The Ebola virus disease (EVD) epidemic in West Africa is the largest on record, responsible for >28,599 cases and >11,299 deaths 1. Genome sequencing in viral outbreaks is desirable in order to characterize the infectious agent to determine its evolutionary rate, signatures of host adaptation, identification and monitoring of diagnostic targets and responses to vaccines and treatments. The Ebola virus genome (EBOV) substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 to 1.42 × 10−3 mutations per site per year. This is equivalent to 16 to 27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic 2-7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought-after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions 8. Genomic surveillance during the epidemic has been sporadic due to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities 9. In order to address this problem, we devised a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. Here we present sequence data and analysis of 142 Ebola virus (EBOV) samples collected during the period March to October 2015. We were able to generate results in less than 24 hours after receiving an Ebola positive sample, with the sequencing process taking as little as 15-60 minutes. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, highmannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.Rotaviruses (RV) are the leading viral etiologic agents of severe pediatric gastroenteritis worldwide, affecting nearly all children before the age of 5, with 2 million cases resulting in 444,000 deaths annually (33,34,40). RV nonstructural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum (ER) transmembrane glycoprotein due to the protein's high-mannose glycosylation and its critical function as an intracellular receptor for the translocation of subviral particles into the ER during virion morphogenesis (2,5,14). However, the identification of NSP4 and NSP4 amino acids (aa) 114 to 135 (NSP4 114-135 ) as enterotoxic and the redistribution of RVencoded proteins upon NSP4 silencing led to a reevaluation of NSP4 function(s) and subcellular localization(s) (4, 31).A cleaved NSP4 fragment, aa 112 to 175, is secreted from RV-infected epithelial cells, indicating that some portion of NSP4 traffics from the ER to the plasma membrane (PM) (65). The colocalization of NSP4 114-135 and the extracellular matrix proteins laminin-␤3 and fibronectin at the basement membrane of small-intestinal epithelia from RV strain EDIM-infected mouse pups also supports NSP4 transport to the PM during host infection (8). While both findings demonstrat...

BackgroundRotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types.MethodsTransport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls.ResultsOnly full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells.ConclusionsThe intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.

BackgroundRotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.MethodsA panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP446-175-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study.ResultsMutations of the hydrophilic face (NSP446-175-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP446-175-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112−140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea.ConclusionsThese data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.

Chemical disinfection and inactivation of viruses is largely understudied, but is very important especially in the case of highly infectious viruses. The purpose of this LDRD was to determine the efficacy of the Sandia National Laboratories developed decontamination formulations against Bovine Coronavirus (BCV) as a surrogate for the coronavirus that causes Severe Acute Respiratory Syndrome (SARS) in humans. The outbreak of SARS in late 2002 resulted from a highly infectious virus that was able to survive and remain infectious for extended periods. For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conducted to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition. The MS-2 and T4 were slightly more resistant than BCV and required a 2 minute exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.4

The SEAMISTTM inverting membrane deployment system has been used successfully at the Mixed Waste Landfill Integrated Demonstration (MWLID) for multipoint vapor sampling, pressure measurement, permeability measurement, sensor integration demonstrations, and borehole lining. Several instruments were deployed inside the SEAMISTTM-lined boreholes to detect metals, radionuclides, moisture, and geologic variations. The liner protected the instruments from contamination, maintained support of the uncased borehole wall, and sealed the total borehole from air circulation. Recent activities included the installation of three multipoint vapor sampling systems * iii MAS Intentionally Left Blank We also wish to acknowledge the DOE Office of Technology Development and the Mixed Waste Landfill Integrated Demonstration for their sponsorship of this project.

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Since the tragic events of September 2001 and the subsequent dissemination of Bacillus anthracis through the US postal system, the international community has begun to focus more intently on the threat of transnational terrorist groups, and, in particular, on the frightening prospect of large‐scale biological terrorism. Various strategies are being applied to combat the biological threat. Most strategies are reactionary in nature. They focus on improving the ability to detect and respond to a biological attack after it has occurred. The international community has also begun employing preventive strategies. One of the principal strategies in this category is laboratory biosecurity, which is defined as the protection of dangerous pathogens and toxins against theft and misuse. By reducing the risk that potential bioterrorists and others could acquire certain dangerous biological materials, biosecurity provides the first line of defense against biological attacks. There are several unique challenges posed by microorganisms that differentiate biosecurity from other forms of high security, and an effective biosecurity effort must take these into account. The first step towards implementing biosecurity is conducting a laboratory risk assessment. Security risk assessments help reduce risks in bioscience laboratories because they prioritize a wide range of risks, and direct management efforts toward the most effective risk reduction measures for the highest risks. Different techniques can be used to mitigate risks in different circumstances to improve security in bioscience laboratories. Although laboratory biosecurity cannot prevent biocrimes or bioterrorism, it is appropriate to take steps that reduce the likelihood that dangerous pathogens and toxins could be stolen and misused from bioscience facilities.