The MinION sequencer (Oxford Nanopore Technologies) is a paradigm shifting device allowing rapid, real time long read sequencing of nucleic acids. Yet external benchmarking of this technologies’ capabilities has not been extensively reported, nor has thorough evaluation of its utility for field-based analysis with sub-optimal sample types been described. The aim of this study was to evaluate the capability of the MinION sequencer for bacterial genomic and metagenomic applications, with specific emphasis placed on the quality, yield, and accuracy of generated sequence data. Two independent laboratories at the National Microbiology Laboratory (Public Health Agency of Canada), sequenced a set of microbes in replicate, using the currently available flowcells, sequencing chemistries, and software available at the time of the experiment. Overall sequencing yield and quality improved through the course of this set of experiments. Sequencing alignment accuracy was high reaching 97% for all 2D experiments, though was slightly lower for 1D sequencing (94%). 1D sequencing provided much longer sequences than 2D. Both sequencing chemistries performed equally well in constructing genomic assemblies. There was evidence of barcode cross-over using both the native and PCR barcoding methods. Despite the sub-optimal nature of samples sequenced in the field, sequences attributable to B. anthracis the target organism used in this scenario, could none-the-less be detected. Together, this report showcases the rapid advancement in this technology and its utility in the context of genomic sequencing of microbial isolates of importance to public health.

Bundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25 to 36%. Despite having been known to the scientific and medical communities for almost 1 decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the order Mononegavirales. To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8 and 9 days, and a petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts and dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological, and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to what was seen with BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates and will accelerate efforts to understand filovirus pathogenesis and transmission as well as the development of specific vaccines and antivirals. IMPORTANCEThe 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genus Ebolavirus and has caused outbreaks in the past but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines and therapies and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection as well as to Ebola virus infection and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to that seen in humans and nonhuman primates. We believe that this will improve the ability to study BDBV and provide a platform to test vaccines and therapeutics.

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay. B otulism, a rare and severe disease characterized by a descending flaccid paralysis, is caused by botulinum neurotoxin (BoNT), the most potent toxin known. BoNT is produced by phylogenetically distinct anaerobic spore-forming bacteria grouped under the taxonomic designation of Clostridium botulinum. Rare botulinogenic strains of related clostridia, such as C. baratii, C. butyricum, and C. argentinense, have also been observed (1, 2). Seven serologically distinct BoNTs (A to G) can be distinguished based on neutralization of toxicity with specific antisera. Recently, a strain of C. botulinum producing botulinum neurotoxin type B (BoNT/B) and another BoNT that is not neutralized by antitoxins to BoNTs A to G has been isolated from a case of infant botulism (3). It has been proposed that this novel neurotoxin is an eighth serotype, BoNT/H (3,4). The botulinum neurotoxins are comprised of three structural domains (translocation [H N ], receptor binding [H C ], and catalytic [LC]). These toxins target different SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins in the neuromuscular junction to block neurotransmitter release (5,6).Heterogeneity within the BoNTs has led to a classification of BoNTs into subtypes. Within a toxin serotype, differences in amino acid sequences can range from 0.9% to 25% (2, 7). Hill and Smith (8) point out that the newer subtypes have been defined based on their DNA sequence and propose the use of the term "subtype/genetic variant" to avoid confusion with the historical use of "subtype," utilized to designate immunological or enzymatic differences among neurotoxins. Two approaches have been used to define new BoNT variants. The first uses a cutoff value of 2.5% difference in amino acid composition (9-11), whereas the second relies on a phylogenetic approach in which variants correspond to clades formed by the clustering of bont sequences (1, 7, 12) Based on these methods, bont/A1 to bont/A5, bont/B1 to bont/ B7, bont/E1 to bont/E9, and bont/F1 to bont/F7 gene variants have been described (1,2,8,10,(12)(13)(14)(15)(16)(17).In Canada, C. botulinum type E is the predominant BoNT serotype associated with food-borne botulism, accounting for 86.2% of all laboratory-confirmed food-borne botulism outbreaks reported between 1985 and 2005 (18). Although variant types E1, E3, and E7 have been identified in t...

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain.