Complementation studies with the novel “Bungowannah” virus provide new insights in the compatibility of pestivirus proteins

AbstractIn recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins Erns, E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1–E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-Erns was only efficient if Bungowannah virus-Erns was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general

Untersuchungen zum Strukturprotein E(rns) des Virus der Bovinen Virusdiarrhoe

Im Rahmen der vorliegenden Arbeit wurden BVDV-Mutanten mit Deletionen, Insertionen oder Substitutionen in der E(rns)-kodierenden Genomregion ausgehend von den infektiösen cDNA-Klonen pA/BVDV und pA/BVDV/Ins- (Meyers et al., 1996) generiert. Die meisten Veränderungen verhinderten die Entstehung infektiöser Virionen, so dass nichtessentielle Regionen im E(rns)-Protein nicht identifiziert werden konnten. Eine Ausnahme stellen die Aminosäuren 105-108 dar, deren Substitution im Zusammenhang mit adaptiven Mutationen toleriert wurde. Mit Hilfe von Zelllinien, die die BVDV-Strukturproteine konstitutiv exprimieren, konnte eine Mutante mit einer kompletten E(rns)-Deletion und einer internen EMCV-IRES im Genom effizient trans-komplementiert und sogenannte DISC-Viren erhalten werden. Außerdem wurden Plasmide für die Expression der BVDV-Strukturproteine (C, E(rns), E1, E2) hergestellt, mit deren Hilfe erstmals ein nicht-prozessiertes E(rns)-E1-Protein mit 60-65 kDa identifiziert werden konnte, das für mindestens 3 h relativ stabil in transfizierten Zellen vorlag. Durch Mutation der P3-Position eines SPP-Motivs gelang es, sowohl in pCITE-2a(+)-Expressionsplasmiden als auch in BVDV-Vollängen-Mutanten die Spaltung dieses E(rns)-E1-Proteins zu verhindern. Dadurch konnte gezeigt werden, dass die E(rns)-E1-Spaltung essentiell für die Bildung infektiöser Viren ist. Bicistronische Mutanten wurden genutzt, um zu zeigen, dass das E(rns)-E1-Protein selbst jedoch nicht notwendig, aber förderlich für Entstehung infektiöser Virionen ist. Weiterhin konnte mittels eines im Rahmen dieser Arbeit generierten polyklonalen Bungowannah Virus-E(rns)-spezifischen Kaninchenserums, das E(rns)-Protein des atypischen Pestivirus Bungowannah Virus in Western Blot-Analysen mit etwa 38 kDa detektiert werden. Da jedoch kein Bungowannah Virus-E(rns)-E1-Protein nachgewiesen werden konnte, spielt E(rns)-E1 möglicherweise keine oder eine untergeordnete Rolle im Bungowannah Virus-Replikationszyklus. BVDV-E(rns)-Deletionen im CP7-Hintergrund konnten erfolgreich mit Bungowannah Virus-E(rns) komplementiert werden, wenn Bungowannah Virus-E(rns) und BVDV-E1 unabhängig von einer E(rns)-E1-Spaltung exprimiert wurden. Ein weiterer Schwerpunkt dieser Arbeit war es schließlich, eine effiziente Methode zur Konzentrierung und Reinigung infektiöser BVDV aus infizierten Zellen zu etablieren. Zum ersten Mal wurden BVDV-Mutanten mit FLAG-markierten E(rns)- und E2-Proteinen generiert, so dass erstmalig BVDV mittels Affinitätschromatografie gereinigt und elektronenmikroskopisch untersucht werden konnten. Mittels Negativkontrast-Elektronenmikroskopie wurden sphärische Partikel mit Durchmessern von 43-58 nm dargestellt. Sowohl durch affinitätschromatografische Virusreinigung als auch durch immunelektronenmikroskopische Untersuchungen konnte eine Assoziation von E(rns)- und E2-Proteinen mit der BVD-Virushülle demonstriert werden. Die in dieser Arbeit vorgestellte Methode kann als Basis für weiterführende Untersuchungen zur Morphogenese von Pestiviren genutzt werden.In this study, BVDV mutants with deletions, insertions or substitutions in the E(rns)-encoding genomic region were generated on the basis of the infectious cDNA clones pA/BVDV and pA/BVDV/Ins- (Meyers et al., 1996). Most of the changes within the E(rns)-encoding region were lethal and therefore non-essential regions for the generation of infectious virions could not been defined. The only exception was the substitution of amino acids 105-108 which was tolerated in combination with adaptive mutations. With the help of recombinant cell lines, constitutively expressing the BVDV structural proteins, a mutant with a complete E(rns) deletion and an internal EMCV-IRES could be successfully trans-complemented and DISC viruses were generated. Furthermore, plasmids for the expression of the BVDV structural proteins (C, E(rns), E1, E2) were generated. With the help of those constructs, an E(rns)-E1 protein with 60-65 kDa, which remained stable for at least 3 h in transfected cells, was identified. Mutation of the P3 position of a putative SPP motif in pCITE-2a(+) as well as in BVDV full length mutants abolished cleavage of the E(rns)-E1 protein. Therefore, it could be demonstrated that cleavage of the E(rns)-E1 protein is essential for the generation of infectious virus progeny. With bicistronic mutants it could further be shown that the E(rns)-E1 protein itself is not essential but beneficial for the generation of infectious virions. Furthermore, with a newly generated polyclonal rabbit serum, an E(rns) protein with about 38 kDa could be detected in Western blot experiments for the atypical pestivirus Bungowannah. An E(rns)-E1 protein was not identified and therefore maybe such a protein plays no or only a minor role in the Bungowannah virus replication cycle. BVDV-E(rns) deletions in a CP7 background could be successfully complemented with Bungowannah virus-E(rns) if Bungowannah virus-E(rns) and BVDV-E1 were expressed without proteolytic cleavage between both proteins. Another key aspect of this study was the establishment of an efficient method for concentration and purification of infectious BVDV. For the first time, BVDV mutants with FLAG-tagged E(rns) and E2 proteins were generated, purified by affinity tag chromatography, and analyzed by electron microscopy. Spherical particles with diameters of 43-58 nm could be observed by negative staining electron microscopy. By immunogold labelling as well as by affinity tag purification an association of E(rns) and E2 with the viral envelope could be demonstrated. This method can serve as a basis for further studies about the morphogenesis of pestiviruses

A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences

International audienceA panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics

Molecular double-check strategy for the identification and characterization of European Lyssaviruses

The “gold standard” for post-mortem rabies diagnosis is the direct fluorescent antibody test (FAT). However, in the case of ante-mortem non-neural sample material or decomposed tissues, the FAT reaches its limit, and the use of molecular techniques can be advantageous. In this study, we developed and validated a reverse transcription PCR cascade protocol feasible for the classification of samples, even those for which there is no epidemiological background knowledge. This study emphasises on the most relevant European lyssaviruses. In a first step, two independent N- and L-gene based pan-lyssavirus intercalating dye assays are performed in a double-check application to increase the method's diagnostic safety. For the second step, characterization of the lyssavirus positive samples via two independent multiplex PCR-systems was performed. Both assays were probe-based, species-specific multiplex PCR-systems for Rabies virus, European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus. All assays were validated successfully with a comprehensive panel of lyssavirus positive samples, as well as negative material from various host species. This double-check strategy allows for both safe and sensitive screening, detection and characterization of all lyssavirus species of humans and animals, as well as the rapid identification of currently unknown lyssaviruses in bats in Europe

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein*

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein Erns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the Erns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of Erns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, ErnsE1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of Erns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase