A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Does Delay Length or Sequence Exposure Affect Repeated Acquisition Performance?

Color poster with text and graphs.Repeated acquisition of response chains is a systematic method used to study learning and memory. Subjects complete sequences of responses in order to earn reinforcers. Subjects must learn the correct sequence of responses for the session through trial-and-error. As the subject completes more correct sequences over session time, the number of errors should be reduced throughout the session. This within-session error reduction is defined as learning. The purpose of this study was to examine learning and memory by establishing baseline error rates for subjects and response sequences.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Effects of Duloxetine in Rats Trained to Discriminate Between 2- and 22- hr Food Deprivation

Color poster with text and graphs.Duloxetine inhibits serotonin and norepinephrine transporters, and is clinically used to treat depression, pain, and generalized anxiety disorder. Duloxetine has been shown to reduce food intake in several species. The purpose of this study was to examine the effects of duloxetine in non-restricted rats trained to discriminate between 22- and 2-hour food deprivation to gain better understanding of neurochemicals mediating the discriminative stimulus effects of 22-hour food deprivation.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Effects of Duloxetine in Rats Trained to Discriminate Between 2 and 22 hr Food Deprivation

Color poster with text, graphs, and tables.Duloxetine inhibits serotonin and norepinephrine transporters, and is clinically used to treat depression, pain, and generalized anxiety disorder. Duloxetine has been shown to reduce food intake in several species. The purpose of this study was to examine the effects of duloxetine in non-restricted rats trained to discriminate between 22- and 2-hour food deprivation to gain better understanding of neurochemicals mediating the discriminative stimulus e ects of 22-hour food deprivation.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Effects of Chlorpromazine in Rats Trained to Discriminate Between 2 and 22 Hr Food Deprivation

Color poster with text, graphs, and tables.Chlorpromazine, a typical antipsychotic, has been shown to have varying effects on food intake. Chlorpromazine is a dopamine antagonist that blocks several dopamine receptors (including D1 and D2). Previous research indicated chlorpromazine (5.0mg/kg) may affect food intake in two time phases. This study looked at the effects of chlorpromazine in a food-deprivation discrimination paradigm that may serve as an animal model of 'hunger'.University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Characterization of ventricular assist device-mediated sensitization in the bridge-to-heart-transplantation patient.

OBJECTIVE:Ventricular assist devices (VADs) are associated with increased anti-human leukocyte antigen antibody production. The purpose of this study is to characterize differences in sensitization patterns in patients receiving axial flow, implantable VADs versus pulsatile, paracorporeal biventricular assist devices (BIVADs) as bridges to transplantation. METHODS:The study is a retrospective review of 68 patients who were bridged to transplantation with either a VAD or a BIVAD, as described, from January 2007 to June 2010, at a university medical center. RESULTS:Five of 15 (33.3%) VAD patients became sensitized during treatment, compared with 30 of 53 (56.6%) BIVAD patients, P = .15. Multivariable analysis comparing BIVAD with VAD, while controlling for previous cardiac surgery, pregnancy, and packed red blood cell transfusion produced an odds ratio of 2.99, P = .14. Of sensitized patients, all 5 (100%) of the VAD patients had pre-existing antibodies before VAD placement, compared with 9 of 30 (30.0%) BIVAD patients, P = .006. Maximum cumulative mean fluorescence intensities for BIVAD were 46,259 ± 66,349 versus 42,540 ± 12,840 for VAD, P = .90. Time to maximum antibody expression was shorter for the VAD group (34 ± 28 days vs 5.8 ± 9 days, P = .04). CONCLUSIONS:Device type was not a factor in patient sensitization after implantation. However, VAD patients required pre-existing sensitization before implantation to produce antibodies during their treatment interval, whereas more than two thirds of BIVAD patients developed de novo antibodies. These data suggest that the mechanism of sensitization between VAD and BIVAD patients may differ, and further mechanistic studies into the impact of device types on patient sensitization are warranted