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qScript XLT 1-Step RT-qPCR ToughMix

Tough-tested 1-step real time PCR
Features & Benefits
  • Tough Tested – Overcomes common inhibitors including polysaccharides, heme/hemoglobin, humic acid, melanin
  • Flexible – Use fast or standard qPCR cycling conditions
  • Broad dynamic range- Reliable data from your precious samples every time
  • Multiplexing enabled – Supports highly sensitive detection for up to four targets

 

qScript XLT 1-Step RT-qPCR ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Product
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Product
Kit Size
Order Info
qScript XLT 1-Step RT-qPCR ToughMix ROX
Request Sample
Kit Size:
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100 x 20 μL rxns (1 x 1 mL)
500 x 20 μL rxns (5 x 1 mL)
2000 x 20 μL rxns (1 x 20 mL)
Product
Kit Size
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qScript XLT 1-Step RT-qPCR ToughMix
Request Sample
Kit Size:
Order Info:
100 x 20 μL rxns (1 x 1 mL)
500 x 20 μL rxns (5 x 1 mL)
2000 x 20 μL rxns (1 x 20 mL)
5000 x 20 µL rxns (1 x 50 mL)
Product
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Order Info
qScript XLT 1-Step RT-qPCR ToughMix Low-ROX
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Kit Size:
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100 x 20 μL rxns (1 x 1 mL)
500 x 20 μL rxns (5 x 1 mL)
2000 x 20 μL rxns (1 x 20 mL)
5000 x 20 µL rxns (1 x 50 mL)

Description

qScript XLT One-Step RT-qPCR ToughMix is a ready-to-use, highly sensitive master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan® 5'-hydrolysis probes. First-strand cDNA synthesis and subsequent PCR amplification are carried out seamlessly in the same reaction mixture with optimized 1-step thermal cycling parameters.

This kit is ideal for highly sensitive quantification of RNA viruses or low abundance RNA targets as well as high throughput gene-expression studies. The system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity in minimal reaction volumes and accelerated thermal cycling rates. The only necessary user-supplied materials for RT-qPCR is RNA sample and probe assay. It is compatible with all types of molecular probe assays including dual-labeling strategies.

Elevating cDNA synthesis reaction temperature to 50-55°C during one-step RT-qPCR improves primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis.

What Customers Say

We used the qScript XLT 1-step RT-qPCR ToughMix to reliably and very quickly identify an experimental drug candidate and demonstrate its efficacy to correct the debilitating effect of an inherited disease. The immediate success and impeccable performance of this mix shortened our labs time to result and confirmation easily by 2-3 months.

TransLab, Children’s Hospital Boston
Lead Scientist / Researcher
Details

Details

Contents

2X concentrated One-Step Master Mix containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP
  • qScript XLT reverse transcriptase
  • RNase inhibitor protein
  • AccuStart II Taq DNA Polymerase
  • Inert AccuVue™ plate loading dye
  • Proprietary enzyme stabilizers and performance-enhancing additives
  • Titrated reference dye (if applicable)
Instrument Capability

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™
  • Agilent AriaMx
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5
Customer Testimonials

Customer Testimonials

qScript XLT 1-Step RT-qPCR ToughMix

"It is an easy kit that contains all the reagents needed for the master mix in one tube. The only components I added were my primers, probe and nuclease free water. The protocol is short and user friendly."

Olivia L. | Senior Research Engineer
qScript XLT One-Step RT-qPCR ToughMix

"qScript XLT One-Step RT-qPCR ToughMix is an easy kit that contains all the reagents needed for the master mix in one tube. The only components I added were my primers, probe and nuclease free water. The protocol is short and user friendly."

