The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. J.K.B., K.J., M.R.H., D.P., L.B., and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: J.H.K., an employee of Tunnell Government Services, Inc.; and M.G.L., an employee of Lovelace Respiratory Research Institute.

This protocol adheres to the following animal care guidelines. Animals were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experimental procedures were approved by the National Institute of Allergy and Infectious Diseases, Division of Clinical Research, Animal Care and Use Committee and were in compliance with the Animal Welfare Act regulations, Public Health Service policy, and the Guide for the Care and Use of Laboratory Animals recommendations.
1. Aerobiology: Animal Biosafety Level 4 (ABSL-4) Suit Laboratory
<ol>
	<li>Laboratory Preparation
	<ol>
		<li>Complete the ABSL-4 suit laboratory entry procedures (outlined in detail in 8).</li>
		<li>Test the functionality of all equipment (e.g., plethysmography equipment, laptop, biohazardous trash cans, biohazardous sharps containers, subject monitoring devices) involved in aerobiology procedures occurring within the ABSL-4 suit laboratory according to manufacturer&#39;s protocol.</li>
		<li>Ensure the transfer cart is biologically clean prior to testing the functionality of the rapid transfer port (RTP), which connects the transport cart through the wall to the Class III BSC).</li>
		<li>Handle and dilute pathogen only within certified BSCs. Prepare the pathogen in the appropriate formulation within a Class II BSC that contains appropriate disinfectants. Transport the pathogen in an air-tight secondary container labeled with a biohazard symbol on wet ice in the transport cart. Pass the pathogen through the RTP into the Class III BSC in the ABSL-4 cabinet laboratory, Figure 1.</li>
	</ol>
	</li>
</ol>
2. Plethysmography: Animal Biosafety Level 4 (ABSL-4) Suit Laboratory
<ol>
	<li>Plethysmography Setup and Calibration
	<ol>
		<li>Determine which method of plethysmography acquisition (head-out plethysmography or respiratory inductance plethysmography [RIP]) will be used and connect equipment components together.</li>
		<li>Calibrate the plethysmograph prior to the experiment using manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Plethysmography Acquisition
	<ol>
		<li>When handling NHPs, don an external pair of latex or nitrile gloves over top of the suit gloves to prevent cross contamination and promote safe practices. When finished handling NHPs, remove these extra gloves and discard in the biohazardous trash can within the room.</li>
		<li>If using head-out plethysmography, attach a new rubber/dental dam to the front of the cylinder. Cut a small hole in the dam for the head of the NHP to fit through the top of the cylinder. When seated, the dam creates a seal around the NHP&#39;s neck.</li>
		<li>If using RIP, check that the RIP bands are properly fitted around the chest and abdomen of the NHP and the electronic connections are snapped tightly.</li>
		<li>Send all data acquired from the plethysmography procedure to the researchers in the ABSL-4 cabinet laboratory. Export the tidal volume and minute volume data for each animal through a compatible program for use during the aerosol process.</li>
	</ol>
	</li>
</ol>
3. Nonhuman Primate Transport and Handling: Animal Biosafety Level 4 Suit Laboratory
<ol>
	<li>NHP Handling
	<ol>
		<li>Monitor and record any physical manipulations, administrations, or routine procedures on charts for each NHP.</li>
		<li>When an aerosol challenge is completed, place the NHP inside the transportation container and return NHP to the home cage located in the animal holding room.</li>
		<li>When handling a live animal, follow the mandatory rule that requires 2 staff members to be present.</li>
	</ol>
	</li>
	<li>NHP Transport
	<ol>
		<li>Determine the type of anesthesia, duration of anesthesia (covers transport, plethysmography acquisition, and aerosol challenge) and corresponding dose of anesthesia prior to administration. Fully anesthetize the NHP based on the process chosen by the Comparative Medicine staff. If additional anesthesia is required, ensure all needles, sharps, syringes, and caps are discarded in a sharps container located in any of the animal procedure rooms. Do not recap any needles after use.</li>
		<li>Transport anesthetized NHPs in clear containers that are secured by a latch on the lid of the transport box.</li>
		<li>Load transport containers onto a mobile cart to allow fully suited researchers to move freely using breathing air lines and through the air pressure resistant (APR) doors, Figure 1.</li>
		<li>As no additional breathing air for the NHP is supplied to the transport container, minimize transport time.</li>
	</ol>
	</li>
</ol>
4. Aerobiology: ABSL-4 Cabinet Laboratory
<ol>
	<li>Class III BSC Setup
	<ol>
		<li>Concurrently with animal preparation performed by Comparative Medicine staff, prepare the Class III BSC. Visually verify that negative pressure in the Class III BSC is maintained within specified range (125 Pa or -0.5 in water gauge (wg) minimum; 250 Pa or -1.0 in wg recommended). Inspect the Class III BSC for any potential leaks or cracks (see Figure 1).</li>
		<li>Physically and visually inspect the Class III BSC synthetic rubber gloves and O-rings attached to the Class III BSC for weak spots, tears, rips, or dry rot. Replace the damaged Class III synthetic rubber gloves and/or O-rings immediately prior to use. At this point, the Class III BSC is not contaminated.</li>
		<li>If a leak occurs while the Class III BSC is contaminated, identify the location of the breach and alert facility management and biosafety personnel. If a Class III BSC integrated glove is torn or breached, replace the damaged glove immediately by following the properly trained technique and internal Class III BSC standard operating procedure.</li>
		<li>To change an integrated glove containing a small tear or breach during an exposure, first spray the tear or breach excessively with the appropriate concentration of a dual quaternary ammonium (n-alkyl dimethyl benzyl ammonium chloride, n-alkyl dimethyl ethyl benzyl ammonium chloride) disinfectant. Do not make excessive movements during this time that create an increase in airflow.</li>
		<li>Carefully, remove the outer O-rings (2 of them) leaving the damaged integrated glove still attached to the Class III BSC. Slightly move the damaged integrated glove cuff away from the port while ensuring the integrated glove seal remains intact. If the seal is compromised, an alarm will sound indicating the procedure was not done correctly. The integrated glove cuff should remain attached to the port after the second O-ring is removed from the Class III BSC.</li>
		<li>Place a new Class III BSC synthetic rubber glove over of the old glove in same orientation. Place this new glove fully over the port similarly to the other Class III BSC glove ports.</li>
		<li>Replace the O-ring closest to the Class III BSC over the new integrated glove. Using an adjacent integrated glove port, carefully pull the damaged Class III BSC synthetic rubber glove inside the Class III BSC. The new Class III synthetic rubber glove will act as the barrier to maintain containment. Once the other damaged Class III synthetic rubber glove is removed (pulled inward), replace the other outer O-ring and continue working.</li>
		<li>Record all details concerning glove tear/breach in the specific Class III BSC log book. If the damaged integrated glove is removed or a breach in containment occurs, the compromised integrated glove/port still maintains an inward air flow of 0.47 m3/sec. This inward airflow is the same airflow used with a Class II BSC, thus maintaining consistency between Class II and Class III BSCs.</li>
		<li>Inspect dunk tank and verify that the dunk tank is filled with disinfectant to the marked level inside the dunk tank, Figure 1. Verify the concentration of disinfectant in the dunk tank is a minimum of 3,500 &#181;S using a conductivity meter. This conductivity is equivalent to 5% concentration of the disinfectant.</li>
		<li>Ensure the Class III BSC autoclave is functional and operational so all contaminated waste and equipment can be autoclaved, Figure 1. Autoclave only equipment known to sustain the rigors of the sterilization process.</li>
		<li>Test the functionality of other aerobiology equipment (e.g., AAMP components, laptop) and air and vacuum lines involved in the experiment, Figure 2.</li>
		<li>Place signs on the Class III BSC indicating the current contamination status of the unit.</li>
	</ol>
	</li>
	<li>Assembly and System Setup of NHP Head-only Exposure Chamber
	<ol>
		<li>Assemble a 16-l NHP head-only exposure chamber by inserting the stainless steel delivery and exhaust lines, Figure 2. Configure the chamber in a push/pull, dynamic configuration by connecting the appropriate air, vacuum, and pressure lines to the AAMP. Connect the AAMP to a power source within the Class III BSC and a laptop computer through a hermetically sealed port located on top of the Class III BSC (Figure 1).</li>
		<li>Inspect the assembled NHP head-only exposure chamber for any leaks or cracks, and ensure that the chamber is properly assembled.</li>
		<li>Attach an aerosol generator and aerodynamic particle size reading instrument to the NHP head-only exposure chamber.</li>
		<li>Open the air and vacuum source to the AAMP.</li>
		<li>Launch the aerosol protocol software on the laptop computer. Enter the appropriate NHP head-only exposure chamber, aerosol generator and biosampler flow rate, and administrative information into the software menus.</li>
		<li>Calculate the aerosol challenge time from the data acquired during the plethysmography procedure, step 2.2.4. If using the head-out plethysmography, calculate the dose prior to the aerosol exposure. If using RIP, calculate the dose simultaneously during the aerosol exposure.</li>
		<li>Fill the aerosol generator with the pathogen.</li>
		<li>Through the aerosol software, turn the aerosol generator &#34;on&#34; and spray the inside of the NHP head-only exposure chamber with the challenge material for 10 min.</li>
		<li>Turn off the aerosol generator, empty the challenge material, and discard the challenge material into a biohazardous trash bag located inside of the Class III BSC.</li>
	</ol>
	</li>
	<li>NHP Head-only Exposure
	<ol>
		<li>Attach a biosampler to the NHP head-only exposure chamber, fill the biosampler with collection media, and attach the appropriate vacuum line to the biosampler.</li>
		<li>Check the depth of anesthesia of the NHP. If the depth of anesthesia is considered adequate (e.g., unresponsive to external stimuli, muscular tone, stable respiratory, and heart rates), pass the anesthetized NHP through the rapid transfer port (RTP) into the Class III BSC. If the depth of anesthesia is inadequate, administer additional anesthesia via IV, direct injection, or through an anesthetic pump prior to passing the NHP through the RTP (Comparative Medicine staff in the suit laboratory). Pass additional anesthetic supplies through the RTP.</li>
		<li>Place the NHP in the supine position onto the NHP exposure ramp.</li>
		<li>Gently pass the NHP&#39;s head through the rubber/dental dam attached to the head portal of the NHP head-only exposure chamber. The rubber/dental dam ensures a seal is created around the NHP&#39;s neck during the aerosol exposure.</li>
		<li>Verify that the NHP&#39;s vital signs are stable visually and with a portable subject monitor.</li>
		<li>Enter the aerosol challenge time calculated from step 4.2.6. and necessary equipment identifiers pertinent to each aerosol run into the aerosol software and begin the aerosol challenge.</li>
		<li>Verify particle size data during each aerosol run with the aerosol particle size analyzer to ensure the desired particle size distribution is achieved. Perform this verification continuously or intermittently throughout the exposure.</li>
		<li>Once the aerosol challenge is complete, remove the NHP from the head-only exposure chamber and wipe the NHP&#39;s face/head off with the appropriate disinfectant to reduce potential contamination to laboratory staff.</li>
		<li>Purge the aerosol chamber or air wash the remaining and lagging particles for 5 minutes by passing air and vacuum through the chamber. This procedure will &#34;clean out&#34; and remove residual particles from the aerosol exposure chamber for subsequent NHP aerosol exposures.</li>
		<li>Pass the NHP back through the RTP to the researchers located on inside the ABSL-4 aerobiology suit laboratory.</li>
		<li>Discard all sharps used within the Class III BSC in a designated sharps container that remains in the BSC. When the sharps container is &#190; full, place into the biohazard trash bag.</li>
		<li>Empty the aerosol generator and any of remaining challenge material into the biohazardous trash bag containing trash, disposable equipment, and/or &#190;-full sharps container if applicable.</li>
		<li>Empty the collection media from the aerosol biosampler into the appropriately labeled collection tubes and place on wet ice.</li>
		<li>Repeat steps 4.3.1 to 4.3.13 until all scheduled test subjects have been challenged.</li>
		<li>Pass all aerosol biosampler samples through the RTP to the researchers for quantification and back titrations of aerosol dose.</li>
		<li>Place the trash and equipment from the aerosol challenge into the pass-through autoclave attached to the Class III BSC and select an applicable sterilization cycle (Figure 3).</li>
		<li>Disassemble the NHP head-only chamber and decontaminate the head-only chamber and the Class III BSC with a paraformaldehyde gas cycle validated with biological indicators.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Alibek, K., Handelman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chilling true story of the largest covert biological weapons program in the world-told from inside by the man who ran it. , (1999).</li><li>Roy, C. J., Pitt, L. M., Swearingen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+disease+aerobiology:+aerosol+challenge+methods.">Infectious disease aerobiology: aerosol challenge methods.</a> Biodefense: research methodology and animal models. , 61-76 (2006).</li><li>Lackemeyer, M. G., 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=ABSL-4+aerobiology+biosafety+and+technology+at+the+NIH/NIAID+integrated+research+facility+at+Fort+Detrick.">ABSL-4 aerobiology biosafety and technology at the NIH/NIAID integrated research facility at Fort Detrick.</a> Viruses. 6 (1), 137-150 (2014).</li><li>Bohannon, J. K., 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=Generation+and+characterization+of+large-particle+aerosols+using+a+center+flow+tangential+aerosol+generator+with+a+non-human-primate,+head-only+aerosol+chamber.">Generation and characterization of large-particle aerosols using a center flow tangential aerosol generator with a non-human-primate, head-only aerosol chamber.</a> Inhal Toxicol. , (2015).</li><li>Chosewood, L. C., Wilson, D. E., eds, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2009).</li><li>Janosko, K., 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=Safety+Precautions+and+Operating+Procedures+in+an+(A)BSL4+Laboratory:+1.+Biosafety+level+4+suit+laboratory+suite+entry+and+exit+procedures.">Safety Precautions and Operating Procedures in an (A)BSL4 Laboratory: 1. Biosafety level 4 suit laboratory suite entry and exit procedures.</a> J Vis Exp. , (2015).</li><li>Mazur, S., 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=Safety+Precautions+and+Operating+Procedures+in+an+(A)BSL4+Laboratory:+2.+General+Practices.">Safety Precautions and Operating Procedures in an (A)BSL4 Laboratory: 2. General Practices.</a> J Vis Exp. , (2015).</li><li>Mortola, J. P., Frappell, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+barometric+method+for+measurements+of+ventilation,+and+its+use+in+small+animals.">On the barometric method for measurements of ventilation, and its use in small animals.</a> Can J Physiol Pharmacol. 76 (10-11), 937-944 (1998).</li><li>Zhang, Z., 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=Development+of+a+respiratory+inductive+plethysmography+module+supporting+multiple+sensors+for+wearable+systems.">Development of a respiratory inductive plethysmography module supporting multiple sensors for wearable systems.</a> Sensors (Basel). 12 (10), 13167-13184 (2012).</li><li>Ingram-Ross, J. L., 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=Cardiorespiratory+safety+evaluation+in+non-human+primates.">Cardiorespiratory safety evaluation in non-human primates.</a> J Pharmacol Toxicol Meth. 66 (2), 114-124 (2012).</li><li>Besch, T. K., Ruble, D. L., Gibbs, P. H., Pitt, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+minute+volume+determination+by+body-only+plethysmography+in+juvenile+rhesus+monkeys.">Steady-state minute volume determination by body-only plethysmography in juvenile rhesus monkeys.</a> Lab Anim Sci. 46 (5), 539-544 (1996).</li></ol>

