We would like to thank S. Werner for providing the crows used in this study and USDA, APHIS, WS, NWRC animal care staff for animal care and monitoring. Mention or use of a product does not imply USDA endorsement. Funding for this study was provided by USDA, APHIS, Veterinary Services.

Our protocol is adapted from one we previously published7. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Wildlife Services (WS), National Wildlife Research Center (NWRC).
1. Crow Gavaging
<ol>
	<li>Estimate passage time of &#39;pseudo brain material&#39; through the alimentary tract of American crows.
	<ol>
		<li>Mix 5 ml of cooked and scrambled whole egg with blue dye and gavage 1 crow using a 2 in&#160;gavage needle (Figure 1).</li>
		<li>Check crow every 30 min until blue/green-stained feces are no longer being excreted.</li>
	</ol>
	</li>
	<li>Obtain uninfected and terminally ill RML (Chandler strain) -infected C57BL/6 mouse brains.</li>
	<li>Generate a 20% wt/vol normal and infected mouse-brain homogenate in a bullet blender homogenizer with 1x phosphate buffered saline (PBS) and glass beads, then centrifuge at 3,000 x g for 1 min to remove larger particulate matter. Remove supernatant and dilute with an equal volume of 1x PBS to generate a 10% wt/vol in sterile PBS. Freeze at -80 &#176;C until needed.</li>
	<li>17 hr prior to gavage remove food, but not water, from crow pens.</li>
	<li>Randomly allocate crows to treatment groups and orally gavage each crow with 5 ml of either normal or infected mouse brain homogenate using a 2 in&#160;gavage needle.</li>
	<li>Transfer crows to individual cages and collect and pool all feces within each cage for 4 hr post gavage (Figure 2).</li>
	<li>Homogenize pooled feces for each crow in a bullet blender homogenizer with glass beads until a uniform texture is achieved. *Crow feces are mostly liquid and can be mixed easily.</li>
	<li>For each crow, dilute 500 &#181;l of fecal homogenate into 9.5 ml 1x PBS for a total volume of 10 ml.</li>
	<li>Centrifuge the fecal homogenate for 15 min at 1,400 x g and extract the supernatant.</li>
	<li>To minimize the threat of a secondary microbial infection, treat supernatant with 1 &#181;l of 100 units/ml penicillin and 100 &#181;g/ml streptomycin (Invitrogen, NY) per 100 &#181;l of homogenate then place under UV light at room temperature for 20 min to reduce risk of viral and bacterial contamination. After UV exposure, sonicate samples in a 3000 Mp sonicator at setting 70 for 30 sec to disrupt the membrane of any remaining microbes.</li>
</ol>
2. Mouse Inoculation
<ol>
	<li>Randomly allocate mice to the following treatment groups (Table 1):
	<ol>
		<li>Group 1 - Positive treatment mice inoculated intraperitoneally (IP) with 1 ml of fecal homogenate from crows orally gavaged with 5ml infected mouse brains.</li>
		<li>Group 2 - Negative treatment mice inoculated IP with 1 ml of fecal homogenate from crows orally gavaged with 5ml normal mouse brains.</li>
		<li>Dilute infected and normal mouse brain homogenate for Groups 3 and 4 to 1:100 w/v in 1x PBS.</li>
		<li>Group 3 - Positive control mice inoculated IP with 1 ml of infected mouse-brain homogenate.</li>
		<li>Group 4 - Negative control mice inoculated IP with 1 ml of normal mouse-brain homogenate.</li>
	</ol>
	</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td>Treatment Group</td>
			<td>Number of Animals</td>
		</tr>
		<tr>
			<td>Group 1</td>
			<td>Scrapie + Crow Feces</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Group 2</td>
			<td>Scrapie - Crow Feces</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Group 3</td>
			<td>Scrapie + Mouse Brain</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Group 4</td>
			<td>Scrapie - Mouse Brain</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1. Scrapie status of inoculum (positive +, negative -) and number of animals used7.
<ol start="2">
	<li>Intraperitoneally inoculate mouse:
	<ol>
		<li>Scruff mouse by dorsal neck fur with thumb and index finger and gently rotate to expose ventral side.</li>
		<li>Elevate posterior end of mouse so head is slightly lower.</li>
		<li>Insert 25 G needle 1 cm through skin, 1 cm lateral of midline, and 1-2 cm anterior to the sacroiliac joint using a needle-locking syringe.</li>
		<li>Inject 1 ml inoculate slowly into mouse body cavity.</li>
	</ol>
	</li>
</ol>
3. Mouse Monitoring
<ol>
	<li>Monitor mice daily until they express clinical signs of mouse scrapie. Clinical signs may include: kyphosis, ataxia, stiff tail, lack of grooming, emaciation, and lethargy.</li>
	<li>Score mice for each of the 6 clinical signs when visible signs are evident, where 0 = none visible, 1 = moderate, and 2 = severe.</li>
	<li>Euthanize mice when total daily scores for each sign reach &#8805;8 for 1 day, &#8805; 6 continuously for 3 days, or at 365 days post inoculation (dpi).</li>
	<li>Harvest brains immediately following euthanasia and store at -70 &#176;C.</li>
	<li>To confirm a scrapie diagnosis, digest brain samples with 3 &#181;l of a 50 &#181;g/ml of proteinase-K solution (PK) diluted as follows: 3.1. &#181;l PK, 12.5 &#181;l of 500 mM EDTA, pH 8, 109.39 &#181;l of 1x PBS for 30 min at 45 &#176;C, then inactivate the PK by adding 8 &#181;l loading buffer and incubating samples at 95 &#176;C for 5 min. Load samples onto a 12% SDS-PAGE gel, electrophorese and transfer to Immobilon PVDF membrane and block with 5% nonfat milk in 0.2% Tween 20 in PBS for 1 hr at room temperature. Then probe with Bar224 anti-PrP monoclonal antibody conjugated to Horseradish peroxidase, diluted in Superblock, for 1 hr at room temperature. Rinse membrane for 1 hr with PBS-0.2% Tween 20. To visualize, incubate Western blot 5 min with chemiluminescent substrate and image on a G-box gel documentation system.</li>
</ol>

