Journal of Virological Methods 153 (2008) 263–265 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Short communication Laboratory tests for evaluating the level of attenuation of bluetongue virus P. Franchi, M.T. Mercante, G.F. Ronchi, G. Armillotta, S. Ulisse, U. Molini, M. Di Ventura, R. Lelli, G. Savini, A. Pini ∗ Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario, 64100 Teramo, Italy Article history: Received 17 April 2008 Received in revised form 3 July 2008 Accepted 17 July 2008 Available online 9 September 2008 Keywords: Attenuation Bluetongue virus Bovine fetal aorta endothelial cell line Newborn mice Vaccine abstract One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK 21c13 or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all ani- mals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained. © 2008 Elsevier B.V. All rights reserved. Bluetongue (BT) is a non-contagious insect borne disease affect- ing ruminants, primarily sheep and to a lesser extent cattle and goats. Buffaloes, camels, deer and antelope could also be infected. The virus causes vascular endothelial damage leading to increased capillary permeability and fragility. Bluetongue virus (BTV) belongs to the Orbivirus genus, Reoviri- dae family, 24 serotypes have been identified so far. The virus is transmitted to ruminants by vectors of the Culicoides genus, Cerato- pogonidae family. It has a worldwide distribution and since 1999 has spread into Southern Europe affecting Greece, Italy, Corsica, Spain, the Balkans, Portugal and more recently Central and North- ern Europe. The following six virus serotypes are circulating at present in Europe: BTV1, BTV2, BTV4, BTV8, BTV9 and BTV16. Although measures such as the ban of livestock movements and/or vector controls could be useful for limiting the spread of infection, vaccination is likely to be the only effective tool to con- trol the disease. A live attenuated polyvalent or monovalent vaccine developed and produced in South Africa, has been used successfully in Africa over the last 60 years and is still used in some European countries (Savini et al., 2007). The vaccine may induce temperature reaction, clinical signs and viraemia. If some of these side effects might have no relevance in the African context, they could repre- ∗ Corresponding author. Tel.: +39 0861 332481; fax: +39 0861 332251. E-mail address: a.pini@izs.it (A. Pini). sent an obstacle to its use in European animal breeds that in some instances are highly sensitive to the virus. Another problem faced in dealing with BTV attenuated vac- cines, is the level of attenuation of the virus serotypes used in their formulation. In this regard, it has to be mentioned that the use of the Ondersterpoort Biological Products live attenuated vac- cine prepared with serotype 16 was banned in Italy because of its pathogenicity for local sheep breeds (Italian Ministry of Health, 2005). A further concern to the use of South African vaccines is the pres- ence of dissimilarities between European and African gene segment sequences of homologous BTV serotypes (Potgieter et al., 2005). Having available attenuated European BTV isolates would indeed solve some of the above-mentioned problems. Under the circumstances, it was decided to proceed with the attenuation of Italian BTV4, BTV9 and BTV16 field isolates. One of the most important steps when preparing a live attenuated vaccine is the assessment of its level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. In vitro and/or in vivo laboratory models, to be used as comple- mentary assays to the target animals’ inoculation, could represent a valid tool in studying BTV attenuation after serial passages in non-conventional hosts. In this study, bovine fetal aorta endothelial cell line (BFA) (Euro- pean Collection of Cell Cultures, Salisbury, Wiltshire, UK) and 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.07.007