The authors would like to thank Dr. James (Zhijian) Chen (UT Southwestern, Molecular Biology) for providing essential reagents, including the Mavs-/- mice, and Dr. Ren Sun (University of California-Los Angeles, Pharmacology and Molecular Medicine) for providing the bacterial artificial chromosome of &gamma;HV68 for this study.

1. Mouse infection with &gamma;HV68
<ol>
	<li>Six-to-eight-week old, gender-matched littermate mice (8 to 12 mice/group) are used for viral infection. Allow mice to acclimate over four full days (96 hours) after shipment.</li>
	<li>Protocol steps using virus should be carried out in a cabinet of biosafety level 2 (BSL2) using standard BSL2 precautions.</li>
	<li>Prepare viral suspension (40 to 1 x 105 plaque-forming units [PFU]) of &gamma;HV68 in 30 &mu;l of sterile PBS per mouse just before the experiment. Keep viral suspension on ice.</li>
	<li>Prepare ketamine/xylazine solution (1.5 mg ketamine and 0.15 mg xylazine/20 g body weight, 100 &mu;l/mouse) for mouse sedation. Ensure that the mice have been sedated by performing a toe pinch.</li>
	<li>Sedate mice with 1.5 mg ketamine and 0.15 mg xylazine per 20 mg body weight (100 &mu;l/mouse) by intra-peritoneal injection.</li>
	<li>Deliver viral suspension (30 &mu;l/mouse) intranasally, in a drop-wise fashion, to one nostril of the sedated mice.</li>
	<li>Lay mice on one side for 5 - 10 min to facilitate the airway delivery of the virus into the lung.</li>
	<li>Place mice back in cage and monitor mice until they have fully recovered from sedation.</li>
	<li>At various days post-infection, sacrifice mice by CO2 asphyxiation and harvest the lungs after assuring death/loss of deep consciousness. Place lungs into sterile 1.5 ml screw-capped tubes containing 500 &mu;l of 1.0 mm Zirconia/Silica beads. Keep tubes on ice. Store samples at -80&deg;C if not proceeding to next step on the same day. The spleen or liver tissue could be collected at this time for the analysis of viral latency depending on time frame of experiment.</li>
	<li>Add 1 ml of cold serum-free DMEM into the tube and homogenize the lungs by bead-beating 30 seconds. Chill tubes on ice for at least 1 min. Repeat this process once.</li>
	<li>Centrifuge lung lysates at 16,000 rcf at 4&deg;C for 1 min and use supernatant to determine viral titer by a plaque assay using a NIH3T3 or BHK21 monolayer (see Section 5 for details).</li>
</ol>
2. Determine &gamma;HV68 multi-step growth kinetics in mouse embryonic fibroblasts
<ol>
	<li>Grow wild-type mouse embryonic fibroblasts (MEFs) and those deficient in a host gene to sub-confluent (approximately 80%) density before plating cells.</li>
	<li>Split MEFs into 24-well plate at 10,000 cells/well for a low multipilicity-of-infection (MOI) on the day before infection. Experiments are normally carried out in triplicates and at a MOI between 0.001 - 0.05 is usually used.</li>
	<li>Prepare &gamma;HV68 suspension containing desired amount of virus (0.5 ml per well).</li>
	<li>Remove medium and cover MEFs with 0.5 ml of &gamma;HV68 suspension per well.</li>
	<li>Incubate the plate in tissue culture incubator, rock every 30 min, and allow incubation to proceed for 2 h.</li>
	<li>Remove viral suspension, and cover cells with 0.5 ml fresh complete DMEM medium containing 8% fetal bovine serum.</li>
	<li>At various days post-infection, harvest the medium and cells into sterile 1.5 ml centrifuge tubes. Immediately freeze tubes at -80&deg;C.</li>
	<li>Release &gamma;HV68 from MEFs by freezing at -80&deg;C , thawing in 37&deg;C water bath and vortexing. Three cycles of freezing and thawing is usually applied to the samples.</li>
	<li>Determine viral titer by a plaque assay using a NIH3T3 or BHK21 monolayer (see Section 5 for details).</li>
	<li>Read viral titer and plot &gamma;HV68 multi-step growth curve on MEFs.</li>
</ol>
3. Molecular dissection of &gamma;HV68 lytic replication in mouse embryonic fibroblasts
<ol>
	<li>Perform viral infection as described in steps 2.1 to 2.6 of section 2.</li>
	<li>At various days post-infection, discard the supernatant. Rinse cells with cold PBS and trypsinize cells. Pellet cells by centrifuge at 1,000 rcf at room temperature for 1 min. Discard the supernatant and store cells at -80&deg;C.</li>
	<li>Extract total DNA (host and viral genome) and total RNA according to previously published methods5,6.</li>
	<li>Perform real-time PCR using total DNA and primers specific for viral lytic transcripts, such as RTA (ORF50), ORF57 and ORF60. Determine the relative quantity of intracellular &gamma;HV68 genome in reference to a host housekeeping gene (e.g., &beta;-actin).</li>
	<li>To remove genomic DNA contamination, treatment with RNase-free DNase is critical for cDNA preparation with total RNA and oligo(dT)11-19 primer. Refer to reference 5 for more details on the RNA extraction including treatment with RNase-free DNase and RT-PCR. Perform real-time PCR using cDNA and primers as above to determine the relative quantity of viral transcripts in reference to that of host housekeeping gene.</li>
</ol>
4. Generating recombinant &gamma;HV68 using bacterial artificial chromosome
The method described here is used to introduce mutations into a &gamma;HV68 gene that is involved in host-virus interaction.
<ol>
	<li>Prepare a DNA fragment (about 1.5 kb) of wild-type sequence or sequence carrying desired mutations in the central region by PCR.</li>
	<li>Prepare bacterial artificial chromosome (BAC) that contains a transposon insertion7 around the mutation site, which specifically inactivates the gene gene of interest, by midi-scale purification (OriGene). Store BAC DNA at 4&deg;C (avoid freezing/thawing of BAC DNA).</li>
	<li>Transfect BAC DNA and PCR product containing desired mutations of the gene of interest into cells (e.g., NIH3T3 or BHK21), which are highly permissive to &gamma;HV68 lytic replication, with Lipofectamine 2000 (Invitrogen).</li>
	<li>Keep splitting cells until cytopathic effect (CPE) shows up in the monolayer. Collect virus-containing supernatant and, if necessary, amplify virus in NIH3T3 or BHK21 cells.</li>
	<li>Infect NIH3T3 cells with recombinant virus by centrifugation at 1,800 rpm, 30&deg;C for 30 min.</li>
	<li>Harvest &gamma;HV68-infected NIH3T3 cells and prepare circularized viral genome using Hirt's protocol8,9.</li>
	<li>Transform Electro-MAX DH10B competent cells (Invitrogen) by electroporation and screen for colonies that are resistance to chloramphenicol (Cm), but sensitive to kanamycin (Kan) (Cm-resistant gene is in BAC backbone, while Kan-resistant gene is in transposon insertion).</li>
	<li>Grow CmRKanS colonies in medium containing chlorophenicol and prepare BAC DNA with mini- or midi-scale purification (OriGene).</li>
	<li>Verify the desired mutation in the target gene by PCR, using primers specific to flanking regions of mutation site, and sequencing.</li>
	<li>Confirm no chromosome rearrangement in BAC by digestion with selected restriction enzymes and pulse-field gel electrophoresis.</li>
	<li>Transfect recombinant BAC into NIH 3T3 or BHK21 cells with Lipofectamine 2000 (Invitrogen) to prepare recombinant &gamma;HV68.</li>
	<li>Keep passaging cells until CPE shows up in the monolayer. Collect virus-containing supernatant and amplify recombinant &gamma;HV68 in NIH3T3 or BHK21 cells.</li>
	<li>Determine titer of the recombinant virus and characterize viral growth properties ex vivo and in vivo as described in sections 1 and 2.</li>
</ol>
5. Determine viral titer by a plaque assay
<ol>
	<li>Grow NIH3T3 cells to sub-confluent (approximately 80%) density before plating cells.</li>
	<li>Split NIH3T3 into 24-well plate at 20,000 cells/well on the day before infection.</li>
	<li>Prepare 10-fold serially-diluted virus supernatants with DMEM medium containing 8% newborn calf serum (NCS).</li>
	<li>Remove medium and cover NIH3T3 cells with 0.5 ml of &gamma;HV68 suspension.</li>
	<li>Incubate the plate in tissue culture incubator, rock every 30 min, and allow incubation to proceed for 2 h.</li>
