral loads [9] and together with mouth lesions serve as the main source of virus for transmission to unin- fected animals. Outbreaks of sheep pox, with very high morbidity ∗ Corresponding author. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: hela.kallel@pasteur.rns.tn (H. Kallel). and mortality rates, have been reported from different parts of the world regularly [4,10]. The mortality rate in young animals can exceed 50%. In naive animals, morbidity and mortality can even approach to 100% [4,10]. Due to the rapid progression of the diseases among livestock animals, treatment is generally not possible or practical, and the emphasis is placed on prevention by vaccination [4,5]. The control of the disease is vital to improve small ruminant productivity. Var- ious kinds of vaccines are available but the live attenuated vaccine is the best choice. Live attenuated vaccines provide long-lasting immunity and, hence, are considered the best choice for use in vac- cination [4,7,11]. The use of inactivated vaccine suffers from several drawbacks such as the use of high doses, and short duration of immunity [1,4,7]. Various cell types such as lamb testes, kidney and thyroid, calf kidney and fetal muscle, fetal sheep kidney, lung and skin tis- sues and continuous cell lines (BHK-21, Vero) have been the most commonly employed systems for virus attenuation and replication [4,11–13]. A live attenuated sheep pox vaccine was locally produced in primary lamb kidney cells for sheep immunization in Tunisia. Nev- ertheless, this vaccine provoked outbreaks of Border Disease and abortion in ewes after vaccine administration. Deep investigations demonstrated the contamination of this vaccine with Pestiviruses http://dx.doi.org/10.1016/j.bej.2014.06.001 1369-703X/© 2014 Elsevier B.V. All rights reserved.