title: Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems.
abstract: BACKGROUND
  Schmallenberg 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.
  
  
  METHODS
  In 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.
  
  
  RESULTS
  All 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Melina
authors.initials: M
authors.lastname: Fischer
authors.forename: Horst
authors.initials: H
authors.lastname: Schirrmeier
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wernike
authors.forename: Anne
authors.initials: A
authors.lastname: Wegelt
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493, Greifswald-Insel Riems, Germany. bernd.hoffmann@fli.bund.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
mesh_terms: D000818:Animals; D002044:Bunyaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D005858:Germany; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology; D012840:Simbu virus / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors