Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems

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. Conclusion 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.


Background
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). The virus caused clinical signs such as fever, diarrhea and decreased milk yield in dairy cattle. It was finally identified via fullgenome sequencing combined with metagenome analysis and named Schmallenberg virus (SBV) after its geographical origin [1]. 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 [2].
Orthobunyaviruses are characterized by a tripartite, single-stranded, negative-sense RNA genome [3,4]. The three segments are termed according to their size as small (S), medium (M) and large (L) [5]. The genus is divided into 18 serogroups defined by serological relationships. One of the largest serogroups, the Simbu serogroup, comprises at least 25 members [6]. 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 [7]. 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 [8].
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. Furthermore, several diagnostic real-time RT-PCR assays for the sensitive and specific detection of the SBV genome were developed and validated. 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. The SBV-M1 assay produced a 137 bp PCR product from the M-segment, whereas the published SBV-S3 assay detected an 88 bp region of the S-segment [9]. All of these PCR-systems were tested with a panel of different Simbu serogroup viruses as well as several SBV field samples from diseased cattle, sheep and goats originating from all over Germany.  Table 1 for species classification) including SBV and three members of the Bunyamwera serogroup (Batai virus BATV, Bunyamwera virus BUNV, Ngari virus NRIV) were tested to define the detection spectrum of the intercalating dye based pan-Simbu real-time RT-PCR system and the TaqMan probe-based SBV real-time RT-PCR assays (Table 1). Although the applied virus titers were partially low (1 × 10 2 , 1 × 10 2.5 or 1 × 10 3 50% tissue culture infectious doses (TCID 50 ) per ml), the pan-Simbu real-time RT-PCR system detected all tested isolates of the Simbu serogroup. DOUV however was recognized with a high quantification cycle (C q ) value of 35.7 presumably caused by mismatches in the binding region of the forward primer ( Figure 1). Also the detection of THIV seems problematic, yielding the highest C q -value (36.2) in this investigation. To further characterize the pan-Simbu real-time RT-PCR system the limit of detection and the PCR efficiency were determined using an SBV L-segment standard. The assay reliably detected 100 copies per reaction and displayed an efficiency of 91.6%.

