DNase SISPA-Next Generation Sequencing Confirms Schmallenberg Virus in Belgian Field Samples and Identifies Genetic Variation in Europe

Abstract

In 2011, a novel Orthobunyavirus was identified in cattle and sheep in Germany and the Netherlands. This virus was named Schmallenberg virus (SBV). Later, presence of the virus was confirmed using real time RT-PCR in cases of congenital malformations of bovines and ovines in several European countries, including Belgium. In the absence of specific sequencing protocols for this novel virus we confirmed its presence in RT-qPCR positive field samples using DNase SISPA-next generation sequencing (NGS), a virus discovery method based on random amplification and next generation sequencing. An in vitro transcribed RNA was used to construct a standard curve allowing the quantification of viral RNA in the field samples. Two field samples of aborted lambs containing 7.66 and 7.64 log10 RNA copies per µL total RNA allowed unambiguous identification of SBV. One sample yielded 192 SBV reads covering about 81% of the L segment, 56% of the M segment and 13% of the S segment. The other sample resulted in 8 reads distributed over the L and M segments. Three weak positive field samples (one from an aborted calf, two from aborted lambs) containing virus quantities equivalent to 4.27–4.89 log10 RNA copies per µL did not allow identification using DNase SISPA-NGS. This partial sequence information was compared to the whole genome sequence of SBV isolated from bovines in Germany, identifying several sequence differences. The applied viral discovery method allowed the confirmation of SBV in RT-qPCR positive brain samples. However, the failure to confirm SBV in weak PCR-positive samples illustrates the importance of the selection of properly targeted and fresh field samples in any virus discovery method. The partial sequences derived from the field samples showed several differences compared to the sequences from bovines in Germany, indicating sequence divergence within the epidemic.

Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

“…However, particularly in complex biological samples (e.g. tissues), the relatively low abundance of viral nucleic acids in comparison to a background of host, bacterial and other contaminating genetic material (Rosseel et al, 2012) remains a challenge. In short, increasing levels of viral genetic material, whilst reducing background signal (host) is the key to a successful metagenomic-based viral discovery pipeline.…”

Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

“…11,12 Using the NGS technology, the whole-genome sequencing of viruses, such as Schmallenberg virus, Dengue virus, enterovirus, and respiratory syncytial virus, has been completed. [13][14][15] This approach is cost effective and highly efficient for the recovery of viral genome sequences, taking advantage of the detection and characterization of viral genomes and viral metagenomics. 16,17 Sequence-independent, single-primer amplification (SISPA) is a methodology to synthesize and enrich viral genomes using random hexamer and SISPA sequences (5′-GCCGGAGCTCTGCAGATATC-3′).…”

Next-Generation Sequencing for Pathogen Detection and Identification

“…There are numerous derivations and examples of this type of technique as applied to environmental and clinical samples in the literature (Ng, Wheeler, et al, 2011;Ng, Willner, et al, 2011;Thurber, Haynes, Breitbart, Wegley, & Rohwer, 2009), and one specific example of a related approach is sequence-independent single-primer amplification (SISPA; Rosseel et al, 2012), which was applied to diagnosis of Schmallenberg virus in lambs and calves. SISPA has also been applied to single plaque sequencing (Depew et al, 2013).…”