scholarly journals Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 562
Author(s):  
Emma K. Loveday ◽  
Kyle S. Hain ◽  
Irina Kochetkova ◽  
Jodi F. Hedges ◽  
Amanda Robison ◽  
...  
Keyword(s):  
High Titer ◽  
Antibody Detection ◽  
Heat Inactivation ◽  
Virion Protein ◽  
Live Virus ◽  
Log Reduction ◽  
Complete Inactivation ◽  
Level 3 ◽  
Virus Research ◽  
The One

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

scholarly journals Effects of inactivation method on SARS-CoV-2 virion protein and structure

2020 ◽  
Author(s):  
Emma K. Loveday ◽  
Kyle S. Hain ◽  
Irina Kochetkova ◽  
Jodi F. Hedges ◽  
Amanda Robison ◽  
...  
Keyword(s):  
High Titer ◽  
Heat Inactivation ◽  
Virion Protein ◽  
Live Virus ◽  
Antibody Recognition ◽  
Log Reduction ◽  
Complete Inactivation ◽  
Level 3 ◽  
Virus Research ◽  
The One

AbstractThe risk posed by Severe Acute Respiratory Syndrome Coronavirus −2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting destructive effects of inactivation. We observed that 15 minutes of incubation at 65°C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 μJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody recognition. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the resulting viral proteins and virions were negatively impacted by inactivation method and time. We outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

scholarly journals Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

2021 ◽  
Author(s):  
Ciaran Richardson ◽  
Sarah Gildea ◽  
Stephen Harkin ◽  
Annie Gallagher ◽  
Eimear Conroy ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
Antibody Detection ◽  
Clinical Samples ◽  
Test Species ◽  
Live Virus ◽  
Clinical Sensitivity ◽  
Protein Receptor ◽  
Laboratory Facilities ◽  
Level 3 ◽  
Routine Clinical Laboratory

The urgent need for a rapid and reliable Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralising antibody detection test compatible with routine clinical laboratory testing currently exists. This is necessary to provide accurate estimates of immunity and to monitor vaccine effectiveness. Utilising Biochip Array Technology (BAT), the Randox SARS-CoV-2 Biochip proxy virus neutralisation test (pVNT) has been developed. Immobilising SARS-CoV-2 Spike RBD on the Biochip surface, innovative assay design enables direct sample addition to the Biochip well without the need for off-board sample pre-incubation step. Results are reported within 1.5 hours and testing is carried out without the prerequisite of live virus or biosafety level 3 (BSL3) laboratory facilities. In this study, assay validation is performed using recombinant antibodies and clinical samples and an excellent correlation against conventional virus neutralisation methods is established (100% clinical specificity and 98% clinical sensitivity). Serial dilution of samples with high neutralising antibody levels demonstrate end-point sample dilutions comparable with those obtained using the SARS-CoV-2 microneutralisation test. Species independent neutralising antibody detection capacity of the SARS-CoV-2 Biochip pVNT is also demonstrated. The findings of this study exemplifying the utility of the SARS-CoV-2 Biochip pVNT as a robust and reliable method for the accurate measurement of neutralising antibodies against SARS-CoV-2 and the availability of this test can now positively impact current testing deficiencies in this area.

scholarly journals Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies

2021 ◽  
Author(s):  
Devon J. Eddins ◽  
Leda C. Bassit ◽  
Joshua Chandler ◽  
Natalie S. Haddad ◽  
Kathryn Musall ◽  
...  
Keyword(s):  
Single Cell ◽  
World Health ◽  
Antibody Detection ◽  
Heat Inactivation ◽  
Viral Inactivation ◽  
The Us ◽  
Control And Prevention ◽  
Level 3 ◽  
Infected Cells ◽  
Health Organization

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Modeling Penicillium expansum Resistance to Thermal and Chlorine Treatments

2009 ◽  
Vol 72 (12) ◽  
pp. 2618-2622 ◽  
Author(s):  
BEATRIZ C. M. SALOMÃO ◽  
JOHN J. CHUREY ◽  
GLÁUCIA M. F. ARAGÃO ◽  
RANDY W. WOROBO
Keyword(s):  
Apple Juice ◽  
Weibull Model ◽  
Superior Performance ◽  
Penicillium Expansum ◽  
Heat Inactivation ◽  
Survival Curves ◽  
Biphasic Model ◽  
Log Reduction ◽  
Weibull Models ◽  
D Values

Apples and apple products are excellent substrates for Penicillium expansum to produce patulin. In an attempt to avoid excessive levels of patulin, limiting or reducing P. expansum contamination levels on apples designated for storage in packinghouses and/or during apple juice processing is critical. The aim of this work was (i) to determine the thermal resistance of P. expansum spores in apple juice, comparing the abilities of the Bigelow and Weibull models to describe the survival curves and (ii) to determine the inactivation of P. expansum spores in aqueous chlorine solutions at varying concentrations of chlorine solutions, comparing the abilities of the biphasic and Weibull models to fit the survival curves. The results showed that the Bigelow and Weibull models were similar for describing the heat inactivation data, because the survival curves were almost linear. In this case, the concept of D- and z-values could be used, and the D-values obtained were 10.68, 6.64, 3.32, 1.14, and 0.61 min at 50, 52, 54, 56, and 60°C, respectively, while the z-value was determined to be 7.57°C. For the chlorine treatments, although the biphasic model gave a slightly superior performance, the Weibull model was selected, considering the parsimony principle, because it has fewer parameters than the biphasic model has. In conclusion, the typical pasteurization regimen used for refrigerated apple juice (71°C for 6 s) is capable of achieving a 6-log reduction of P. expansum spores.

Effect of Heat Processing and Ultrasonication Treatment on Custard Apple Peroxidase Activity and Vitamin C

2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Mugdha P. Dabir ◽  
L. Ananthanarayan
Keyword(s):  
Vitamin C ◽  
Arrhenius Plot ◽  
Enzyme Inactivation ◽  
Heat Processing ◽  
Heat Inactivation ◽  
Ultrasonic Power ◽  
First Order ◽  
Complete Inactivation ◽  
Heat Stable ◽  
Custard Apple

Abstract: Peroxidase isolated from custard apple (Annona squamosa L.) being a heat stable enzyme can be inactivated by heat processing and ultrasonication treatment as well. Heat processing took up to 22 min at 91 °C for complete inactivation of peroxidase. The ultrasonication treatment was performed at 40, 55, 70, 85 and 100 W ultrasonic power and was found to achieve complete inactivation at 85 W as well as at 100 W power within 5 min. The heat inactivation followed first-order reaction kinetics and the activation energy (Ea) calculated from Arrhenius plot was found to be 7.03 × 104 J/mol, which was near the reported range for peroxidases. Heat processing caused 52.7 % loss of vitamin C while achieving complete inactivation of peroxidase at 91 °C for 22 min, whereas ultrasonication caused 21.6 % vitamin C loss at 85 W power within 5 min. Ultrasonication was thus found to be efficient in retaining vitamin C while achieving complete enzyme inactivation.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings

2020 ◽  
Author(s):  
Sandra Westhaus ◽  
Marek Widera ◽  
Holger F. Rabenau ◽  
Sebastian Hoehl ◽  
Denisa Bojkova ◽  
...  
Keyword(s):  
Culture Media ◽  
Heat Inactivation ◽  
The Novel ◽  
Safety Issues ◽  
Rapid Spread ◽  
Level 3 ◽  
Global Concern ◽  
The Stability ◽  
Novel Coronavirus ◽  
Uv C

SummaryThe novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3.In this study, the stability of the virus in cell culture media at 4°C and on touch panel surfaces used in laboratory environment was analyzed. In addition, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation.In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.

scholarly journals Resistance of L. monocytogenes and S. Typhimurium towards Cold Atmospheric Plasma as Function of Biofilm Age

2018 ◽  
Vol 8 (12) ◽  
pp. 2702 ◽  
Author(s):  
Marlies Govaert ◽  
Cindy Smet ◽  
Maria Baka ◽  
Branimir Ećimović ◽  
James L. Walsh ◽  
...  
Keyword(s):  
Cell Density ◽  
Atmospheric Plasma ◽  
Inactivation Kinetics ◽  
Bacterial Cells ◽  
Cold Atmospheric Plasma ◽  
Complete Inactivation ◽  
Innovative Methods ◽  
Food Contact Surfaces ◽  
Different Ages ◽  
The One

The biofilm mode of growth protects bacterial cells against currently applied disinfection methods for abiotic (food) contact surfaces. Therefore, innovative methods, such as Cold Atmospheric Plasma (CAP), should be investigated for biofilm inactivation. However, more knowledge is required concerning the influence of the biofilm age on the inactivation efficacy in order to comment on a possible application of CAP in the (food) processing industry. L. monocytogenes and S. Typhimurium biofilms with five different ages (i.e., 1, 2, 3, 7, and 10 days) were developed. For the untreated biofilms, the total biofilm mass and the cell density were determined. To investigate the biofilm resistance towards CAP treatment, biofilms with different ages were treated for 10 min and the remaining cell density was determined. Finally, for the one-day old reference biofilms and the most resistant biofilm age, complete inactivation curves were developed to examine the influence of the biofilm age on the inactivation kinetics. For L. monocytogenes, an increased biofilm age resulted in (i) an increased biomass, (ii) a decreased cell density prior to CAP treatment, and (iii) an increased resistance towards CAP treatment. For S. Typhimurium, similar results were obtained, except for the biomass, which was here independent of the biofilm age.

