This work was supported by funding from the CDC/NIOSH High Plains Intermountain Center for Agricultural Health and Safety (5U54OH008085) and the Colorado Bioscience Discovery Evaluation Grant Program (14BGF-16).

1. Setup of the Bioaerosol Chamber (See Figure 2)
<ol>
	<li>Pre-load the liquid impingers with 20 mL of 0.01 M phosphate buffered saline, pH 7.5 (PBS).
	<ol>
		<li>Add 0.5 g of anion exchange resin and suspend within the PBS of one of the liquid impingers, with another liquid impinger serving as a control.</li>
	</ol>
	</li>
	<li>Position liquid impingers in parallel inside the bioaerosol chamber using clamp stands with aerosol inlets facing the nebulizer. 
	NOTE: See Figure 2 for additional detail.</li>
	<li>Co-locate a direct-read aerosol monitor near the liquid impingers to measure mass concentration (mg/m3) of the bioaerosol.</li>
	<li>Co-locate additional direct-read instrumentation (indoor air quality monitor and thermal anemometer) near the liquid impingers to monitor environmental variables, such as temperature, relative humidity and wind speed, within the bioaerosol chamber.</li>
	<li>Run axial fans in the corners of the bioaerosol chamber to facilitate mixing of aerosol. 
	NOTE: Axial fans run at a fixed speed and are not adjustable.</li>
</ol>
2. Model Bioaerosolization Experiments (see Figure 3)
<ol>
	<li>Perform serial, 10-fold dilutions of MS2 bacteriophage and influenza vaccine to desired concentrations in phosphate-buffered saline (PBS). 
	NOTE: To demonstrate the method here, 103.5 FFU/mL of the type A and B influenza virus vaccine strains and 105 PFU/mL of the MS2 bacteriophage are utilized. To determine titers for the inocula, bacteriophages are propagated in Escherichia coli HS (pFamp)R and enumerated as previously described20. The commercial vaccine contains known quantities of four live attenuated influenza virus strains, two type A influenza viruses and two type B influenza viruses, expressed in fluorescent focus units or FFU.</li>
	<li>Prepare inocula within the glass vessel of a 6-jet collision nebulizer by creating suspensions of desired concentrations of viral particles in 100 mL of PBS and install within the custom bioaerosol chamber.</li>
	<li>Initiate instrumentation to measure variables within the bioaerosol chamber, such as temperature, % relative humidity, carbon dioxide concentration, and wind speed (generated using an axial fan). Use a direct-read aerosol monitor to track mass concentration within the bioaerosol chamber and to establish consistency within and between trials.</li>
	<li>Seal the bioaerosol chamber and purge for 10 min with HEPA-filtered air.</li>
	<li>Deliver filtered, dried air to the nebulizer operating at 6.9 kPa.</li>
	<li>Generate a target concentration of viral bioaerosols (via nebulizer) in each bioaerosol chamber trial. For this demonstration, bioaerosols with a concentration of 5 mg/m3 are generated.</li>
	<li>Once the target mass concentration of the aerosol has been reached, stop nebulization.</li>
	<li>Operate an air sampling pump for each liquid impinger that has been calibrated to a flow rate of 12.5 L/min. Conduct calibration using a primary flow standard before and after each sampling event to ensure consistency in sampling volume. Supply dilution air using a HEPA-filtered line located near the nebulizer and maintain constant pressure in the chamber.</li>
	<li>Actively sample the bioaerosol chamber atmosphere for a period of 40 min to achieve a sampling volume of 500 L per sample. 
	NOTE: Sampling for 40 min at 12.5 L/min with two pumps in parallel allows for sampling of 1,000 L of the bioaerosol. Thus, at the completion of the experiment, it is expected that the majority of the air present in the bioaerosol chamber has been sampled and released into the environment via a HEPA-filtered system. This is evidenced by the decrease in particle concentration. Post-experiment, surfaces are decontaminated with 10% household bleach and 70% ethanol. Indicators are used in this demonstration; however, if known pathogenic organisms are used in this system, additional biosafety measures may be implemented as needed.</li>
</ol>
3. Nucleic Acid Extraction and qRT-PCR Detection
<ol>
	<li>Isolate nucleic acids directly from anion exchange resin and, for comparative purposes, from the liquid used within the liquid impingers. In this example, a RNA isolation procedure is described, but a similar protocol could be used for DNA viruses.
	<ol>
		<li>Nucleic acid isolation from the anion exchange resin.
		<ol>
			<li>Transfer all of the anion exchange resin-containing liquid of the liquid impinger to a sterile 50 mL conical tube.</li>
			<li>Allow the resin to settle, and slowly decant the liquid sample from the anion exchange resin. Use a 1 mL pipette tip to remove all of the remaining liquid from the anion exchange resin.</li>
			<li>From a viral RNA isolation kit, add 560 &#181;L of viral lysis buffer containing carrier RNA directly to the anion exchange resin and incubate for 10 min at room temperature with periodic mixing.</li>
			<li>Using a 1 mL pipette tip, transfer the viral lysate (the viral lysis buffer) to a sterile 1.5 mL conical tube and proceed with RNA isolation using a viral RNA isolation kit (see Table of Materials) in accordance with the manufacturer&#39;s instructions, except that RNA is eluted in a total volume of 60 &#181;L of elution buffer.</li>
		</ol>
		</li>
		<li>Nucleic acid isolation from liquid impingers.
		<ol>
			<li>Pipette 140 &#181;L of the liquid sample into a sterile 1.5 mL conical tube and perform RNA isolation using the viral RNA isolation kit in accordance with the manufacturer&#39;s instructions, with the exception of eluting RNA in a total volume of 60 &#181;L of elution buffer. 
			NOTE: 140 &#181;L is the maximum sample volume that can be processed with the specific viral RNA isolation kit employed here in a single-step preparation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform qRT-PCR for detection of MS2 and influenza A and B viruses as previously described23,24. In this demonstration, 5 &#181;L of nucleic acid eluates are used for qRT-PCR.</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+airborne+transmission+of+Norwalk-like+virus+(NLV)+in+a+hotel+restaurant.">Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.</a> Epidemiology and Infection. 124 (3), 481-487 (2000).</li><li>Marks, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+school+outbreak+of+Norwalk-like+virus:+Evidence+for+airborne+transmission.">A school outbreak of Norwalk-like virus: Evidence for airborne transmission.</a> Epidemiology and Infection. 131 (1), 727-736 (2003).</li><li>Corzo, C. A., Culhane, M., Dee, S., Morrison, R. 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The authors have nothing to disclose.

This protocol outlines a method for sensitive viral capture from bioaerosols using modified liquid impingers. The method is optimized for detection and quantification of the viral load in bioaerosols. The specific modification demonstrated here involves the addition of anion exchange resin to liquid contained within a common liquid impinger. This method was developed for its simplicity in downstream sample processing, whereas other sample processing techniques such as centrifugation, filtration, and precipitation-based m...

The purpose of this method is to provide a highly sensitive bioaerosol sampling platform to facilitate molecular detection of negatively-charged viruses from bioaerosols. Microorganisms, including viral particles, can survive in bioaerosols for extended periods of time1. Bioaerosols can travel over relatively long distances and maintain viability and infectivity, as evidenced by an outbreak of Legionnaires&#39; disease that originated from industrial cooling towers located at a distance of 6 km from the affected individuals and resulted in 18 fatalities2. Indirect transmission of viruses to humans mediated by bioaerosols can occur in multiple settings and has been demonstrated for norovirus outbreaks in schools and restaurants3,4. Similarly, bioaerosol transmission of viruses can occur in agricultural settings such as in swine and poultry farms, with this transmission route being considered as a major factor in the movement of viruses between production facilities5,6,7,8,9.
Effective sampling of virus-laden bioaerosols allows for improvement in rapid diagnostics and preparedness for outbreak prevention, as shown in demonstrations in which H5 influenza A viruses were detected from bioaerosols in live animal markets in China and the United States10,11. Current bioaerosol sampling technologies involve a number of different particle capture principles, and can be broadly categorized into impingers, cyclones, impactors, and filters12. It is beyond the scope of this protocol to exhaustively cover all advantages and disadvantages of these platforms for sampling of viruses from bioaerosols; however, it can be stated that the majority of these sampling devices have not been optimized for the collection of viruses and bacteriophages13. Furthermore, infectivity of viral particles is often negatively affected, with liquid impingers considered to maintain viral infectivity more effectively than sampling devices such as solid impactors or filters14. However, one disadvantage of liquid impingement is the target dilution effect, which occurs because viruses are collected in relatively large volumes (typically &#8805;20 mL) of liquid in the collection vessel. Another important disadvantage involves the suboptimal efficiency of liquid impingers to concentrate particles &#60;0.5 &#181;M in size15. However, capture efficiency of these devices can be improved by immobilization on solid matrices, as immobilization can enhance preservation of viral nucleic acids and viral infectivity16,17.
We have previously demonstrated that anion exchange resin is an effective tool for the capture and concentration of viruses from liquid matrices, including F-RNA bacteriophages, hepatitis A virus, human adenovirus, and rotavirus18,19,20. As defined by the manufacturer, the anion exchange resin utilized in this work is a macroreticular polystyrene strong base anion exchange resin in which functionalized quaternary amine groups mediate attraction and capture of anions in a liquid medium21. Consequently, the anion exchange resin is expected to capture viruses with net-negative surface charges, including many enteric viruses, influenza viruses and other viruses relevant to public and animal health.
The current protocol involves the addition of anion exchange resin to a liquid impinger. In this system, the resin serves as a secondary concentration step for viral particles captured in the impinger liquid. Nucleic acids can then be directly eluted in small volumes, providing a concentrated sample for molecular analyses. Thus, the main advantage of this method is the improvement in viral detection sensitivity, primarily through reduction in sample volume. Additionally, due to the inherent non-specific capture of negatively-charged viruses, the method is likely applicable for detection of a large number of viruses of interest. Here, the method is demonstrated for vaccine strains of type A and type B influenza viruses and the FRNA coliphage MS2 (MS2). These viruses are subsequently detected using standard qRT-PCR assays as previously described22. The end-point user should not expect to encounter difficulties in performing this method, because modifications to currently existing equipment do not constitute major disruptions to the conventional flow of bioaerosol sampling and analysis.

<table><tbody><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; bacteriophage MS2 (ATCC 15597-B1)</td><td>American Type Culture Collection</td><td>ATCC 15597-B1</td><td></td></tr><tr><td>FluMist Quadrivalent</td><td>AstraZeneca</td><td>Contact manufacturer</td><td>Viral constitutents of this vaccine are subject to change on an annual basis</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad</td><td>1855195</td><td></td></tr><tr><td>Primers and probes</td><td>Integrated DNA Technologies</td><td>NA</td><td></td></tr><tr><td>0.2 &amp;micro;M sterile filter</td><td>NA</td><td>NA</td><td></td></tr><tr><td>1 L pyrex bottles or equivalent</td><td>NA</td><td>NA</td><td></td></tr><tr><td>1 mL pipet tips</td><td>NA</td><td>NA</td><td></td></tr><tr><td>1 mL pipettor</td><td>NA</td><td>NA</td><td></td></tr><tr><td>50 mL serological pipet</td><td>NA</td><td>NA</td><td></td></tr><tr><td>PCR tubes</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Pipet-aid or equivalent</td><td>NA</td><td>NA</td><td></td></tr><tr><td>QIAamp Viral RNA Mini Kit</td><td>Qiagen</td><td>52904</td><td></td></tr><tr><td>QuantiTect Probe RT-PCR Kit</td><td>Qiagen</td><td>204443</td><td></td></tr><tr><td>Amberlite IRA-900 chloride form</td><td>Sigma-Aldrich</td><td>216585-500G</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>Water (molecular biology grade)</td><td>Sigma-Aldrich</td><td>W4502-1L</td><td></td></tr><tr><td>Eppendorf DNA LoBind Microcentrifuge Tubes</td><td>ThermoFisher</td><td>13-698-791</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes&amp;nbsp;</td><td>ThermoFisher</td><td>14-432-22</td><td></td></tr><tr><td>Falcon Polypropylene Centrifuge Tubes</td><td>ThermoFisher</td><td>05-538-62</td><td></td></tr><tr><td>SuperScript III Platinum One-Step qRT-PCR Kit w/ROX</td><td>ThermoFisher</td><td>11745100</td><td></td></tr><tr><td>SKC Biosampler 20 mL, 3-piece glass set</td><td>SKC Inc.</td><td>225-9593</td><td></td></tr><tr><td>Vac-u-Go sample pumps</td><td>SKC Inc.</td><td>228-9695</td><td></td></tr><tr><td>Collison nebulizer (6-jet)</td><td>BGI Inc.</td><td>NA</td><td></td></tr><tr><td>HEPA capsule</td><td>PALL</td><td>12144</td><td></td></tr><tr><td>Q-TRAK indoor air quality monitor 8554</td><td>TSI Inc.</td><td>NA</td><td></td></tr><tr><td>Alnor velometer thermal anemometer AVM440-A</td><td>TSI Inc.</td><td>NA</td><td></td></tr><tr><td>SidePak AM510 personal aerosol monitor</td><td>TSI Inc.</td><td>NA</td><td></td></tr><tr><td>Bioaerosol chamber</td><td>NA</td><td>NA</td><td></td></tr></tbody></table>

detection of viruses from bioaerosols using anion exchange resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

Figure 1 demonstrates the principle behind charge-based capture of viruses from bioaerosols via inclusion of resin in liquid-based impingers. Figure 2 shows the setup of the custom-built bioaerosol chamber. Figure 3 describes the steps involved in setting up the aerosolization experiment and measures to ensure quality control. Figure 4 shows amplification curves for qRT-...

Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

detection-of-viruses-from-bioaerosols-using-anion-exchange-resin

Research

JoVE Journal

Environment

8.1K Views.  Colorado State University. Een anion wisselen hars gebaseerde methode, aangepast aan vloeistof impingement gebaseerde bioaerosol bemonstering van virussen is aangetoond. Wanneer gekoppeld aan downstream moleculaire detectie, zorgt de methode voor facile en gevoelige detectie van virussen van bioaerosols.

Video: Detection of Viruses from Bioaerosols Using Anion Exchange Resin

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Immunology and Infection

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Chemistry

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Biochemistry

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Bioengineering

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Medicine

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Biology

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

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