Results and discussion
The potential of a subsequent species classification via sequencing was investigated for AINOV, DOUV, PEAV, SABOV, SANV, SATV, SHAV, SHUV and SIMV. Each sequence fragment displayed 100% sequence identity with the respective Simbu serogroup virus in GenBank, whereas 60-88% sequence identity was determined among the amplified products, facilitating a reliable differentiation. In silico analyses indicated that this pan-Simbu system is probably able to detect also members from other serogroups, e.g. several members of the California serogroup: Tahyna-, Chatanga-, La Crosse-, Jamestown Canyon-, Snowshoe hare-and Inkoo virus [11] (Figure 1). This assumption was supported by detection of the Bunyamwera serogroup viruses BATV, BUNV and NRIV (Table 1). However, it cannot be excluded that some untested members could be only poorly or not detected at all. Previously published real-time RT-PCR systems are available e.g. for the specific detection of OROV [12] as well as for the simultaneous detection of AKAV and AINOV [13], but to our knowledge this pan-Simbu PCR is the first generic real-time PCR approach for the detection of orthobunyaviruses, predominantly members of the Simbu serogroup.
The different Simbu serogroup viruses and the three Bunyamwera serogroup viruses were also analyzed with SBV-specific real-time RT-PCR assays. With this approach, only the SBV-M1 assay scored negative for the Bunyamwera serogroup viruses and for all Simbu serogroup viruses, with the exception of SBV (Table 1). This assay seemed to be highly specific for SBV. In addition to SBV, the SBV-L1 and the SBV-L1.4 assays also recognized RNA from a SHAV (species Shamonda virus, reassortant comprising the SBV S-and L-segments and the M-segment from an unclassified virus [7]) sample. Compared to each other, these assays displayed markedly different C q -values for the detection of SHAV presumably due to differences in the primer binding capability caused by a slight adjustment of the detection region for the SBV-L1.4 assay. The SBV-S3 assay was able to detect RNA from SBV, SHAV and DOUV (species Sathuperi virus; see Table 1 for species classification). In this case, the detection region of the SBV-S3 assay on the S-segment is highly similar between SBV and the reassortant SHAV and further seems to be rather similar between the two members of the Sathuperi virus species SBV and DOUV which consequently enables their recognition. neg.: negative result; -: not available; pan Simbu: pan-Simbu real-time RT-PCR system; TCID 50 : 50% tissue culture infectious doses; M1: highly specific SBV-M1 assay; L1: SBV-L1 assay, L1.4: SBV-L1.4 assay; S3: SBV-S3 assay; *species classification according to [10], SBV [7]; # 1:100 dilution of original RNA; columns 5-9 represent mean C q -values from duplicates. To evaluate the sensitivity of the different assays a log 10 dilution series (10 -2 to 10 -8 dilution) of SBV-RNA extracted from a cell culture sample (BH80/11-4, initial titer 1 × 10 6.3 TCID 50 /ml) was tested ( Table 2). The maximum detection rate of the pan-Simbu real-time RT-PCR system was seen at the 10 -4 dilution; due to recognition up to this dilution step, the system seems sufficient for a preliminary screening of undiluted field samples of good quality. The SBV-S3 assay provided the best performance with a reliable recognition (detection of duplicate samples) of every dilution step up to the 10 -7 dilution in combination with the lowest C q -values compared to the other assays. Furthermore, the SBV-L1 and SBV-L1.4 assays displayed comparable C q -values for the dilution series with a reliable detection up to a dilution of 10 -6 . The SBV-specific SBV-M1 assay allowed a positive detection up to the dilution step 10 -5 . Despite its lower sensitivity, the SBV-M1 assay is a valuable tool for distinguishing SBV from other orthobunyaviruses to yield a precise diagnosis in regions where multiple orthobunyaviruses occur.
In addition to the sensitivity analysis, the pan-Simbu real-time RT-PCR system, the SBV-M1, the SBV-L1, the SBV-L1.4 and the SBV-S3 assays were tested using 47 RNA samples from SBV-suspicious field material as well as a defined sample from an SBV challenge experiment [1] (Table 3). In this approach, the SBV-S3 assay showed the lowest C q -values among the tested assays and recognized 40 positive samples, while eight isolates scored negative (pre-tested as negative by SBV-L1 assay).
The pan-Simbu real-time RT-PCR system detected 32 positive samples out of the 40 SBV-S3-positive samples. Three samples which had C q -values above 28 using the SBV-S3 assay scored doubtful with only one positive duplicate using the pan-Simbu real-time RT-PCR system. Eight samples defined as SBV-negative with the SBV-S3 assay, and additional five samples with a genome load of C q -values >28 using the SBV-S3 assay, scored negative in the pan-Simbu real-time RT-PCR system. In general, the pan-Simbu real-time RT-PCR system produced higher C q -values than the SBV-specific assays (Table 3). Therefore, the sensitivity of the pan-Simbu system presumably was not sufficient for samples containing a low viral genome load. Nevertheless, the pan-Simbu real-time RT-PCR system was able to detect field samples with C qvalues below 28 reliably measured by the SBV-S3 assay as well as the animal trial sample (Table 3).
Furthermore, the SBV-M1 assay revealed 39 positive and nine negative samples (Table 3). One SBV-S3 positive sample (C q 37) scored negative, presumably due to the reduced sensitivity of the SBV-M1 assay. The SBV-L1 assay detected 37 positive and eleven negative samples. Three samples tested positive with C q -values >31 in the SBV-S3 assay scored negative. The SBV-L1.4 assay yielded comparable C q -values to the SBV-L1 assay, but recognized 38 positive samples, and two doubtful results (SBV-S3 C qvalues >34) with only one positive duplicate displaying a Cq-value higher than 38. Eight isolates scored negative (in concordance with SBV-S3) with both SBV-L assays.
Finally, the analytical sensitivity of the SBV-S3 singletarget assay in comparison to two different SBV-S3 duplex assays including internal amplification controls was evaluated ( Table 4). The single-target assay detected a dilution of 2 × 10 -1 copies/μl, which corresponded to one RNA copy per reaction. Both duplex assays were able to detect at most 2 × 10 0 copies/μl (10 copies per reaction), while simultaneous co-amplification of either IC2-RNA (~C q 26) or the housekeeping gene beta-actin (~C q 29) was performed reliably.

