BackgroundSchmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, was first identified in October 2011 in dairy cattle in Germany, where it caused fever, diarrhea and a drop in milk yield. Since then, SBV additionally has been detected in adult sheep and goats. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. In view of the current situation and the possible emergence of further Simbu serogroup members, a pan-Simbu real-time reverse transcriptase (RT) PCR system for the reliable detection of Simbu serogroup viruses should be developed.MethodsIn this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. All PCR-systems were tested using a panel of different Simbu serogroup viruses as well as several field samples from diseased cattle, sheep and goats originating from all over Germany. Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses.ResultsAll tested members of the Simbu serogroup (n = 14) as well as most of the field samples were successfully detected by the pan-Simbu real-time RT-PCR system. The comparison of this intercalating dye assay with different TaqMan probe-based assays developed for SBV diagnostics confirmed the functionality of the pan-Simbu assay for screening purposes. However, the SBV-TaqMan-assay SBV-S3 delivered the highest analytical sensitivity of less than ten copies per reaction for duplex systems including an internal control. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established.ConclusionThe pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup, most of the SBV field samples as well as three tested Bunyamwera serogroup viruses with a suitable sensitivity. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity.
A 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.
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.
Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins E rns , E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged E rns or E2 proteins, respectively. The expression of the epitope-tagged E rns and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 10 8.75 TCID 50 ml "1 , spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both E rns and E2 proteins at the virus membrane.Bovine viral diarrhea virus (BVDV) belongs together with classical swine fever virus (CSFV) and border disease virus to the genus Pestivirus within the family Flaviviridae (Collett et al., 1988a;Fauquet et al., 2005;Korn, 1977;Pringle, 1998). BVDV infections cause worldwide enormous economic losses in cattle production with symptoms ranging from respiratory disorders and diarrhoea to abortions (Ahn et al., 2005;Houe, 1999;Ross et al., 1986). The pestivirus genome is a ssRNA of positive polarity with a size of about 12.3 kb. One large polyprotein is co-and post-translationally cleaved by viral and cellular proteases into the 12 mature structural and non-structural proteins N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Collett et al., 1988b; Rümenapf et al., 1993). In the 59 and 39 UTR, elements important for viral replication are located (Collett et al., 1988a;Yu et al., 1999Yu et al., , 2000. Within the 59 UTR an internal ribosomal entry site allows the cap-independent translation of the viral polyprotein (Pestova & Hellen, 1999; Poole et al., 1995;Yu et al., 2000). It is supposed that the viral RNA is packaged into capsids composed of the basic protein C (Gray & Nettleton, 1987;Thiel et al., 1991), but studies by Rümenapf et al. (2008) suggested that the capsid protein might be dispensable for virus assembly. Riedel et al. (2010) showed the high plasticity of the capsid protein and support the idea of a histone-like protein-RNA interaction. The three glycosylated proteins E rns , E1 and E2 are supposed to be located in the lipid membrane of cellular origin (Chu & Zee, 1984;Coria et al., 1983;Fetzer et al., 2005). For E rns and E2 homodimers and for E1 heterodimers with E2 can be detected in infected cells (Rümenapf et al., 1993;Weiland et al., 1990). The envelope protein E rns lacks a typical membrane anchor and seems to be important for a first cell contact by binding to glycosaminoglycans (Fetzer et al., 2005;Iqbal et al., 2000). E1-E2 heterodimers are essential for virus entry by binding to the bovine CD46 molecule ...
In 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 E(rns), 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-E(rns) was only efficient if Bungowannah virus-E(rns) 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.