The authors have nothing to disclose.

We outline the aerobiology procedures used at the IRF-Frederick for working with highly hazardous (Risk Group 4) pathogens. One purpose of visualizing the bioaerosol procedures is to emphasize the safety of staff when using a Class III BSC during experimentation with such pathogens to avoid laboratory-acquired infections. Class III BSCs maintain an inward directional airflow that exhausts into double HEPA filters to ensure that pathogens are contained within the laboratory (Figure 1).
<p class="jove_...

Transmission of viruses generally occurs by direct or physical contact, but many important viral diseases (e.g., measles, chickenpox, influenza) are caused by pathogens that are transmitted by aerosol or respiratory droplets. Such pathogens have the potential to cause a pandemic with consequences ranging from widespread mild disease associated with loss of work (e.g., common cold) to rarer serious disease with high lethality (e.g., smallpox). High-consequence pathogens that spread naturally by aerosol or by intentional aerosol release (biological weapons) are of particular interest to aerobiology1. Humans may become rapidly infected with some of these pathogens by large respiratory droplets or small-particle nuclei and easily spread these pathogens to others through salivary secretions, coughing, and sneezing2. In the US biodefense community, high-consequence pathogens (e.g., filoviruses or other NIAID Category A-C Priority Pathogens and CDC Bioterrorism Agents) are the focus of aerosol research programs due to high lethality of associated infections3,4. Significant scientific strides within the aerobiology field have been made within the past decade due to technological advances in aerosol equipment and high containment facilities5,6. Research at the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH/NIAID), Integrated Research Facility at Fort Detrick located in Frederick, MD, USA (IRF-Frederick) focuses on high-consequence emerging pathogens that require animal biosafety level 4 (ABSL-4) containment. The overall mission of the IRF-Frederick is to evaluate and facilitate the development of candidate vaccines and therapeutics (medical countermeasures).
Research with high-consequence pathogens at the IRF-Frederick is governed by stringent biosafety and animal care and use requirements. These requirements are outlined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) manual7 and the federal animal welfare regulations. These necessary requirements may restrict the type of research that can be performed and impact overall study design. As we previously described in this journal, all research conducted in an ABSL-4 environment requires particular caution, highly specialized training, and a robust and redundant facility infrastructure8,9.
Entry into the IRF-Frederick ABSL-4 suit laboratory requires donning a positive-pressure encapsulating suit8. Positive-pressure encapsulating suits are not required for entering the ABSL-4 cabinet laboratory. Donning a scrub suit, rubber or nitrile gloves, and close-toed shoes is appropriate when manipulating Risk Group 4 infectious material within a certified Class III biosafety Cabinet (BSC) in an ABSL-4 cabinet laboratory7.
At the IRF-Frederick, aerosol equipment is engineered, assembled, and maintained in two hermetically sealed, stainless steel, air-tight, negative-pressure Class III BSCs, Figure 1. The IRF-Frederick Aerobiology Core employs an automated aerosol management Platform (AAMP) to control and monitor aerosol experimentation within these BSCs, Figure 2. A previous publication outlined the specific functions of the Class III BSCs at the IRF-Frederick and the connection to the suit laboratory via a pass-through port5. The procedure of preparing the Class III BSC prior to experimentation is specific to the IRF. Other Class III BSCs used at other institutions function similarly to the Class III BSC in use at the IRF, but may have different mechanisms for transport, access, or docking.
To further understand how high-consequence pathogens remain infectious and spread through aerosol transmission, safe aerobiological experimentation must be conducted in these Class III BSCs according to a specific workflow procedure. Researchers have been carefully and thoroughly trained to ensure this workflow is followed in a safe and consistent manner. Prior to nonhuman primate (NHP) aerosol challenge, several aerosol characterization or sham aerosol runs are performed to test the stability and viability of an agent when in aerosol form. The aerosol characterization process mimics the actual aerosol challenge, and the researcher evaluates the variables associated with aerosol studies.
Another part of the workflow is to record physical manipulations, administration or anesthetics or other agents, or routine procedures on charts for each NHP. These subject charts are analyzed thoroughly to ensure procedural consistency and standardization. Subjects are anesthetized prior to aerosol exposure. Example anesthetics include tiletamine/zolazepam, ketamine/acepromazine, and ketamine. Anesthetics are chosen based on minimizing respiratory suppression and promotion of controlled, steady-state breathing. Additional anesthesia supplies are maintained in the animal procedure rooms and transported on the transfer cart with the NHP to the aerobiology ABSL-4 cabinet laboratory.
Within the ABSL-4 suit laboratory, NHPs undergo plethysmography via one of two methods (i.e., head-out plethysmography, respiratory inductive plethysmography [RIP]) to determine inspiratory tidal volume and breathing rate changes10-12. These derived parameters are used for accurate calculation of the estimated inhaled dose of the pathogen immediately prior to or during an aerosol exposure. Head-out plethysmography uses a long, cylindrical chamber that houses the NHP13. The pressure drop created when an animal is in the cylinder is captured by a pneumotachograph, relayed to the amplifier, processed by the alternating current/direct current converter, and integrated into the software to derive the above pulmonary parameters. RIP uses sensors made of inductive coiled copper wires that are embedded in elastic bands around the subject&#39;s chest and abdomen11,12. An inductive-capacitor generates a magnetic field in the sensor. Breathing changes the magnetic field, and the resulting voltage changes are relayed from a transmitter next to the elastic band to a receiver in the computer via short-wavelength ultra-high frequency radio waves. Dedicated software determines breathing rate and tidal volume from total thoracic displacement.
The minute volume (MV) obtained through plethysmography is used in the calculation of the estimated inhaled dose (D). In generating and sampling an aerosol, the aerosol concentration (AC) is calculated by multiplying the biosampler concentration (BC) by the volume of media (V) and dividing by result of multiplying the flow rate of the biosampler (FL) by the exposure time (T). The simplified formula is represented as AC = BC x V &#247; FL x T. In turn, for the actual aerosol challenge in NHPs, D is calculated by multiplying AC by MV and the exposure duration (time = T). The simplified formula is represented as D = AC x MV x T.
The purpose of this article is to visually demonstrate the entire aerosol challenge procedure using NHPs from two viewpoints, the ABSL-4 suit laboratory side and the ABSL-4 cabinet laboratory side. Although these procedures may be general in nature for several practices mentioned, they are specific to the IRF-Frederick Aerobiology Core and represent the actual practices used at this institution. This article focuses on the biosafety procedures needed to safely perform an aerosol challenge, not the actual aerosol challenge itself. In these procedures, we are using a dummy subject to show biosafety practices, due to the risk associated with anesthetizing an NHP. However, the process of performing an aerosol challenge is written in a general way because the procedure is the same regardless of high-consequence pathogen used. We aim to enhance knowledge and understanding of scientists about the rigors of conducting aerosol studies of high consequence pathogens under maximum containment conditions.