Senior Research Engineer | Umeå University

Details

Contents

2X concentrated One-Step Master Mix containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP
  • qScript XLT reverse transcriptase
  • RNase inhibitor protein
  • AccuStart II Taq DNA Polymerase
  • Inert AccuVue™ plate loading dye
  • Proprietary enzyme stabilizers and performance-enhancing additives
  • Titrated reference dye (if applicable)

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™
  • Agilent AriaMx
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5

Resources

Customer Profile Stories

Flyers

Product Manuals

Technical Notes

CofA (PSFs)

Click here to see all CofA (PSFs)

SDSs

Publications

A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings
Kathleen Gärtner - 2022
Abstract
Background To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. Methods Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. Results In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. Conclusion SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.
SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish
Dragan Brnić - 2022
Abstract
The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.
Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens
Alexander Y. Trick - 2022
Abstract
The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses
Brigitte Sigrist - 2021
Abstract
Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay w
Innate and Adaptive Immune Genes Associated with MERS-CoV Infection in Dromedaries
Sara Lado - 2021
Abstract
The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.
Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds
Jennifer Weidhaas - 2021
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is shed in feces and the viral ribonucleic acid (RNA) is detectable inwastewater. A nine-weekwastewater epidemiology study of tenwastewater facilities, serving 39% of the state of Utah or 1.26Mindividuals was conducted in April andMay of 2020. COVID-19 cases were tabulated fromwithin each sewershed boundary. RNA from SARS-CoV-2 was detectable in 61% of 126 wastewater samples. Urban sewersheds serving >100,000 individuals and tourist communities had higher detection frequencies. An outbreak of COVID-19 across two communities positively correlated with an increase in wastewater SARS-CoV-2 RNA, while a decline in COVID-19 cases preceded a decline in RNA. SARS-CoV-2 RNA followed a first order decay rate in wastewater, while 90% of the RNAwas present in the liquid phase of the influent. Infiltration and inflow, virus decay and sewershed characteristics should be considered during correlation analysis of SAR-CoV-2 with COVID-19 cases. These results provide evidence of the utility of wastewater epidemiology to assist in public health responses to COVID-19.
COVID-19 infection among emergency department healthcare providers in a large tertiary academic medical center following the peak of the pandemic
Eric Murakami - 2021
Abstract
The COVID-19 pandemic has spread through the US during the last few months exposing healthcare providers to possible infection. Here we report testing of emergency department (ED) healthcare providers (HCP) for exposure to COVID-19 through lateral flow point of care (POC) and lab-based enzyme-linked immunosorbent assay (ELISA), and RTq-PCR for evidence of acute infection. 138 ED HCP were tested between May 26th (approximately one month after the peak of COVID-19 first wave of cases) and June 14th. Enrolled ED HCP represented about 70% of the total ED HCP workforce during the study period. Subjects were tested with a POC COVID-19 antibody test, and standard ELISA performed by a university-based research lab. Subjects also provided a mid-turbinate swab and a saliva specimen for RTq-PCR. All subjects provided demographic information, past medical history, information about personal protective equipment (PPE) use, COVID-19 symptoms, as well as potential COVID-19 exposures during the previous 4 weeks, both in the ED, and outside the clinical setting. None of the HCP had positive RT-PCR results; 7 HCP (5%) had positive IgG for COVID-19; there was strong agreement between the lab-based ELISA (reference test) and the POC Ab test (P ≤ 0.0001). For the POC Ab test there were no false negatives and only one false positive among the 138 participants. There was no significant difference in demographic/ethnic variables, past medical history, hours worked in the ED, PPE use, or concerning exposures between seropositive and seronegative individuals. Moreover, there was no significant difference in reported symptoms between the two groups during the previous four weeks. The rate of COVID-19 seroconversion in our ED was 5% during the month following the pandemic's first wave. Based on questionnaire responses, differences in demographics/ethnicity, medical history, COVID-19 exposures, and PPE use were not associated with ED HCP having been infected with SARS-CoV-2. In the setting of our limited cohort of subjects the COVID-19 POC Ab test performed comparably to the ELISA lab-based standard.