scholarly journals Use of Sindbis/Eastern Equine Encephalitis Chimeric Viruses in Plaque Reduction Neutralization Tests for Arboviral Disease Diagnostics

2011 ◽  
Vol 18 (9) ◽  
pp. 1486-1491 ◽  
Author(s):  
B. W. Johnson ◽  
O. Kosoy ◽  
E. Wang ◽  
M. Delorey ◽  
B. Russell ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Slight Reduction ◽  
Wild Type ◽  
Regulatory Requirements ◽  
Live Virus ◽  
Eastern Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Disease Diagnostics ◽  
Level 3 ◽  
Chimeric Viruses

ABSTRACTEastern equine encephalitis virus (EEEV) is a highly virulent, mosquito-borne alphavirus that causes severe and often fatal neurological disease in humans and horses in eastern North American, the Caribbean, and Mexico and throughout Central and South America. EEEV infection is diagnosed serologically by anti-EEEV-specific IgM detection, with confirmation by the plaque reduction neutralization test (PRNT), which is highly specific for alphaviruses. Live virus is used in the PRNT procedure, which currently requires biosafety level 3 containment facilities and select agent security in the case of EEEV. These requirements restrict the ability of public health laboratories to conduct PRNTs. Sindbis virus (SINV)/EEEV recombinant constructs have been engineered to express the immunogenic structural proteins from 2 wild-type EEEV strains in an attenuated form. These SINV/EEEVs, which are not classified as select agents, were evaluated as alternative diagnostic reagents in a PRNT using human, equine, and murine sera. The results indicate that the chimeric viruses exhibit specificity comparable to that of wild-type EEEV, with only a slight reduction in sensitivity. Considering their benefits in increased safety and reduced regulatory requirements, these chimeric viruses should be highly useful in diagnostic laboratories throughout the Americas.

scholarly journals A Rapid Bead-Based Assay For Screening Of SARS-CoV-2 Neutralising Antibodies

2021 ◽  
Author(s):  
Santhik SL ◽  
Pramod Darvin ◽  
Aneesh Chandrasekharan ◽  
Shanakara Narayanan Varadarajan ◽  
Soumya Jaya Divakaran ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Life Time ◽  
Cost Effective ◽  
Fluorescent Imaging ◽  
Antibody Detection ◽  
Screening Assay ◽  
Live Virus

Quantitative determination of neutralizing antibodies against SARS CoV2 is of paramount importance in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead based screening assay for S1 neutralization using recombinant fluorescent proteins of hACE2 and SARS CoV2 S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS CoV2 Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single color fluorescent imaging of ACE2 GFPspark binding to His tagged S1 Receptor Binding Domain (RBD His) immobilized beads. The second approach is dual color imaging of soluble ACE2 GFPSpark to S1 Orange Fluorescent Protein (S1 OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life time imaging of the ACE2 GFPSpark confirmed the interaction of ACE2 and S1 OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.

scholarly journals Literature Review on Fingerprint Level 1 and Level 2 Features Enhancement to Improve Quality of Image

2017 ◽  
pp. 8-19 ◽  
Author(s):  
Krishna Prasad K. ◽  
P. S. Aithal
Keyword(s):  
Recognition System ◽  
Fingerprint Recognition ◽  
Human Beings ◽  
Global Features ◽  
Basic Premise ◽  
Level 3 ◽  
Level 1 ◽  
The One ◽  
Level 2

Biometrics is the one most popular property in human distinguishing proof based on physical or behavioral features. The different physiological characteristics are Fingerprint, DNA, Face, hand, retina, ear features, and odor, where as behavioral characteristics or features are typing rhythm, gait, gesture, and voice with the basic premise that all are unique and all human beings are identified by these intrinsic traits. In the physiological traits, Fingerprint is most commonly utilized the biometric feature in diverse fields for identification and verification purpose. Fingerprint features can be separated into three noteworthy classifications in view of the granularity at which they are removed as level 1, level 2, and level 3 features. Level 1 feature contains macro details, which are easily extractable and include orientation filed, ridge frequency filed and pattern configuration. Only these global features or Level 1 features are not sufficient to uniquely identify or recognize, but if these features are used along with level 2 or level 3 features, that can make the fingerprint recognition system more robust and secure. Level1 features are used for image enhancement and orientation purpose. In this paper, we made a survey of existing literature on Level 1 features and try to analyze other researcher's contribution to this field.

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