These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

<ol>
	<li>Domingo, E., Holland, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+virus+mutations+and+fitness+for+survival.">RNA virus mutations and fitness for survival.</a> Annu Rev Microbiol. 51, 151-178 (1997).</li><li>Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+viral+emergence.">Mechanisms of viral emergence.</a> Veterinary research. 41 (6), 38 (2010).</li><li>Martinez-Picado, J., Martinez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+reverse+transcriptase+inhibitor+resistance+mutations+and+fitness:+a+view+from+the+clinic+and+ex+vivo.">HIV-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo.</a> Virus Res. 134 (1-2), 104-123 (2008).</li><li>Troyer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+fitness+impact+of+HIV-1+escape+mutations+to+cytotoxic+T+lymphocyte+(CTL)+response.">Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.</a> PLoS Pathog. 5 (4), e1000365 (2009).</li><li>Rolland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+co-variation+in+HIV-1+Gag+subtype+C:+HLA-mediated+selection+pressure+and+compensatory+dynamics.">Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics.</a> PLoS One. 5 (9), e12463 (2010).</li><li>Abraha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR5-+and+CXCR4-tropic+subtype+C+human+immunodeficiency+virus+type+1+isolates+have+a+lower+level+of+pathogenic+fitness+than+other+dominant+group+M+subtypes:+implications+for+the+epidemic.">CCR5- and CXCR4-tropic subtype C human immunodeficiency virus type 1 isolates have a lower level of pathogenic fitness than other dominant group M subtypes: implications for the epidemic.</a> J Virol. 83 (11), 5592-5605 (2009).</li><li>Quinones-Mateu, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+infection/competition+assay+shows+a+correlation+between+ex+vivo+human+immunodeficiency+virus+type+1+fitness+and+disease+progression.">A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression.</a> J Virol. 74 (19), 9222-9233 (2000).</li><li>Ball, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+ex+vivo+fitness+of+CCR5-tropic+human+immunodeficiency+virus+type+1+isolates+of+subtypes.">Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes.</a> B and C. J Virol. 77 (2), 1021-1038 (2003).</li><li>Lanxon-Cookson, E. 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Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes. Les opinions exprimées sont celles des auteurs et ne doivent pas être interprétées comme officiel ou représentant les opinions du ministère américain de la Défense ou du ministère de l&#39;Armée.

Les protocoles présentés se composait de deux parties principales: la construction du VIH-1 clones moléculaires recombinantes et des essais de compétition de croissance. Afin de distinguer les deux virus dans une culture de cellules doublement infectées, il est important que les clones moléculaires concurrentes contiennent des marqueurs de séquences, qui peuvent être détectés par un dosage amorce-sonde RT-qPCR ou par séquençage de Sanger. Ce protocole utilise les Vifa et VifB étiquettes, qui occupent la même région de HIV-1 NL4-3 vif et codent pour la séquence d&#39;acides aminés identique, mais diffèrent par six mutations synonymes. Ces mutations ont été présentés pour ne pas affecter la réplication virale remise en forme 20. La méthode de clonage par PCR utilisée dans ce protocole prévoit une plus grande flexibilité dans le choix des sites de clonage, par rapport à des sites de restriction clonage basé. Cependant, l&#39;efficacité de clonage PCR diminue à mesure que la taille augmente insert. La limite de courant de la taille de l&#39;insert est 34 ~ 5 kb. Pour le clonage basé sur la PCR et du site dirigé mutagenesis, l&#39;utilisation d&#39;une ADN polymerase haute fidélité est essentiel de réduire la probabilité de mutations supplémentaires. L&#39;utilisation du nombre minimal de cycles PCR nécessaires pour obtenir des quantités suffisantes de produits est également recommandé. Dans notre expérience, nous ne détectons aucune mutation supplémentaire après le clonage et dirigée vers le site étapes de mutagenèse basées sur la PCR décrites ici. Néanmoins, les produits de PCR doivent être séquencés pour vérifier toutes les mutations indésirables. Idéalement, la région codante entière du VIH doit être reséquencé. Au moins trois points de temps échantillonnage doivent être examinées dans la phase de croissance exponentielle 9. La cinétique de croissance virales doivent d&#39;abord être établies en utilisant échantillonnage quotidien pour déterminer la période de culture et de temps d&#39;échantillonnage des points appropriés pour la compétition de croissance. Un facteur qui affecte la cinétique de croissance virale est la multiplicité d&#39;infection (MOI). Ce protocole utilise un total de 0,005 MOI pour les mono-infection et de la concurrence de la croissance, comme il a été montré pour donner plus de roRésultats buste que IAM inférieur 9. Néanmoins, IAM inférieures peuvent être utilisées pour obtenir une phase de croissance exponentielle plus si nécessaire, mais au détriment de la cohérence résultat. Ce protocole indique un rapport de 50:50 de l&#39;infection initiale, en supposant que la différence de forme physique des virus sont généralement inconnue à l&#39;avance. Cependant, l&#39;utilisation de ratios d&#39;entrée inégales sont appropriées quand il ya des données préliminaires suggérant des différences significatives dans la cinétique de la réplication virale. Dans ces cas, un rapport d&#39;infection de 70:30 est recommandé afin de permettre la détection d&#39;une grande différence de remise en forme où le virus moins en forme est mis en excès neuf. Le protocole pour déterminer la DICT 50, la cinétique de croissance virales, et pour effectuer la croissance des essais de compétition ont été optimisés en utilisant le VIH-1 sous-type B, NL4-3 COTB-p24, et PBMC provenant d&#39;un seul donneur dont CMSP ont démontré la sensibilité cohérente pour le VIH une infection in vitro. La période de cultureet les points de temps d&#39;échantillonnage présentés dans ce protocole sont susceptibles d&#39;être adapté à l&#39;étude des virus VIH-1 groupe M dans les CMSP humaines. Utilisation CMSP d&#39;une source unique est fortement recommandé d&#39;obtenir des résultats cohérents que la réplication virale peut varier dans différentes cellules du donneur 35. Bien que moins souhaitable, des PBMC mises en commun provenant de plusieurs donneurs peuvent être utilisés comme une substitution à condition que le même pool est utilisé dans toutes les expériences. Une autre alternative est l&#39;utilisation de lignées cellulaires. Le protocole présenté ici a été utilisé avec succès par la lignée de cellules T CEMx174 23,36. Cependant, il est important que le nombre de cellules ensemencées sont ré-optimisées pour atteindre une croissance cellulaire cohérente. La cinétique de croissance virales doivent être rétablis, car il est susceptible de varier dans différentes lignées cellulaires, afin de déterminer les points de temps d&#39;échantillonnage appropriés dans les étapes de compétition de croissance. Deux méthodes différentes pour déterminer le rapport virale pour le calcul de remise en forme sont inclus dans le protocol. La première utilise RT-qPCR pour mesurer virale nombre de copies d&#39;ADNc à chaque point de temps d&#39;échantillonnage. La réplication virale remise en forme a ensuite été calculée à partir du rapport virale, sur la base de l&#39;ADNc nombre de copies. En variante, le rapport viral peut être déterminée sur la base du rapport de la hauteur de pic de chromatogramme sur les sites d&#39;étiquette VifAB. Les deux méthodes ont donné des résultats comparables (Figure 4). Le procédé de la hauteur de pic chromatogramme peut être appliquée à d&#39;autres souches du VIH-1 sans un marqueur de séquence d&#39;ingénierie. Pour RT-qPCR, l&#39;utilisation des amorces ou des sondes pour distinguer les variants viraux doit d&#39;abord être évaluée avec soin (voir 20). Néanmoins, RT-qPCR fournit une meilleure sensibilité pour des échantillons contenant une petite quantité de l&#39;ARN viral, telles que celles du premier point de temps à l&#39;intérieur de la phase de croissance exponentielle. La mesure directe des ADNc viral permet également de détecter les problèmes techniques qui peuvent survenir de l&#39;extraction de l&#39;ARN et la synthèse d&#39;ADNc. Utilisation de clones moléculaires avec des étiquettes de séquence fournit une solution rentable to les méthodes de RT-qPCR, car seuls deux couples d&#39;amorces et sondes sont nécessaires pour étudier les virus multiples. Cette stratégie évite également le problème de la variation en hauteur de pic séquence chromatogrammes en raison de bases voisines 37, comme le séquençage est réalisé sur le même site à travers tous les virus dans l&#39;étude. Les produits de PCR obtenus pour la RT-PCR quantitative et séquençage de Sanger peut également être utilisé avec d&#39;autres méthodes pour déterminer le rapport viral tel que le séquençage des clones individuels en vrac de 12,13, 4,7,17,19 HTA, ou le dosage de ligation d&#39;oligonucléotides (OLA) 38 .