Conclusion
In conclusion, the pan-Simbu real-time RT-PCR system was able to detect all tested members of the Simbu serogroup as well as most of the SBV field samples and three Bunyamwera serogroup viruses with an acceptable sensitivity for screening diagnostics. Although we were not able to test all 25 members of the Simbu serogroup, we investigated key-members with clinical or economic importance like OROV and AKAV [6]. According to in silico analyses the system seems to be able to detect a broad spectrum of orthobunyaviruses ( Figure 1) as supported by detection of members from the Bunyamwera serogroup (Table 1). The pan-Simbu real-time RT-PCR system is based on two highly conserved sequence regions of the L-segment which were selected as primer binding sites. As an additional feature of the novel system, these primers enclosed a variable sequence region, which is suitable for subsequent species classification via sequencing, avoiding misdiagnosis. This opportunity was confirmed exemplarily for AINOV, DOUV, PEAV, SABOV, SANV, SATV, SHAV, SHUV and SIMV. The here  described pan-Simbu PCR system is therefore the first available tool for a broad screening of samples predominantly for Simbu serogroup virus genomes and could also allow the identification of related orthobunyaviruses in mammalian but also insect vector samples.
For the specific detection of SBV-genome, the SBV-S3 assay turned out to be the most suitable system with the highest sensitivity and reliability. The analytical sensitivity of the SBV-S3 single-target assay as well as the two duplex assays including an internal amplification control (IC2-RNA, beta-actin respectively) were determined as one copy per reaction performing the single-target assay and ten copies per reaction using both duplex systems. Furthermore, the SBV-S3 assay was also able to detect the highly related Sathuperi virus species member DOUV as well as the reassortant SHAV apart from SBV.

Samples and RNA isolation
Viral RNA from field samples, from blood of an experimentally infected calf [1] (Table 3) as well as from cell culture material of the Simbu serogroup viruses namely AINOV, AKAV, DOUV, OROV, PEAV, SABOV, SANV, SATV, SHAV, SHUV, SIMV, THIV, TINV [6] and from the Bunyamwera serogroup viruses BATV [14], BUNV, NRIV was extracted manually using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions or using the MagNA Pure LC 2.0 in combination with the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's recommendations. All samples were stored at −20°C until use.

Oligonucleotides and primer design
The sequences of primers and probes are listed in Table 5. For the pan-Simbu assay, primers were selected by analyzing the consensus sequence of the L-segment of 70 orthobunyaviruses available in GenBank. The locations of the primers and potential mismatches with the L-segment of different orthobunyaviruses are depicted in Figure 1.
The selection of primers and probes of the SBV realtime RT-PCR assays was based on the first available sequence data of the SBV isolate BH80/11-4 [1] and was supported by the software package Beacon Designer 7.0 (PremierBiosoft, Palo Alto, USA).
All oligonucleotides were synthesized by metabion (metabion international AG, Martinsried, Germany) and stored at −20°C until use.

Pan-Simbu real-time RT-PCR
In order to allow a broad detection of members of the Simbu serogroup a pan-Simbu real-time RT-PCR was     Table 5) and 5 μl RNA template or RNase free water for the no template control (NTC) was used. The following thermal program was applied 1 cycle of 50°C for 30 min and 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and 78°C for 15 s. The collection of the fluorescence data was performed during the 78°C elongation step.

PCR efficiency and limit of detection for pan-Simbu real-time RT-PCR
The PCR efficiency and the limit of detection for the pan-Simbu real-time RT-PCR were determined using an SBV L-segment plasmid (L-segment sequence according to GenBank: HE649912 in X8δT backbone). The exact number of plasmid molecules was calculated (http:// www.molbiol.edu.ru/eng/scripts/01_07.html) and a log 10 dilution series (2 × 10 6 to 2 × 10 -1 copies/μl) was prepared. This dilution series was tested with the pan-Simbu real-time RT-PCR in four replicates. PCR efficiency was calculated by the CFX manager software 3.0 (Bio-Rad Laboratories Inc., Hercules, USA).

Sequencing of pan-Simbu real-time RT-PCR products
For species classification of members of the Simbu serogroup (i.e. AINOV, DOUV, PEAV, SABOV, SANV, SATV, SHAV, SHUV and SIMV) obtained PCR products of the pan-Simbu real-time RT-PCR were analyzed on a 1.5% agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Direct sequencing was carried out in both directions by termination cycle sequencing using the Big Dye Terminator Mix 1.1 (Applied Biosystems, Carlsbad, USA) with the same primers used for PCR product amplification. The assay was optimized by using a total reaction volume of 10 μl. Briefly, for one reaction 1 μl RNase-free water, 1 μl of 5x Sequencing Buffer, 2 μl Big Dye Terminator Mix 1.1 and 1 μl of the according primer (5 μM). The following thermal program was applied 1 cycle of 96°C for 1 min followed by 26 cycles of 95°C for 15 s, 53°C for 10 s, and 60°C for 4 min. After that, cycle sequencing products were purified with DyeEx 2.0 Spin Kit (Qiagen, Hilden, Germany). The nucleotide sequences were resolved in an ABI 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, USA). Sequences were aligned and analyzed in BioEdit [15] (version 7.1.7).