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Journal of Virology, December 2009, p. 12415-12423, Vol. 83, No. 23
0022-538X/09/$08.00+0 doi:10.1128/JVI.01487-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity 
Alexander Pankraz,1,
Simone Preis,1
Heinz-Jürgen Thiel,1
Andreas Gallei,1,
and
Paul Becher1,2*
Institute of Virology, Justus-Liebig University, D-35392 Giessen, Germany,1 Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine, D-30559 Hannover, Germany2
Received 17 July 2009/ Accepted 15 September 2009
For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.
* Corresponding author. Mailing address: Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany. Phone: 49 511 953 8452. Fax: 49 511 953 828452. E-mail: paul.becher{at}tiho-hannover.de
Published ahead of print on 23 September 2009.
Present address: Biocontrol Ingelheim, Konrad-Adenauer-Strasse 17, D-55218 Ingelheim am Rhein, Germany.
Present address: BioScreen European Veterinary Disease Management Center GmbH, Mendelstrasse 11, D-48149 Münster, Germany.
Journal of Virology, December 2009, p. 12415-12423, Vol. 83, No. 23
0022-538X/09/$08.00+0 doi:10.1128/JVI.01487-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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