<table><tbody><tr><td>Micro-Chem Plus</td><td>National Chemical Laboratories</td><td>255</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>BP2818500</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>441244</td><td></td></tr><tr><td>Class III BSC</td><td>Germfree</td><td>DGB-10</td><td></td></tr><tr><td>Integrated BSC gloves</td><td>Piercan</td><td>10UY2032-9</td><td></td></tr><tr><td>Aerosol Management Platform (AeroMP)</td><td>Biaera Technologies</td><td>NA</td><td></td></tr><tr><td>Head-out plethysmography</td><td>Buxco/Data Sciences International</td><td>NA</td><td></td></tr><tr><td>Respriatory inductive plethysmography</td><td>Data Sciences International</td><td>NA</td><td></td></tr><tr><td>Centered flow tangential aerosol generator (CenTAG)</td><td>CH Technologies</td><td>NA</td><td></td></tr><tr><td>Collison nebulizer</td><td>BGI Inc.&amp;nbsp;</td><td>CN25</td><td></td></tr><tr><td>Autoclave</td><td>Getinge</td><td>GEB 2404 AMB-2</td><td></td></tr><tr><td>Sperian positive-pressure suit</td><td>Honeywell Safety Products</td><td>BSL 4-2</td><td></td></tr><tr><td>Outer suit gloves (latex, Ansell Canners and Handlers)</td><td>Fisher</td><td>19-019-601</td><td></td></tr><tr><td>Outer suit gloves (nitrile/rubber, MAPA)</td><td>Fisher</td><td>2MYU1</td><td></td></tr><tr><td>Scrubs</td><td>Cintas</td><td>60975/60976</td><td></td></tr><tr><td>Socks</td><td>Cintas</td><td>944</td><td></td></tr><tr><td>Duct tape</td><td>Pack-N-Tape</td><td>51131069695</td><td></td></tr><tr><td>Towels</td><td>Cintas</td><td>2720</td><td></td></tr><tr><td>O-rings</td><td>O-ring warehouse</td><td>AS568-343</td><td></td></tr><tr><td>Overshoes</td><td>Amazon</td><td>B0034KZE22</td><td></td></tr><tr><td>Zip lube</td><td>Amazon</td><td>B000GKBEJA</td><td></td></tr></tbody></table>

