The authors have no acknowledgements to make.

1. Sample Collection
<ol>
	<li>Collect 70 samples from different environments and regions. In this case, use the following: 5 dirty water samples from the village of Saint Pierre de Meyzoargues (France), 15 samples from the lake in Parc Bor&#233;ly in Marseille (France); 15 sea water samples with sediment from the rocky inlets at Samena in Marseille (France), 25 river water samples from the Alps (France), and finally, 10 samples from sewage in La Ciotat (France).</li>
	<li>Vortex samples for homogenization before inoculating, for one minute at room temperature without any treatment.</li>
</ol>
2. Isolation Procedure
<ol>
	<li>Host Preparation for Blind Culture Procedures and Sample Inoculation
	<ol>
		<li>Use V. vermiformis (strain CDC19) as a cell support for the co-culture.</li>
		<li>Maintain the amoebae at 28 &#176;C, in a 75 cm2 cell culture flask with 30 ml of protease-peptone-yeast extract-glucose medium (PYG). Please check PYG composition in Table 1. 
		Note: After 48 hr, amoebae are quantified by a normal count using counting slides (e.g., Kovaslides).</li>
		<li>Harvest cells at a concentration of 5 x 105 to 106 cell/ml, and pellet amoebae by centrifugation at 720 x g for 10 min.</li>
		<li>Remove the supernatant by aspiration, and re-suspend the amoebae pellet in 30 ml of sterile Page&#39;s amoebal saline (PAS) Table 1. Repeat this rinsing procedure.</li>
		<li>Re-suspend amoebae in 30 ml of starvation medium with an antibiotic and antifungal mixture at a concentration of approximately 106 amoeba/ml. The starvation medium is a survival medium for the amoeba, enabling it to stay alive without encystment or multiplication. Check composition in Table 1. 
		Note: The antimicrobial agent mix contained 10 &#181;g/ml vancomycin, 10 &#181;g/ml imipenem, 20 &#181;g/ml ciprofloxacin, 20 &#181;g/ml doxycycline, and 20 &#181;g/ml voriconazole. This mix served to reduce the bacterial and fungal contamination that can decrease or alter viral multiplication.</li>
		<li>Directly inoculate 25 &#181;l of each sample onto the 200 &#181;l amoeba monolayer in a 96-well plate flat bottom favoring the amoeba adherence and incubate at 30 &#176;C in a humid environment (a humid environment consists of placing a wet compress inside the bag containing the plate in order to avoid evaporation). This is the primo-culture. Leave two wells of amoebae without any sample inoculation; this acts as the negative control. Incubate this primo-culture for three days.</li>
		<li>Three days after the first inoculation, sub-culture the primo-culture plate on fresh amoebae as described above without any microscopic observation. Incubate the new plate for three days under the same conditions as the primo-culture.</li>
		<li>Perform the final culture after these three days of incubation the same way as for the primo- and sub-culture. Incubate the final culture for two days. Then proceed to detection.</li>
	</ol>
	</li>
	<li>Detection of Lysis by Automated Flow Cytometry 
	Note: Blind culture coupled with flow cytometry differs from the previous standard system of isolation. It is more sensitive, precise and automated. For detection purposes, we applied a new technique developed to detect lysis at the highest speed and without microscopic observation. This technique is advantageous for screening samples and has better sensitivity than older techniques.7-9
	<ol>
		<li>Use the 96-well plate of the final co-culture to detect lysis.</li>
		<li>Turn on the cytometer and run a clean and rinse cycle in order to obtain lower background noise according to manufacturer&#39;s protocol.</li>
		<li>Launch the High Throughput Sampler (HTS) combined with flow cytometer to automatically load samples from the 96-well plate and perform a completely automated acquisition within 40 min according to manufacturer&#39;s protocol.</li>
		<li>Set up the HTS as follows: sample volume 150 &#181;l, mixing volume 50 &#181;l, mixing speed 180, number of mixes 5, washing volume between each sample 400 &#181;l, flow rate 2.5 &#181;l/sec, number of recorded events 10,000.</li>
		<li>Assess the negative control wells of the final co-culture micro-plate first in order to gate the amoeba population and to determine the percentage of these cell hosts according to their physical characteristics. Be sure to have a homogeneous amoeba population and a second population of a lower events corresponding to debris and noise. 
		Note: This gating procedure distinguishes subcellular debris and clumps from single cells by size, estimated by forward scatter. It is therefore important to accurately determine the percentage of each population using the best possible gating procedure. These percentages may vary, especially if many types of protozoa are used, so it is important always to have a negative control, which can be used as the reference population for the rest of the samples.</li>
		<li>Start the gating by clicking acquire sample.</li>
		<li>Record the number of events not less than 10,000 events.</li>
		<li>Localize the amoeba population of interest using the arrow. This is how to define the gates.</li>
		<li>After gating, let the HTS spot or locate the plate by clicking &#39;Home&#39;, then run the entire plate automatically. Perform data acquisition and analysis according to the size and structure parameters (respectively, FSC (forward scatter) and SSC (side scatter)). 
		Note: Detecting the loss of the amoeba-gated population signals the presence of a pathogenic agent responsible for amoeba lysis. Protozoa lysis associated with virus infection is detected with an arbitrarily designed threshold of 50% lysis of the amoeba population gated by the analyzer and compared to the same non-infected population used as a reliable negative control.</li>
	</ol>
	</li>
</ol>
3. Characterization of the New Isolates after Lysis Detection
<ol>
	<li>Preliminary Staining to Reveal the Presence of Giant Viruses
	<ol>
		<li>After lysis detection by flow cytometry, pipette the lysed co-cultures to suspend the remaining amoebae. Then directly cytocentrifuge 50 &#181;l of the suspension at 800 x g for 10 min.</li>
		<li>After centrifugation, fix the slides with a pure methanol solution. Stain the slides using a commercial rapid staining of Eosin/blue Azur and DAPI staining and observe under a light microscope at 1,000X magnification in order to check for the presence of virus factories.
		<ol>
			<li>For rapid staining, plunge the slides into the first eosin solution (4 sec repeated three times) then pass directly to plunge slides into the second fixing solution containing blue Azur for 6 sec, and finally rinse the slides for 45 sec with a phosphate buffer solution pH 7.2. Leave the slides to dry at room temperature, then proceed to observation.</li>
			<li>For DAPI staining, deposit 15 &#181;l of the DAPI commercial stock solution onto the fixed dry slide. Protect from light then proceed to observation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Electron Microscopy
	<ol>
		<li>After the first identification on the slides, assess the presence of the giant virus through electron microscopy observation of supernatant cultures.</li>
		<li>Deposit 5 &#181;l of the positive sample onto the glow-discharged grid. Wait for approximately 20 min at room temperature.</li>
		<li>Dry the grid carefully and deposit on it a small drop of 1% ammonium molybdate for 10 sec. Leave the grid to dry for 5 min.</li>
		<li>Proceed to electron microscopy at 200 keV.</li>
		<li>Place the grid on the holder; introduce the holder into the microscope. Check the vacuum overview by clicking on the corresponding icon. Close valves and begin observation at a spot size 5, and magnification of x 25,000.</li>
		<li>Measure the giant virus using ImageJ or directly on the microscope using the measure tool of the microscope located on the acquisition screen. 
		Note: Classify the sample into the Faustovirus group with a capsid size of approximately 200 nm.</li>
	</ol>
	</li>
	<li>Molecular Biology 
	Note: Following this identification, molecular biology tests were crucial for the confirmation in order to distinguish Faustovirus from Marseillevirus, which has the same capsid size and appearance under electron microscopy, although they do not have the same host specificity. The Marseillevirus cannot grow on Vermamoeba, the Faustovirus is specific to Vermamoeba and all attempts to grow it on other amoeba such as Acanthamoeba have failed to date. The design and application of the specific primer/probe systems listed in the works of Reteno et al. enabled a more precise preliminary classification of the newly isolated viruses through amplification and sequencing of the viral genes.
	<ol>
		<li>Extract DNA from the positive culture samples using an automated extraction system according to the manufacturer&#39;s protocol.</li>
		<li>Perform Real-time PCR using a thermocycler as described in Reteno et al.19</li>
		<li>Sequence using the same primers that were used for amplification. Use the primers targeting DNA-directed RNA polymerase subunit 1 gene: Fstv_S2F 5&#39;-CCA GGA CAT GAT GGT CAC ATA G-3&#39; (forward) and Fstv_S2R 5&#39;-TTG CAC CTC CGC AGT TAA A-3&#39; (reverse) with the Fstv_S2P FAM-TATGCTCCAATGGCCTTCAACGACA-TAMRA probe.</li>
	</ol>
	</li>
</ol>
4. Virus Production, Purification and Genome Sequencing
<ol>
	<li>Subculture the single virus amoeba culture for end-point dilution cloning.</li>
	<li>For the production: prepare 20 flasks containing 30 ml of PYG, 10 ml of Vermamoeba vermiformis and 5 ml of the isolated virus already transferred from wells to small flasks.</li>
	<li>Incubate at 30 &#176;C until complete lysis of the amoeba. Observe every day by inverted optical microscopy until all amoeba are lysed.</li>
	<li>After complete lysis, pool all flasks into one recipient. Filter with a 0.45 &#181;m filter to eliminate debris.</li>
	<li>Start the purification by ultracentrifugation at 89,800 x g for 75 min. Make sure to calibrate the weight of the tubes before starting centrifugation.</li>
	<li>After centrifugation, remove the supernatant from each tube by aspiration and re-suspend the pellet in PAS with the same volume used for calibration, before repeating centrifugation.</li>
	<li>As above, remove the supernatant and re-suspend all the pellets in 1 ml of phosphate buffered saline (PBS).</li>
	<li>Purify the virus that is produced, using 25% sucrose (27.5 g of sucrose in 100 ml of PBS, filtered on a 0.22 &#181;m filter).
	<ol>
		<li>Use 8 ml of sucrose and 2 ml of the viral suspension. Centrifuge at 89,800 x g for 1 hr 15 min. Re-suspend the pellet in 1 ml of PBS. Store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Raoult, D., 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=The+1.2-megabase+genome+sequence+of+Mimivirus.">The 1.2-megabase genome sequence of Mimivirus.</a> Science. 306 (5700), 1344-1350 (2004).</li><li>Claverie, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+viruses+in+the+oceans:+the+4th+Algal+Virus+Workshop.">Giant viruses in the oceans: the 4th Algal Virus Workshop.</a> Virol J. 2, 52-52 (2004).</li><li>Monier, A. A., 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=Marine+mimivirus+relatives+are+probably+large+algal+viruses.">Marine mimivirus relatives are probably large algal viruses.</a> Audio, Transactions of the IRE Professional Group on. 5, 12-12 (2007).</li><li>Monier, A., Claverie, J. M., Ogata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxonomic+distribution+of+large+DNA+viruses+in+the+sea.">Taxonomic distribution of large DNA viruses in the sea.</a> Genome Biol. , (2008).</li><li>Claverie, J. -. M., 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=Mimivirus+and+Mimiviridae:+Giant+viruses+with+an+increasing+number+of+potential+hosts,+including+corals+and+sponges.">Mimivirus and Mimiviridae: Giant viruses with an increasing number of potential hosts, including corals and sponges.</a> J Invertebr Pathol. 101 (3), 9-9 (2009).</li><li>Colson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+the+megavirome+in+humans.">Evidence of the megavirome in humans.</a> J Clin Virol. 57 (3), 191-200 (2013).</li><li>La Scola, B., 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=Tentative+characterization+of+new+environmental+giant+viruses+by+MALDI-TOF+mass+spectrometry.">Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry.</a> Intervirology. 53 (5), 344-353 (2010).</li><li>Boughalmi, M., 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=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ Microbiol. , (2012).</li><li>Pagnier, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+improvements+in+mimiviridae+and+marseilleviridae+isolation+from+amoeba.">A decade of improvements in mimiviridae and marseilleviridae isolation from amoeba.</a> Intervirology. 56 (6), 354-363 (2012).</li><li>Philippe, N., 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=Pandoraviruses:+Amoeba+Viruses+with+Genomes+Up+to+2.5+Mb+Reaching+That+of+Parasitic+Eukaryotes.">Pandoraviruses: Amoeba Viruses with Genomes Up to 2.5 Mb Reaching That of Parasitic Eukaryotes.</a> Science. 341 (6143), 281-286 (2013).</li><li>Legendre, M., 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=Thirty-thousand-year-old+distant+relative+of+giant+icosahedral+DNA+viruses+with+a+pandoravirus+morphology.">Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.</a> PNAS. , (2014).</li><li>Saadi, H., 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=First+Isolation+of+Mimivirus+in+a+Patient+With+Pneumonia.">First Isolation of Mimivirus in a Patient With Pneumonia.</a> Clin Infect Dis. , (2013).</li><li>Saadi, H., 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=Shan+virus:+a+new+mimivirus+isolated+from+the+stool+of+a+Tunisian+patient+with+pneumonia.">Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia.</a> Intervirology. 56 (6), 424-429 (2013).</li><li>Claverie, J. -. M., Abergel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimivirus:+the+emerging+paradox+of+quasi-autonomous+viruses.">Mimivirus: the emerging paradox of quasi-autonomous viruses.</a> Trends Genet : TIG. 26 (10), 431-437 (2010).</li><li>Colson, P., 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=Viruses+with+more+than+1,000+genes:+Mamavirus,+a+new+Acanthamoeba+polyphaga+mimivirus+strain,+and+reannotation+of+Mimivirus+genes.">Viruses with more than 1,000 genes: Mamavirus, a new Acanthamoeba polyphaga mimivirus strain, and reannotation of Mimivirus genes.</a> Genome Biol Evol. 3, 737-742 (2010).</li><li>Claverie, J. -. M., Abergel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+questions+about+giant+viruses.">Open questions about giant viruses.</a> Adv Virus Res. 85, 25-56 (2013).</li><li>Fischer, M. G. M., Allen, M. J. M., Wilson, W. H. W., Suttle, C. A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+virus+with+a+remarkable+complement+of+genes+infects+marine+zooplankton.">Giant virus with a remarkable complement of genes infects marine zooplankton.</a> PNAS. 107 (45), 19508-19513 (2010).</li><li>Van Etten, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+really,+really+big+virus.">Another really, really big virus.</a> Viruses. 3 (1), 32-46 (2011).</li><li>Reteno, D. 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=Faustovirus,+an+asfarvirus-related+new+lineage+of+giant+viruses+infecting+amoebae.">Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.</a> J.Virol. , (2015).</li><li>Co&#351;kun, K. A., Oz&#231;elik, S., Tutar, L., Elald&#305;, N., Tutar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+free-living+amoebae+from+tap+water+in+Sivas,+Turkey.">Isolation and identification of free-living amoebae from tap water in Sivas, Turkey.</a> Biomed res Int. 2013, 675145 (2013).</li><li>Bradbury, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-living+amoebae+recovered+from+human+stool+samples+in+Strongyloides+agar+culture.">Free-living amoebae recovered from human stool samples in Strongyloides agar culture.</a> J Clin microbiol. 52 (2), 699-700 (2014).</li><li>Pagnier, I., Valles, C., Raoult, D., La Scola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Vermamoeba+vermiformis+and+associated+bacteria+in+hospital+water.">Isolation of Vermamoeba vermiformis and associated bacteria in hospital water.</a> Microb Pathog. 80, 14-20 (2015).</li><li>Ogata, H., Toyoda, 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=Remarkable+sequence+similarity+between+the+dinoflagellate-infecting+marine+girus+and+the+terrestrial+pathogen+African+swine+fever+virus.">Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus.</a> Virol J. 6, 178-178 (2008).</li><li>Tarutani, K., Nagasaki, K., Itakura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+virus+infecting+the+novel+shellfish-killing+dinoflagellate+Heterocapsa+circularisquama.">Isolation of a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circularisquama.</a> Aquat Microb Ecol. 23, 103-111 (2001).</li></ol>