La réplication virale remise en forme est définie comme la capacité d&#39;un virus à produire une descendance infectieuse dans un environnement donné 1 et est un facteur important dans la détermination de la prévalence d&#39;une variante du virus au niveau de la population au fil du temps 2. Comme les études in vivo de remise en forme ne sont pas réalisables avec des virus pathogènes humains, telles que le VIH-1, divers in vitro et ex vivo tests de conditionnement physique de réplication ont été développés pour étudier les effets sur la condition physique découlant de la résistance aux médicaments et des mutations d&#39;échappement immunitaire, épistasie et de l&#39;évolution des populations virales 3-6. Parmi les différents tests de conditionnement physique, des essais de compétition de croissance sont reconnus pour obtenir des mesures plus sensibles et valides de différences de forme physique, comme deux ou plusieurs variantes virales en compétition pour la même population cellulaire exactement dans les mêmes conditions environnementales, comme cela se produit in vivo 1,7,8. Avant de commencer des expériences de compétition de croissance, plusieurs variables besoin d&#39;être déterminée, y compris l&#39;utilisation de différentes multiplicités d&#39;infection (MOI), le ratio d&#39;entrée virale, et le calendrier de l&#39;échantillonnage pour analyse. Nous avons étudié les effets de ces paramètres sur la cinétique de croissance virales et sur ​​les résultats des expériences de compétition, et nous avons identifié des facteurs clés nécessaires pour les mesures robustes de VIH-1 de remise en forme dans la culture de cellules 9. En plus des variables d&#39;analyse, il existe une variété de méthodes pour la quantification de variants viraux dans des expériences de compétition de croissance. Bulk 10,11 ou 12,13 clonale séquençage a été utilisé pour déterminer le rapport des virus concurrentes sur la base des fréquences de nucleotides au niveau du site (s) d&#39;intérêt. Remise en forme relative est dérivé de variations de ce ratio au fil du temps. Cette méthode est pratique que des services de séquençage d&#39;ADN sont largement disponibles. Le séquençage spécifique d&#39;allèle parallèle méthode (PASS) permet le séquençage à des sites multiples et la détection de recombinants 14 </sup&gt;, Mais elle exige également des réactifs développés spécifiquement et des systèmes de détection. Essentiellement, ces méthodes ont été développées pour étudier les souches virales avec un petit nombre de différences de nucleotides dans une région d&#39;intérêt. D&#39;autres méthodes utilisent une petite gène rapporteur 15 ou synonymes mutations 4,16-18 que des étiquettes pour distinguer les virus concurrents par séquençage, tests de suivi hétéroduplex (ETS) 4,7,17,19 ou inverser PCR quantitative transcription quantitative (RT-qPCR) 15,16,18,20, qui peuvent tous être rendue applicable à étudier souches concurrence indépendamment de similarité de séquence. Une étape supplémentaire est nécessaire pour introduire les mots-clés dans les génomes viraux et le dosage par RT-PCR quantitative nécessite également des réactifs et des instruments spécifiques. Nous avons constaté que vrac séquençage Sanger donne des résultats comparables 9. Suite à la concurrence de la croissance, la réplication virale remise en forme est présentée comme remise en forme relative, ou un rapport de remise en forme entre two variants viraux. L&#39;aptitude relative d&#39;un virus peut être défini comme la proportion finale d&#39;une variante virale normalisée par sa proportion dans l&#39;inoculum initial ou à la différence nette du taux de croissance entre les deux virus concurrentes. Nous avons constaté que cette dernière méthode, en utilisant les points de données longitudinales que dans la phase exponentielle de croissance, a donné les résultats les plus robustes 9,20. Les essais in vitro de remise en forme sont utilisés principalement pour étudier clones biologiques 6-8 et clones moléculaires infectieux du VIH-1. Ce dernier, se prêter à la manipulation génétique, sont souvent employés pour étudier l&#39;effet sur ​​l&#39;aptitude de certaines mutations ou des séquences spécifiques d&#39;intérêt 3-5,21,22. Les protocoles suivants décrivent un flux de travail du point de construire de nouvelles pleine longueur infectieuses VIH-1 clones moléculaires du VIH-1 en utilisant des vecteurs contenant un marqueur de séquence, l&#39;introduction de mutations d&#39;intérêt, faisant stocks viraux et d&#39;établir la cinétique de croissance virales, De réaliser le dosage de la concurrence de la croissance et de calcul physique relative (Figure 1). Grâce à nos procédures optimisées, nous avons créé trois mutants recombinants du VIH-1 et déterminé leur aptitude de réplication. Le clone moléculaire recombinant a été construit en remplaçant la région des gènes du VIH-1 gag-p24 de pNL4-3, un plasmide contenant une longueur génome complet infectieuse du VIH-1 souche de laboratoire NL4-3, avec un COTB synthétique (Centre-of Arbre, sous-type B) séquence gag-p24 23 pour créer la souche prototype. Acides changements simples (T186M, T242N et I256V) ont ensuite été introduits pour créer trois clones mutants. Chaque mutant a été disputé contre le virus prototype d&#39;observer l&#39;impact de fitness de chaque mutation dans le fond génétique donné. Les trois mutants ont démontré des niveaux de réplication fsitness variant de légère à significativement plus faible que le virus prototype. La mutation a été précédemment rapporté T242N d&#39;avoir un Fitne modéréess coûte 24 à 26, similaire au résultat montré dans cette étude. Le coût de la remise en forme des deux autres mutations avait pas été signalé précédemment.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name of Material/ Equipment</td>
			<td style="width:484px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:196px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Construction of recombinant clones</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Chimeric primer/Mutagenic/Sequencing primers</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom DNA oligos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pNL4-3VifA and pNL4-3VifB plasmid</td>