safety precautions and operating procedures in an (a)bsl-4 laboratory: 3. aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

The Class III biosafety cabinet (BSC) is a hermetically sealed stainless steel cabinet containing an ABSL-4 environment under negative pressure within an ABSL-4 cabinet laboratory (Figure 1). Materials can be introduced into the BSC by staff working in the ABSL-4 cabinet laboratory through an under-cabinet-mounted stainless steel tank (commonly referred to as a &#34;dunk tank&#34; in ABSL-4 or BSL-4 settings) containing a 5% dual quaternary ammonium (n-alkyl dimethyl benz...

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Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

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JoVE Journal

Immunology and Infection

14.1K Views.  National Institutes of Health (NIH). As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Video: Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

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

Operational Protocols for Handling Wildling Mice in Research Facilities

Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science due to their close resemblance to the human superorganism. However, the breeding and maintenance of these mice, which harbor a diverse microbiome&#160;including bacteria, viruses, and parasites, pose significant challenges for animal husbandry facilities at research institutions. To address these challenges, a specialized facility concept was developed for housing &#34;Wildling mice&#34; at Charit&#233; - Universit&#228;tsmedizin Berlin. This approach involved designing a facility with specific structural features and operational protocols to effectively contain the natural microbiome, thereby protecting areas with higher hygiene standards.
A methodology for blood sampling from both specified pathogen-free (SPF) and &#34;Wildling mice&#34; for immunophenotyping is demonstrated, highlighting the workflow and biocontainment measures implemented in the facility. Remarkable results reveal that &#34;Wildling mice&#34; exposed to a natural microbiome develop distinct immune cell populations, which are significantly reduced in mice bred and maintained under stringent hygiene conditions.
The significance of this study lies in its potential to provide researchers with access to mice that possess a natural microbiome and a mature immune system similar to that of human adults. This approach could enhance the translatability of preclinical findings into clinical practice, thereby advancing the field of biomedical research.

Here, we describe the structure and operating procedures, including microbial containment measures of a facility for "Wilding mice" using blood sampling for immunophenotyping as an example.

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

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