The authors declare that they have no competing financial interests.

The possibility that Faustovirus could be the first member of a new Megavirales family close to ASFV was first suggested by Reteno et al.,19 but some differences can still be distinguished. It appears unclear whether Faustovirus should join the Asfarviridae family or whether it should instead form a new putative viral family. This issue will require further investigation, in particular a more comprehensive characterization of its morphology, host range, replication cycle and gene repertoire. More Faus...

The discovery of giant viruses, especially those belonging to the Megavirales order, completely changed the world of viruses in terms of particle size and genome complexity. Viruses were previously thought to be small entities, and the Mimivirus appeared to break all the rules.1 Metagenomic data suggests the ubiquity of giant viruses not only in the environment, 2-5 but also in humans.6 Therefore, there is still a need to search for these viruses on a large scale. The diversity of these giant viruses was assessed by sampling not only a variety of aquatic environments and their associated sediments worldwide,7-11 but also by screening a variety of human samples12,13 and environmental samples.7,9 The Acanthamoeba polyphaga mimivirus was isolated by co-culture using phagocytic protists, primarily Acanthamoeba spp.14-16 An entire collection of giant viruses were then also isolated from this specified protist host, which made the scientific community restrict its research and isolation procedure for Acanthamoeba spp. Clearly this reliance on a single host species has resulted in a large fraction of viruses being overlooked. The fact that the giant virus, CroV, was isolated with the highly motile marine protozoa Cafeteria roenbergensis,17,18 demonstrates the need to use a wider range of protozoa in order to discover new lineages or families of giant viruses. Reteno et al. managed to select other protozoa as cell hosts which had never previously been used, and isolated the new Asfar-related lineage of giant viruses (the newly named Faustovirus).19
In an attempt to isolate new Faustovirus genotypes in order to expand the members of this viral lineage, we modified our isolation procedures and used them to screen environmental samples capable of harvesting new Faustoviruses. We then described the entire protocol to characterize the new isolates. We assessed Vermamoeba vermiformis, the most common free-living protist found in human environments,20-22 which is already used in the isolation of the first Faustovirus prototype E12.19 This protist is currently still host-specific for Faustovirus. We knew that none of the known giant viruses was pathogenic for this amoeba, because no attempts to grow other giant viruses in our lab showed amoeba lysis or viral growth. For this reason, we believe that V. vermiformis is the best and most unique cell support known to isolate new Faustoviruses.