			<td style="width:484px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-Fidelity DNA polymerase</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-fidelity buffer</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems&#174; GeneAmp&#174; PCR System 9700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA loading dye</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td style="width:137px;">R0631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1kb Plus DNA ladder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAquick PCR Purification Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick gel extraction kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">DpnI enzyme</td>
			<td style="width:484px;">New England Biolabs</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">TOP10 chemically competent Escherichia coli</td>
			<td style="width:484px;">Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Luria Broth base powder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">12795-084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Carbenicillin</td>
			<td style="width:484px;">Research Products International</td>
			<td style="width:137px;">C46000-25.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep kit&#160;</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">27104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">12362</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">X-tremeGENE 9 DNA transfection reagent</td>
			<td style="width:484px;">Roche</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HEK 293T-17</td>
			<td style="width:484px;">ATCC</td>
			<td style="width:137px;">CRL-11268</td>
			<td>http://www.atcc.org/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.22um filter top tube</td>
			<td style="width:484px;">VWR International</td>
			<td>89220-716&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#8242;s Modified Dulbecco&#8242;s Medium (IMDM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>31980-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RPMI-1640 media</td>
			<td style="width:484px;">Life Technologies</td>
			<td>61870-036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phytohemagglutinin (PHA)</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>R30852801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Human Interleukin-2</td>
			<td style="width:484px;">Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum</td>
			<td style="width:484px;">JR Scientific</td>
			<td style="width:137px;">43640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicillin and Streptomycin</td>
			<td style="width:484px;">Corning Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">73520-906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">62407-338</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat-bottomed plate</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td style="width:137px;">T-3015-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well round-bottomed plate</td>
			<td style="width:484px;">BD Falcon</td>
			<td style="width:137px;">353077</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 ml Microcentrifuge Tube</td>
			<td style="width:484px;">Mt. Baker Bio</td>
			<td style="width:137px;">MBD-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 mL tubes w/ O-ring</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">89004-290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">50 mL conical tube</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td>C-3317-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Triton-X 100</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phosphate buffer saline (PBS)</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum (FBS)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>F0392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">glycerol</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td style="width:137px;">G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tween 20</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-HIV-1 SF2 p24 antiserum</td>
			<td style="width:484px;">NIH AIDS Reagent Program</td>
			<td style="width:137px;">4250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit HRP</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">474-1516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMB Microwell Peroxidase Substrate</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">52-00-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">H2SO4</td>
			<td>Fisher Scientific</td>
			<td>A300-500</td>
			<td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 minutes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HIV p24 standard</td>
			<td style="width:484px;">AIDS and Cancer Virus program (NCI-Frederick)</td>
			<td style="width:137px;">N/A</td>
			<td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microplate reader</td>
			<td style="width:484px;">Molecular Devices</td>
			<td style="width:137px;">N/A</td>
			<td>VMax Kinetic ELISA Microplate Reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA isolation cDNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxtractor</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">N/A</td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAamp Viral RNA Mini Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">52906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">SuperscriptIII</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">First-strand buffer&#160;</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Rnase inhibitor</td>
			<td style="width:484px;">Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RnaseH</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18021-071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Real-time qPCR machine</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems 7300 Real-Time PCR&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan&#174; Gene Expression Master Mix</td>
