Generation of molecular tools for Bluetongue virus (BTV) diagnosis and immunization

ABSTRACT Blutongue virus (BTV), the prototype member of the genus Orbivirus in the family Reoviridae, is an insect-borne virus that infects domestic and wild ruminants, causing a non contagious infectious disease spread by biting midges of some species of Culicoides genus, known as Bluetongue (BT). Historically, BT occurs most commonly and seriously in sheep and in some species of wild ruminants, occasionally in goats and rarely in cattle. In 2006, BTV serotype 8 (BTV-8) emerged in Norhern Europe followed by a still ongoing epidemic; the BTV-8 occurrence is remarkable for several reasons and, particularly, for its virulence not only in sheep between domestic ruminants. Currently, BTV, and particularly BTV-8, is responsible of greet farming industries losses; for this reason it is critical to have effective molecular tools to define standardized, specific and sensitive serodiagnostic assay and successful and economic prophilatic immunization plans. BTV is a double-stranded RNA (dsRNA) virus and its genome consists of 10 dsRNA segments that encode 4 nonstructural proteins (NS1-NS3 and NS3A) and 7 structural proteins (VP1-VP7). Among the 7 structural proteins, VP2, the outermost viral protein, is the cellular binding protein, elicitis virus-neutralizating antibody, and is responsible for hemagglutinin activity and serotype specifity. Although VP2 has been expressed successfully throught many systems and particularly with insect cell-baculovirus expression system, its paracrin expression as a soluble form in mammalian cells represents a difficult task. In this work we have investigared a mammalian expression platform for BTV VP2 production as a soluble form, generating several expression vector plasmids with BTV-8 VP2 sequence, whole or fragmented, by fusion peptides strategy. Starting from the assumption that VP2 is strongly cell associated, the full length of VP2 ORF was sub-cloned in frame with the immunoglobulin kappa light chain (IgK) signal peptide specifying secretion of heterologous proteins, generating pIgkVP2. In order to attempting the expression of BTV-8 VP2 with pIgkVP2 vector in mammalian cells and due to the lack of a suitable monoclonal antibody against BTV-8 VP2, a tag soluble epitope peptide belonging to the Bovine herpesvirus 1 (BoHV-1) glycoprotein D (gD) ectodomain was generated, obtaing pIgkVP2gD106. After electroporation of HEK 293T cells, we found that VP2 was well expressed from the tranfected cells, but not secreted into the medium, showing that introduction of a heterologous signal peptide in the amino-terminus of VP2 is not sufficient to specify its secretion. In previous studies was demonstrated that BTV VP2 associates with Vimentin cytoskeleton protein, and deletions of amino acids between residues 65 and 114 into VP2 sequence have shown to disrupt VP2-vimentin association. This aspect could be the cause of the missing VP2 secretion. Thus, new plasmid vectors was generated with different fragments of VP2 in frame upstream with Igk signal peptide, and downstream with gD106 tag; although all VP2 fragments are found in cell fraction, none is secreted into the medium. Based on the fact that BVDV gE2 ectodomain was very well expressed and secreted by pIgK-E2, gE2 ectodomain was exploited as a leader sequence to get VP2 and derived fragments secreted into the medium of transfected cells. Therefore, VP2 and derived fragments were sandwiched between IgkE2 and gD106 tag. We found that two constructs, pIgkE2VP2720-1425gD106 and pIgkE2VP22070-2883gD106, successfully expressed and secreted the VP2 fragments. Finally, BoHV-4 U ΔORF50 strain cloned as a bacterial artificial chromosome (BAC) was engineered to express VP22070-2883. Thus, we inserted the IgkE2VP22070-2883gD106 expression cassette into IE2 gene of a mutant BoHV-4 U ΔORF50 strain, in which IE2 locus is duplicated and one of them is inactivated by the insertion of 2004 bp KanaGalK DNA sequence stuffer double selecting cassette. So we obtained a recombinant BoHV-4 strain virus-vector able to yeald hight quantity of soluble VP22070-2883gD106 fragment from infected cells, which could be employed for immunodiagnostic assay development or vaccine purposes.