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">LSR FORTESSA cytometer&#160;</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:119px;">France&#160;</td>
			<td style="width:167px;">649225B4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TECNAI G2 F20</td>
			<td style="width:231px;">FEI</td>
			<td style="width:119px;">Germany&#160;</td>
			<td>5027/11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Optical inverted microscope</td>
			<td style="width:231px;">leica&#160;</td>
			<td style="width:119px;">France</td>
			<td>72643</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">DNA extraction</td>
			<td style="width:231px;">Qiagen EZ1 Advanced XL Extraction Robot</td>
			<td style="width:119px;">France&#160;</td>
			<td>L106A0452</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PCR Cycler CFX96</td>
			<td style="width:231px;">Bio rad</td>
			<td style="width:119px;">France</td>
			<td>785BR06298</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PYG medium , PAS, Starvation medium</td>
			<td style="width:231px;">In house laboratory production&#160;</td>
			<td style="width:119px;">Marseille URMITE</td>
			<td>x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Amoeba strain CDC-19</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:119px;">France</td>
			<td>50237</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Plates</td>
			<td style="width:231px;">Cellstar</td>
			<td style="width:119px;">France</td>
			<td>655180</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PCR materials, primers.&#160;</td>
			<td style="width:231px;">eurogentec</td>
			<td style="width:119px;">France</td>
			<td>Primers cited in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">glasstic slide 10 with grids</td>
			<td style="width:231px;">Kova&#160;</td>
			<td style="width:119px;">USA</td>
			<td style="width:167px;">H899871441F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Eosin/ blue Azur-Hemacolor stain</td>
			<td style="width:231px;">Merck milipore&#160;</td>
			<td style="width:119px;">France</td>
			<td>111955,6,57,109468</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum driven filters</td>
			<td style="width:231px;">Thermo scientific</td>
			<td style="width:119px;">France</td>
			<td>BPV4550 / 20170115</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Phosphate-Buffered Saline</td>
			<td style="width:231px;">Thermo Fisher scientific&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DAPI stain</td>
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			<td height="21" style="height:21px;width:235px;">Single cytology tunnel</td>
			<td style="width:231px;">Biomedical polymers inc.</td>
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			<td>BMP-cyto-S50</td>
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			<td>FCF400NI</td>
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			<td height="21" style="height:21px;width:235px;">Ammonium molibdate</td>
			<td style="width:231px;">VWR internationanl&#160;</td>
			<td style="width:119px;">France&#160;</td>
			<td>21276185</td>
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			<td height="21" style="height:21px;width:235px;">Flasks&#160;</td>
			<td style="width:231px;">SARSTEDT</td>
			<td style="width:119px;">Germany</td>
			<td>833911</td>
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			<td height="21" style="height:21px;width:235px;">0.22&#956;m filters&#160;</td>
			<td style="width:231px;">Milex millipor&#160;</td>
			<td style="width:119px;">France</td>
			<td>SE2M229104</td>
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			<td height="21" style="height:21px;width:235px;">Ultracentrifuge Sorval WX 80</td>
			<td style="width:231px;">Thermo scientific</td>
			<td style="width:119px;">France</td>
			<td>9102448</td>
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			<td height="21" style="height:21px;width:235px;">Rapid-flow filters</td>
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			<td style="width:119px;">France</td>
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a rapid strategy for the isolation of new faustoviruses from environmental samples using vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

The system studied in this manuscript validated its proof of concept by isolating two new Faustoviruses. Of the 70 samples tested, two episodes of lysis were detected, in contrast to our reliable negative controls. The negative control for lysis contained an 86% amoeba population. By contrast, the positive samples (ST1 for Saint Pierre de Meyzoargues), and (LC9 for the La Ciotat Sample 9) showed a dramatic decline in gated amoebae; more than 60% of amoebae were lysed with the highest perc...

Watch this Scientific Journal Video about A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis at JoVE.com

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

a-rapid-strategy-for-isolation-new-faustoviruses-from-environmental

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Aix Marseille University. We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba. 

Video: A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...