			<td style="width:484px;">Life Technologies</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Forward, reverse primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Probe</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">optical 96 well reaction plate</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">I19N3Q216</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR+sequencing</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Taq DNA polymerase (Biolase)</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NH4 buffer&#160;</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Sequencing primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxcel</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;"></td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA sequencing service provider</td>
			<td>http://www.htseq.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GRC web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/grc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChromatQuan web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Pour étudier l&#39;impact de remise en forme des changements d&#39;acides aminés uniques dans le VIH-1 Gag p24, nous avons utilisé oligonucléotide mutagenèse dirigée pour introduire des mutations dans un plasmide contenant pNL4-3 du VIH-1 COTB-p24 gène 23,32,33. Les stocks viraux ont été générés par transfection de cellules 293T et récoltées après 48 heures. Nous avons estimé la dose de 50% de la culture de tissus infectieux (DICT 50) de chaque stock virale par la méthode de Reed-Muench 31. La TCID50 du virus mutants prototype et variaient entre 10 avril à 10 mai UI / ml (Figure 3A). La cinétique de virus recombinants de croissance ont été établis dans les CMSP à partir d&#39;un seul donneur à une MOI de 0,005. RT-qPCR a été utilisé pour mesurer virale nombre de copies d&#39;ADNc par jour pendant six jours. Tous les virus mutants et prototypes ont augmenté de façon exponentielle entre le jour 2 et le jour 4, après quoi a ralenti la croissance virale, comme indiqué par une diminution de la pente de l&#39;augmentation du nombre de copies virales d&#39;ARN (&lt;strong&gt; figure 3B). La diminution du nombre de copies d&#39;ADNc entre le jour 0 (correspondant à l&#39;inoculum) et jour 2 est due à l&#39;absorption du virus à des cellules et l&#39;élimination des virions non liés par lavage le jour 1 (étape 5.1.6). Dans la phase de croissance exponentielle, les trois mutants ont un taux de croissance plus lent (g) que le virus prototype (figure 3C). Tous les trois mutants ont été en compétition contre le virus prototype dans des tests de compétition de croissance à un total de 0,005 MOI. La cinétique de la croissance virale dans des cultures doublement infectées étaient similaires à celle de la mono-infection; la phase exponentielle de croissance virale était entre les jours 2 et 4, et de la croissance virale a atteint un plateau autour de cinq jours (figure 4A). Les différences de taux de croissance virales ont été obtenues à partir de la variation du rapport viral dans le temps. Le rapport viral a été calculé sur la base du nombre de copies d&#39;ADNc de la référence et les virus mutants à l&#39;aide de RT-qPCR, et en comparant les hauteurs de picchromatogrammes en séquence au niveau de sites de nucleotides qui distinguent les deux virus (figure 4B). Les différences de taux de croissance déterminées en utilisant le pic-hauteur et copie de l&#39;ARN viraux méthodes numériques ont donné des résultats similaires (figure 4C). Tous les trois mutants avaient inférieure remise en forme de réplication que les virus des prototypes avec le I256V mutant ayant l&#39;aptitude la plus faible (figure 4C). <img alt="Figure 1" src="/files/ftp_upload/52610/52610fig1.jpg" /> Figure 1: Schéma de principe des protocoles présentés dans le présent document Le Virus protocoles de préparation concernent la construction du VIH-1 clones recombinants et la génération de stocks viraux.. Les protocoles de remise en forme dosages sont pour établir la cinétique de croissance virales et déterminer l&#39;aptitude virale relative. Les lignes pointillées représentent les flux alternatifs pour les protocoles. Par exemple, une mutation peut être introduite directement dans le VIH-1 NL4-3clone moléculaire sans générer un clone recombinant. <img alt="Figure 2" src="/files/ftp_upload/52610/52610fig2.jpg" /> Figure 2:. Construction du VIH-1 NL4-3 COTB Gag p24-clones moléculaires recombinantes en utilisant l&#39;extension de PCR de chevauchement (A) Concevoir les amorces chimériques. La 5 &#39;moitiés des amorces contiennent la séquence du vecteur NL4-3 et 3&#39; contiennent des moitiés des extrémités de la séquence de l&#39;insert. (B) amorces chimériques sont utilisés pour amplifier le fragment d&#39;insertion, COTB Gag p24. (C) Les fragments d&#39;insertion sont utilisés comme amorces pour une seconde PCR pour générer un nouveau plasmide recombinant. <img alt="Figure 3" src="/files/ftp_upload/52610/52610fig3.jpg" /> Figure 3:. Caractéristiques de croissance virale dans les CMSP (A) Connexion 10 DICT 50 </sub&gt; des stocks viraux. (B) de la cinétique de la croissance virale dans monoinfections commencé à un MOI = 0,005. (c) Les taux de croissance virale de plus de six jours, y compris la phase exponentielle de croissance (jours 2-4) dérivé de panneau (B). Les valeurs indiquées représentent la moyenne de trois répétitions d&#39;une expérience. Les barres d&#39;erreur représentent les intervalles de 95% de confiance. <img alt="Figure 4" src="/files/ftp_upload/52610/52610fig4.jpg" /> Figure 4: croissance virale et de conditionnement physique par rapport déterminations (A) de la croissance virale dans les cultures de CMSP doublement infectés.. (B) Les ratios de virus T242N et prototypes déterminés par RT-qPCR ou la méthode de séquençage de pointe hauteur. (C) des différences nettes virales du taux de croissance (d) entre le mutant et les virus dans des cultures de prototypes doublement infectées, comme représenté en (A). d valeurs ont été calculées fROM Le viraux données de rapport indiqués dans (B). Les valeurs indiquées représentent la moyenne de trois répétitions d&#39;une expérience. Les barres d&#39;erreur représentent les intervalles de 95% de confiance.