Investigation of pooling strategies using clinical COVID-19 samples for more efficient diagnostic testing
Samantha H. Adikari - 2020
Abstract
When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.
Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay
David Cate - 2020
Abstract
The global COVID-19 pandemic has created an urgent demand for accurate rapid point of care diagnostic tests. Antigen-based assays are suitably inexpensive and can be rapidly mass-produced, but sufficiently accurate performance requires highly optimized antibodies and assay conditions. An automated liquid handling system, customized to handle lateral flow immunoassay (LFA) arrays, was used for high-throughput antibody screening of anti-nucleocapsid antibodies that will perform optimally on an LFA. Six hundred seventy-three anti-nucleocapsid antibody pairs were tested as both capture and detection reagents with the goal of finding those pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 while also performing optimally in an LFA format. In contrast to traditional antibody screening methods (e.g. ELISA, bio-layer interferometry), the methods described here integrate real-time LFA reaction kinetics and binding directly on nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.
Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR tests
Samantha A. Byrnes - 2020
Abstract
The rapid onset of the global COVID-19 pandemic has led to multiple challenges for accurately diagnosing the infection. One of the main bottlenecks for COVID-19 detection is reagent and material shortages for sample collection, preservation, and purification prior to testing. Currently, most authorized diagnostic tests require RNA extraction from patient samples and detection by reverse transcription polymerase chain reaction (RT-PCR). However, RNA purification is expensive, time consuming, and requires technical expertise to perform. Additionally, there have been reported shortages of the RNA purification kits needed for most tests. With these challenges in mind, we report on extraction-free amplification of SARS-CoV-2 RNA directly from patient samples. In addition, we have developed a multiplex RT-PCR using the CDC singleplex targets. This multiplex has a limit of detection of 2 copies/µL. We have demonstrated these improvements to the current diagnostic workflow, which reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
An efficient, reproducible and accurate RT-qPCR based method to determine mumps specific neutralizing antibody
Chisha T. Sikazwe - 2020
Abstract
Introduction A resurgence of mumps among fully vaccinated adolescents and young adults globally has led to questions about the longevity of vaccine derived specific immunity. Unfortunately, the ideal serological correlate of immunity to mumps has yet to be identified. However, neutralising antibody titres in serum are used extensively as a surrogate marker of immunity to mumps. Conventional neutralisation tests are technically challenging, thus we developed and validated a high throughput, RT-qPCR microneutralisation (RT-qPCR-MN) method to determine serum neutralising antibody levels to mumps virus strains which avoids a number of the technical limitations of existing methods. Methods The qPCR-MN assays were thoroughly validated using human serum samples from patients with prior exposure to mumps infection or vaccination. Results Each sample of pooled sera neutralised virus at a constant rate and without significant changes when tested against genotype A (MuV-A) and G (MuV-G) mumps virus concentrations from 200 to 3200 TCID50. The within run and between run variation of the RT-qPCR-MN assays for both genotypes were less than 3 % and 9 % for low and high titre samples, respectively. The correlation between the focus reduction neutralisation test and RT-qPCR-MN was excellent for both MuV-G (r2 = 0.80, 95CI: 0.67–1.00, p < 0.0001) and MuV-A genotypes (r2 = 0.88, 95 %CI 0.69–1.00, p < 0.0001) endpoint determinations. Conclusions We have developed a RT-qPCR MN assay for mumps virus that is simple, fast, scientifically objective and has high throughput. The assay can be used to provide key insights into the efficacy of mumps vaccination, to help explain the causes for the resurgence of mumps infection in vaccinated populations.
Characterization of Experimental Oro-Nasal Inoculation of Seba’s Short-Tailed Bats (Carollia perspicillata) with Bat Influenza A Virus H18N11
Marco Gorka - 2020
Abstract
