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Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. Its single-stranded RNA encodes a polyprotein that is cleaved co-and posttranslationally by viral and cellular proteases. However, the cleavage between the envelope proteins E rns and E1 is still unexplained. In this study, an E rns -E1 protein could be identified and characterized with a new E1-specific antiserum. With bicistronic constructs bearing a deletion in the E rns -encoding region and expressing E rns or the E rns -E1 protein, it could be shown that this protein is not essential for virus replication. Furthermore, two putative cleavage sites were mutated in eukaryotic expression plasmids, as well as in full-length cDNA constructs. The mutation of position P3 of a potential signal peptide peptidase site abolished cleavage completely and no infectious virus progeny could be observed, indicating that cleavage of the E rns -E1 protein is indispensable for virus growth.Bovine viral diarrhea virus (BVDV), the causative agent of an economically important disease of cattle worldwide, belongs, like classical swine fever virus and border disease virus, to the genus Pestivirus within the family Flaviviridae (Collett et al., 1988a;Fauquet et al., 2005;Houe, 1999;Korn, 1977;Pringle, 1998). The genome is a singlestranded RNA of positive polarity with a size of about 12.3 kb. One large open reading frame (ORF) encodes a polyprotein that is cleaved co-and post-translationally by viral and cellular proteases (Collett et al., 1988b; Rümenapf et al., 1993). The ORF is flanked by 59 and 39 untranslated regions (UTRs) that are important for virus replication (Collett et al., 1988a;Yu et al., 1999Yu et al., , 2000. An internal ribosomal entry site (IRES) within the 59 UTR allows capindependent translation of the virus polyprotein (Pestova & Hellen, 1999; Poole et al., 1995;Yu et al., 2000). The virions consist of four structural proteins (C, E rns , E1 and E2). The basic protein C was believed to form a capsid that surrounds the virus genome, but recent studies by Rümenapf et al. (2008) have shown that it is dispensable for virus assembly (Gray & Nettleton, 1987;Thiel et al., 1991). The three glycosylated proteins E rns , E1 and E2 are located in the lipid membrane of cellular origin (Chu & Zee, 1984;Coria et al., 1983;Fetzer et al., 2005). The envelope protein E rns , which lacks a typical membrane anchor, seems to be important for first cell contact by binding to glycosaminoglycans (Fetzer et al., 2005;Iqbal et al., 2000). The cellular receptor for BVDV is the bovine CD46 molecule and it has recently been shown that E1-E2 heterodimers are essential for virus entry (Maurer et al., 2004;Ronecker et al., 2008). The envelope proteins E rns , E1 and E2 are processed from the polyprotein in a hierarchical way, starting with the translocation of the C-terminal signal peptide downstream of the capsid protein into the endoplasmic reticulum (ER), followed by the release of the capsid and E2 protein (Heimann et al., 2006; Rümenap...

Please cite this article as: Zemke, J., König, P., Mischkale, K., Reimann, I., Beer, M., Novel BVDV-2 mutants as new candidates for modified live vaccines, Veterinary Microbiology (2008Microbiology ( ), doi:10.1016Microbiology ( /j.vetmic.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Please cite this article as: Mischkale, K., Reimann, I., Zemke, J., König, P., Beer, M., Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants, Veterinary Microbiology (2008Microbiology ( ), doi:10.1016Microbiology ( /j.vetmic.2009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.