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No conflicts of interest declared.

We demonstrate a procedure to document passage of PrPRes material through the digestive system of crows. We used conventional methods to determine if crows have the ability to translocate PrPRes material to disease-free geographic areas. Others have evaluated resistance of PrPRes to ruminant19-21 and rodent22,23 digestive fluids, both of which failed to eliminate it. Future application of these techniques should be applied to other carnivores24 as they cou...

Transmissible spongiform encephalopathies (TSE) are fatal infectious neurodegenerative disorders that affect wildlife, domestic animals, and humans. The infectious agent of TSE diseases appears to be misfolded or pathogenic isoforms (PrPRes) of prion proteins1. Animal TSE diseases include chronic wasting disease (CWD) in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), elk (Cervus elaphus), and moose (Alces alces); scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) in domestic cattle; transmissible mink encephalopathy in farmed mink; feline spongiform encephalopathy in cats; exotic ungulate spongiform encephalopathy in exotic zoo ruminates of the family Bovidae; and spongiform encephalopathy in nonhuman primates8. The single human TSE disease, variant Creutzfeldt-Jakob disease, is rare and thought to be acquired by consuming PrPRes-contaminated food9. Similarly, BSE can infect humans if contaminated beef is consumed10. Of all the TSE diseases, scrapie and CWD are the only two with self-sustaining epidemics and sources for infection are presumed to be from animal to animal2,3,11 as well as from environmental sources4-6. Research suggests that most TSE diseases require notable extended incubation periods from natural exposure events of PrPRes material to manifestation of clinical signs2-4,6,8 and apparent species barriers minimize, but do not eliminate the potential for, interspecies transmission12-14.
Identifying mechanisms for the spread of infectious prion (PrPRes) material is extremely important for answering questions about how TSE diseases move across the landscape. Experimental investigations have suggested that insects15,16, poultry and pigs17, and American crows (Corvus brachyrhynchos)7,18 are passive carriers or dispersers of PrPRes material. Passage of PrPRes material through the digestive system of crows has recently been documented, demonstrating the role they may play in dispersal of TSE diseases7. These results make it plausible that crows, a scavenger, could encounter, consume, and transport infectious material via feces deposition, to disease-free areas.
The procedures we demonstrate here were used to document passage of PrPRes material through the digestion system of crows and will greatly facilitate the application of these methods to other scavenger and carnivore species-specific models in related future research. In this study conventional methods were used to investigate an unconventional means of trafficking PrPRes material, which could contribute to the spread and overall burden of PrPRes material.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

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.

The procedures used demonstrate that the digestive system of crows does not eliminate PrPRes infectivity 4 hr after oral gavage of scrapie brain homogenate7. All twenty crows that were gavaged with PrPRes material subsequently transmitted PrPRes material via feces to mice. Diseased mice were identified by manifestation of clinical mouse-scrapie signs and disease confirmation was completed by Western blot analysis.
Investigation of the retention time ...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

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

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 ...

procedures-for-identifying-infectious-prions-after-passage-through

Research

JoVE Journal

Immunology and Infection

11.0K Views.  USDA. 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.

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

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 ...

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.

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.

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

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...