In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat (Artibeus planirostris), respectively. The pathogenesis caused by these viruses in their respective host species is currently insufficiently understood, which is primarily due to the inability to obtain and keep these bat species under appropriate environmental and biosafety conditions. Seba’s short-tailed bats (Carollia perspicillata), in contrast, are close relatives and a natural H18N11 reservoir species, with the advantage of established animal husbandry conditions in academic research. To study viral pathogenesis in more detail, we here oro-nasally inoculated Seba’s short-tailed bats with the bat IAV H18N11 subtype. Following inoculation, bats appeared clinically healthy, but the histologic examination of tissues revealed a mild necrotizing rhinitis. Consistently, IAV-matrix protein and H18-RNA positive cells were seen in lesioned respiratory and olfactory nasal epithelia, as well as in intestinal tissues. A RT-qPCR analysis confirmed viral replication in the conchae and intestines as well as the presence of viral RNA in the excreted feces, without horizontal transmission to naïve contact animals. Moreover, all inoculated animals seroconverted with low titers of neutralizing antibodies.
Different dynamics of Usutu virus infections in Austria and Hungary, 2017–2018
Pia Weidinger - 2020
Abstract
Usutu virus (USUV), a mosquito‐borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass‐mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV‐related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT‐PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV‐associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future.
Respiratory viral infections in Western Australians with cystic fibrosis
Brian Brestovac - 2020
Abstract
Background Viral respiratory infections (VRI) in people living with Cystic fibrosis (CF) is less well understood than respiratory bacterial infections, particularly adults with CF and few studies have compared children with adults. This study evaluated the frequency of respiratory viruses in patients with cystic fibrosis (CF) in Western Australia (WA). We determined the VRI in CF and compared them with non-CF patients. Further, we compared CF patients that were hospitalised with those that were not. Patients/methods Nucleic acid from sputum of 157 CF and 348 non-CF patients was analysed for influenzavirus A (Flu A) and B, (Flu B), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human rhinovirus (RV), and parainfluenza viruses (PIV 1-3) by RT-PCR, during the 2016 winter respiratory season. Results No significant difference in the frequency of respiratory virus detection between CF and non-CF patients was found. RV was the most frequently detected virus in CF patients, and in hospitalised CF. RSV and hMPV were found less frequently in CF patients and RSV was not found in any hospitalised CF patient. A trend for fewer influenzavirus detections in adult CF patients was observed, however the trend was opposite for paediatric patients. RV and Flu A were the most common viruses detected in hospitalised CF patients. Conclusion There was no significant difference in VRI between CF and non-CF patients. RV and influenza A were most commonly found in hospitalised CF patients, suggesting that infection with these viruses may contribute to hospitalisation for CF respiratory exacerbations.
Human Norovirus Neutralized by a Monoclonal Antibody Targeting the Histo-Blood Group Antigen Pocket
Anna D. Koromyslova - 2019
Abstract
Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The persistence of GII.4 is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolates (in 1974) and a more recent epidemic GII.4 variant (in 2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities but similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivity with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus virus-like particles (VLPs) from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide the first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies. IMPORTANCE The emergence of new epidemic GII.4 norovirus variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between the 1974 and 2012 GII.4 variants, whereas these variants showed various levels of reactivity against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket, blocked norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4 1974 isolate but was able to neutralize the newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggest that the amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for infection.
Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of -Lactams and a Novel Regulatory Circuit (MmnRS) Controlling Mex
Srijan Ranjitkar - 2019
Abstract
Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13- 9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a 150-fold upregulation of MexMN pump gene expression and a 50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several -lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.
Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblasts
Pascal Morissette Martin - 2019
Abstract
There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed “bead foams”, comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings. Statement of Significance Biological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.
The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteins
Bernat Blasco-Moreno - 2019
Abstract
The highly conserved 5’–3’ exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins.
Urine RNA Processing in a Clinical Setting: Comparison of 3 Protocols
Megan S. Bradley, MD - 2019
Abstract
Objective: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols. Methods: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses. Results: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9). Conclusions: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.
Storage-Dependent Generation of Potent Anti-ZIKV Activity in Human Breast Milk
Carina Conzelmann - 2019
Abstract
Zika virus (ZIKV) causes congenital neurologic birth defects, notably microcephaly, and has been associated with other serious complications in adults. The virus has been detected in human breast milk and possible transmissions via breastfeeding have been reported. Breast milk is rich in nutrients and bio-active substances that might directly affect viral infectivity. Thus, here, we analyzed the effect of human breast milk on ZIKV infection. We observed that fresh human breast milk had no effect on ZIKV, but found that upon storage, milk effectively suppressed infection. The antiviral activity is present in the fat-containing cream fraction of milk and results in the destruction of the structural integrity of viral particles, thereby abrogating infectivity. The release of the factor is time dependent but varies with donors and incubation temperatures. The viral titer of milk that was spiked with ZIKV decreased considerably upon storage at 37 °C for 8 h, was lost entirely after 2 days of 4 °C storage, but was not affected at −20 °C. This suggests that cold storage of milk inactivates ZIKV and that the antiviral factor in milk may also be generated upon breastfeeding and limit this transmission route of ZIKV.
Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus
Yi Ni - 2019
Abstract
Human hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Approximately 15–25 million people are chronically infected with HDV. As a satellite virus of the human hepatitis B virus (HBV), HDV uses the HBV-encoded envelope proteins for egress from and de novo entry into hepatocytes. So far, in vitro production of HDV particles is restricted to co-transfection of cells with HDV/HBV encoding cDNAs. This approach has several limitations. In this study, we established HuH7-END cells, which continuously secrete infectious HDV virions. The cell line was generated through stepwise stable integration of the cDNA of the HDV antigenome, the genes for the HBV envelope proteins and the HBV/HDV receptor NTCP. We found that HuH7-END cells release infectious HDV particles up to 400 million copies/milliliter and support virus spread to co-cultured cells. Due to the expression of NTCP, HuH7-END cells are also susceptible to de novo HDV entry. Virus production is stable for >16 passages and can be scaled up for preparation of large HDV virus stocks. Finally, HuH7-END cells are suitable for screening of antiviral drugs targeting HDV replication. In summary, the HuH7-END cell line provides a novel tool to study HDV replication in vitro.
Targeting the HIV-infected brain to improve ischemic stroke outcome
Luc Bertrand - 2019
Abstract
HIV-associated cerebrovascular events remain highly prevalent even in the current era of antiretroviral therapy (ART). We hypothesize that low-level HIV replication and associated inflammation endure despite antiretroviral treatment and affect ischemic stroke severity and outcomes. Using the EcoHIV infection model and the middle cerebral artery occlusion as the ischemic stroke model in mice, we present in vivo analysis of the relationship between HIV and stroke outcome. EcoHIV infection increases infarct size and negatively impacts tissue and functional recovery. Ischemic stroke also results in an increase in EcoHIV presence in the affected regions, suggesting post-stroke reactivation that magnifies pro-inflammatory status. Importantly, ART with a high CNS penetration effectiveness (CPE) is more beneficial than low CPE treatment in limiting tissue injury and accelerating post-stroke recovery. These results provide potential insight for treatment of HIV-infected patients that are at risk of developing cerebrovascular disease, such as ischemic stroke.
Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings
Shivani G. Beall - 2019
Abstract
Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.
Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings
Jason L. Cantera - 2019
Abstract
Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.
Arbovirus surveillance using FTATM cards in modified CO2‐baited encephalitis virus surveillance traps in the Northern Territory, Australia
Nina Kurucz - 2019
Abstract