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Pairwise croissance de la concurrence analyse pour déterminer la condition physique réplication du virus de l&#39;immunodéficience humaines

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

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JoVE Journal

Immunology and Infection

11.0K vues.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Vidéo: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Détermination de la réactivité et le titre du sérum en utilisant un dosage d&#39;hémagglutination

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

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Immunologie et infection

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Rentable méthode de suivi des sources microbiennes spécifiques utilisant des virus humains et animaux

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Détermination de l&#39;activité cytotoxique CD8 spécifiques optimale des cellules T humaines Her2neu en comparant les Cr51 Essai de libération dans le système de xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

Déterminer transmis par le sol Statut helminthiase et la condition physique des enfants d&#39;âge scolaire

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Un test visuel pour surveiller T6SS à médiation bactérienne concurrence

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

L&#39;établissement d&#39;une culture liquide recouvert de Airway humaines épithéliales polarisées cellules Calu-3 pour étudier la réponse cellulaire aux agents pathogènes respiratoires hôte<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

Une enzyme de restriction basée sur le clonage méthode pour évaluer la<em&gt; In vitro</em&gt; Capacité de réplication du VIH-1 sous-type C Gag-MJ4 chimériques virus

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Administration intranasale de vaccins contre la grippe recombinant chimérique dans les modèles de souris pour étudier l&#39;immunité muqueuse

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basées sur la fluorescence neuraminidase Essai d&#39;inhibition pour évaluer la sensibilité des virus de la grippe à la neuraminidase Inhibiteur Classe de Antiviraux

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.