2/25/2023 0 Comments Whm template for 010 editor![]() ![]() Within the SBV L-segment the SBV-L1 and the SBV-L1.4 assays detected an amplification fragment of 144 bp and 107 bp in length, respectively. ![]() Furthermore, several diagnostic real-time RT-PCR assays for the sensitive and specific detection of the SBV genome were developed and validated. In this study a pan-Simbu real-time reverse transcriptase (RT) PCR system was developed based on two conserved sequence regions of the L-segment facilitating the amplification of a 279 base pair (bp) fragment for the reliable detection of viruses predominantly from the Simbu serogroup. Sequencing also revealed a complementarity of the 3 prime and the 5 prime end of each segment, a typical feature of the members of the family Bunyaviridae, which leads to the formation of panhandle structures. Full-genome sequencing as well as Sanger sequencing and subsequent phylogenetic analyses revealed that SBV is a Simbu serogroup virus, closely related to viruses of the species Sathuperi virus. One of the largest serogroups, the Simbu serogroup, comprises at least 25 members. The genus is divided into 18 serogroups defined by serological relationships. The three segments are termed according to their size as small (S), medium (M) and large (L). Orthobunyaviruses are characterized by a tripartite, single-stranded, negative-sense RNA genome. Subsequently, SBV was detected in samples from diseased cattle in the Netherlands and in malformed sheep and goat lambs as well as in calves from several European countries. It was finally identified via full-genome sequencing combined with metagenome analysis and named Schmallenberg virus (SBV) after its geographical origin. The virus caused clinical signs such as fever, diarrhea and decreased milk yield in dairy cattle. In October 2011, a novel orthobunyavirus (family: Bunyaviridae) related to the Simbu serogroup viruses affected dairy cattle on a farm near the city of Schmallenberg (North Rhine-Westphalia, Germany). Regarding SBV diagnostics, the performance of the S-segment targeting SBV-S3 assay was superior with respect to the analytical sensitivity. As an additional feature of the pan-Simbu real-time RT-PCR system, subsequent species classification via sequencing is feasible. According to in silico analyses, this system seems to be able to detect a broad orthobunyavirus spectrum. 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. In addition, for confirmation of SBV-genome detection the highly specific SBV-M1 assay was established. 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. 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. ResultsĪll 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. Furthermore, in silico analyses were performed to investigate suitability for the detection of further orthobunyaviruses. ![]() Several pan-Simbu real-time RT-PCR products were sequenced via Sanger sequencing. 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. In this study a pan-Simbu real-time RT-PCR system was established and compared to several SBV real-time RT-PCR assays. 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. Although symptoms of acute infection were not observed, infection during a vulnerable phase of pregnancy caused congenital malformations and stillbirths. Since then, SBV additionally has been detected in adult sheep and goats. It'd probably be better if each struct were defined separately and then I could just refer to them in a "main" struct.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. I've tried to apply color to it to make it easier to pick out which part is what, but adding one color for an entire struct does not seem too useful in the long-run. Here, there are structs defined within structs, all defined within the File struct which seems kind of silly. ![]()
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