In 2016, modified CO2‐baited encephalitis virus surveillance (EVS) traps were evaluated for flavivirus surveillance in the Northern Territory, Australia. The traps were fitted with honey‐soaked nucleic acid preservation cards (FTATM) for mosquitoes to expectorate virus while feeding on the cards. Cards were tested for the presence of selected arboviruses, with two cards testing positive for Kunjin virus and Alfuy, while sentinel chickens tested in parallel also showed Kunjin virus activity at the same time. The results from the cards and vector mosquito feeding rates indicate that CO2‐baited EVS traps coupled with honey‐baited FTATM cards are an effective tool for broad‐scale arbovirus surveillance.
Experimental Infection and Transmission Competence of Sindbis Virus in Culex torrentium and Culex pipiens Mosquitoes from Northern Sweden
Olivia Wesula Lwande - 2019
Abstract
Introduction: Sindbis virus (SINV) is a mosquito-borne Alphavirus known to infect birds and cause intermittent outbreaks among humans in Fenno-Scandia. In Sweden, the endemic area has mainly been in central Sweden. Recently, SINV infections have emerged to northern Sweden, but the vectorial efficiency for SINV of mosquito species in this northern region has not yet been ascertained. Objective: Mosquito larvae were sampled from the Umea˚ region in northern Sweden and propagated in a laboratory to adult stage to investigate the infection, dissemination, and transmission efficiency of SINV in mosquitoes. Materials and Methods: The mosquito species were identified by DNA barcoding of the cytochrome oxidase I gene. Culex torrentium was the most abundant (82.2%) followed by Culex pipiens (14.4%), Aedes annulipes (1.1%), Anopheles claviger (1.1%), Culiseta bergrothi (1.1%), or other unidentified species (1.1%). Mosquitoes were fed with SINV-infected blood and monitored for 29 days to determine the viral extrinsic incubation period. Infection and dissemination were determined by RT-qPCR screening of dissected body parts of individual mosquitoes. Viral transmission was determined from saliva collected from individual mosquitoes at 7, 14, and 29 days. SINV was detected by cell culture using BHK-21 cells, RT-qPCR, and sequencing. Results: Cx. torrentium was the only mosquito species in our study that was able to transmit SINV. The overall transmission efficiency of SINV in Cx. torrentium was 6.8%. The rates of SINV infection, dissemination, and transmission in Cx. torrentium were 11%, 75%, and 83%, respectively. Conclusions: Cx. torrentium may be the key vector involved in SINV transmission in northern Sweden.
Indications for a vertical transmission pathway of piscine myocarditis virus in Atlantic salmon (Salmo salar L.)
Britt Bang Jensen - 2019
Abstract
Losses due to cardiomyopathy syndrome (CMS) keep increasing in salmon‐producing countries in the North‐Atlantic. Recently, Piscine myocarditis virus (PMCV) has been detected in post‐smolts shortly after sea‐transfer, indicating a possible carry‐over from the hatcheries. In addition, there are reports of prevalences of PMCV as high as 70%–90% in certain groups of broodfish, and a recent outbreak of CMS in the Faroe Islands has been linked to the importation of eggs from a CMS‐endemic area. Thus, there is a need to investigate whether PMCV can be transmitted vertically from infected broodstock to their progeny. In the present study, samples from eggs, larvae, fingerlings and presmolt originating from PMCV‐positive broodstock from two commercial Atlantic salmon producers were tested for PMCV. The prevalence of PMCV in the broodstock was 98% in the hearts, 69% in the roe and 59% in the milt. Piscine myocarditis virus was detected in all stages of the progeny until and including the 40 g stage. Piscine myocarditis virus was also detected in presmolt sampled for tissue tropism. This provides farmers with several options for minimizing the risk of transfer of PMCV from broodstock to progeny, including screening of broodstock and aiming to use only those that are negative for PMCV or have low levels of virus.
Monitoring infection with Piscine myocarditis virus and development of cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.) in Norway
Julie Christine Svendsen - 2019
Abstract
An epidemiological study was carried out in Norway in 2015–2018, investigating the development of infection with Piscine myocarditis virus (PMCV) and development of cardiomyopathy syndrome (CMS) in farmed Atlantic salmon. Cohorts from 12 sites were followed and sampled every month or every other month from sea transfer to slaughter. PMCV was detected at all sites and in all sampled cages, and fish in six sites developed clinical CMS. The initial infection happened between 1 and 7 months post‐sea transfer, and the median time from infection with PMCV until outbreak of CMS was 6.5 months. Generally, fish from sites with CMS had higher viral titre and a higher prevalence of PMCV, compared to sites that did not develop clinical CMS. The virus persisted until the point of slaughter at most (11 out of 12) of the sites. The detection of PMCV in all sites suggests that PMCV is more widespread than previously known. Screening for PMCV as a tool to monitor impending outbreaks of CMS must be supported by observations of the health status of the fish and risk factors for development of disease.
Rifamycin SV exhibits strong anti-inflammatory in vitro activity through pregnane X receptor stimulation and NFκB inhibition
Caridad Rosette - 2019
Abstract
Rifamycin SV (rifamycin), is a member of the ansamycin family of antimicrobial compounds which kills bacteria commonly associated with infectious diarrhea and other enteric infections. Rifamycin has been found to be effective in experimental animal models of gut inflammation and its efficacy in these settings has been attributed partially to immunomodulatory non-bactericidal activities. This study aimed to further evaluate the anti-inflammatory activities of rifamycin by analyzing its effect on two key regulators of inflammation: PXR and NFκB. Rifamycin stimulated PXR transcriptional activity in two PXR reporter cell lines and induced expression of two genes known to be regulated by PXR and are directly involved in cellular detoxification: CYP3A4 and PgP. Moreover, CYP3A4 metabolic activity was induced by rifamycin in HepG2 cells. Rifamycin also antagonized TNFα and LPS-induced NFκB activities and inhibited IL1β-induced synthesis of inflammatory chemokine, IL8. Although reciprocal regulation of PXR and NFkB by rifamycin was not directly addressed, the data suggest that in the absence of PXR, inhibition of NFκB by rifamycin is not dependent on PXR stimulation. Thus, rifamycin exhibits potent anti-inflammatory activities, characterized by in vitro PXR activation and concomitant CYP3A4 and PgP induction, in parallel with potent NFκB inhibition and concomitant IL8 inhibition.
Development of a PPRV challenge model in goats and its use to assess the efficacy of a PPR vaccine
Francois Enchery - 2019
Abstract
Peste des Petits Ruminants (PPR) is a severe disease of small ruminants and has high economic impacts in developing countries. Endemic in Africa, the Middle East and Asia, the disease is currently progressing with occurrences reported in North Africa, Turkey and in Georgia, and now threatens Europe. Much remains unknown about the infection dynamics, the virulence of the different strains and species/breed susceptibility. Robust experimental challenge models are needed to explore these fields and to confirm the efficacy of currently sold vaccines. We first assessed virulence of two PPR virus strains (CI89 and MA08) in Saanen goats. Whereas the MA08 strain led to classical severe clinical signs of PPR, the CI89 strain appeared to cause a mild disease in Saanen goats, highlighting the difference in virulence between strains in this animal model. We further demonstrated the importance of the inoculation route in the appearance of clinical signs and that ocular excretion is a better choice than blood for viral detection. After developing a robust challenge model, we assessed the efficacy of a vaccine (PPR-VAC®, BVI Botswana) against the MA08 strain and demonstrated that this vaccine blocked viral excretion and significantly reduced clinical signs. These results reinforce the paradigm that a strain from one lineage could protect against strains from other lineages.
The ubiquitin-protein ligase E6AP/UBE3A supports early encephalomyocarditis virus replication
Marybeth Carmody - 2018
Abstract
Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6 AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.
Xenobiotic Nuclear Receptor Signaling Determines Molecular Pathogenesis of Progressive Familial Intrahepatic Cholestasis
Kang Ho Kim - 2018
Abstract
Abstract. Progressive familial intrahepatic cholestasis (PFIC) is a genetically heterogeneous disorder of bile flow disruption due to abnormal canalicular tran
Synthesis and antiviral evaluation of novel peptidomimetics as Norovirus protease inhibitors
Franck Amblard - 2018
Abstract
A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.
The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification - viruses-09-00207-v2.pdf
Leanne M. Bilawchuk - 2017
Abstract
Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition ( p=0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p=0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition;p=0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses.
Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method
Irit Davidson - 2016
Abstract
The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, dropping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA was now upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the env and the NS5 genes, increasing the sensitivity by 10-100 fold compared to the previously existing assays. Based on the recent complete sequences of 5 TMEV isolates we now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009-2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least, in experimentally-infected turkeys and in 3 cases of disease encountered in commercial flocks.
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