	<li>Remove viral suspension and cover cells with 0.5 ml DMEM medium containing 2% NCS and 0.75% methylcellulose (Sigma).</li>
	<li>Incubate the plate in tissue culture incubator. Count plaques at day 6 post-infection. Staining of the monolayer, e.g. with crystal violet, may facilitate plaque counting.</li>
	<li>Calculate the viral titer in the undiluted tissue lysates or cell lysates using the following formula: Titer (PFU/ml) = D x N x 1000 &mu;l/ml &divide; V &mu;l. N, the mean of plaque number at an appropriate dilution; D, dilution fold (such as 5, 10, 100&hellip;..) V (&mu;l), volume of serially-diluted supernatant added per well.</li>
</ol>
6. Representative Results:
Three representative figures are shown here, including &gamma;HV68 lytic replication in the lung of wild-type and Mavs-/- mouse10, &gamma;HV68 lytic replication phenotypes in mouse embryonic fibroblasts (MEF), and recombinant &gamma;HV68 carrying mutations within the phosphorylation sites that are modulated by the MAVS-dependent IKK&beta;. These three corroborating experiments constitute a scheme to define the roles of innate immune components in &gamma;HV68 lytic replication in vivo and ex vivo.
<img alt="Figure 1" src="/files/ftp_upload/3140/3140fig1.jpg" /> 
Figure 1. &gamma;HV68 loads in the lungs of Mavs+/+ and Mavs-/- mice. A) Two main innate signaling pathways downstream of MAVS. The MAVS adaptor molecule relays signaling from cytosolic RIG-I-like receptors to activate NF&kappa;B and interferon regulatory factors (IRFs) that, in turn, up-regulate the gene expression of proinflammatory cytokines and interferons. B) Mavs+/+ and Mavs-/- mice were infected with 40 PFU &gamma;HV68 intranasally and viral loads in the lung at indicated time points were determined by a plaque assay using NIH3T3 monolayer. Each symbol represents a mouse.
<img alt="Figure 2" src="/files/ftp_upload/3140/3140fig2.jpg" /> 
Figure 2. &gamma;HV68 lytic replication kinetics in mouse embryonic fibroblasts (MEFs). The lytic replication of &gamma;HV68 on Mavs+/+ and Mavs-/- MEFs was assessed by multi-step growth curves (A) and quantitative real-time PCR (B). For both experiments, equal number of MEFs and amount of &gamma;HV68 were used for viral infection at a multiplicity-of-infection (MOI) of 0.01. (A) Cells and supernatants were harvested at indicated time points and subject to a plaque assay to determine viral titers. (B) Total RNA was extracted from &gamma;HV68-infected MEFs and analyzed by quantitative real-time PCR with primers specific for selected lytic transcripts (RTA, ORF57, and ORF60).
<img alt="Figure 3" src="/files/ftp_upload/3140/3140fig3.jpg" /> 
Figure 3. The lytic replication kinetics of recombinant &gamma;HV68 carrying mutations within the RTA transactivation domain that abolish phosphorylation by IKK&gamma;. (A) Multi-step growth curves of recombinant wild-type virus (&gamma;HV68.wt) and mutant virus (&gamma;HV68.mut) in Mavs+/+ and Mavs-/- MEFs cells (MOI=0.01). MEFs were infected with &gamma;HV68 at an MOI of 0.01. Cells and supernatants were harvested at indicated time points and viral titers were determined by a plaque assay using NIH 3T3 monolayer. (B) &gamma;HV68 RTA mRNA level in &gamma;HV68-infected NIH3T3 cells (MOI=0.01). At 30 h post-infection, total RNA was extracted from &gamma;HV68-infected NIH 3T3 cells and analyzed by quantitative real-time PCR.

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+microorganisms+and+activation+of+the+immune+response.">Recognition of microorganisms and activation of the immune response.</a> Nature. 449, 819-826 (2007).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Speck, S. H., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+latency+and+its+regulation:+lessons+from+the+gamma-herpesviruses.">Viral latency and its regulation: lessons from the gamma-herpesviruses.</a> Cell Host Microbe. 8, 100-115 (2010).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. 6, e1001001-e1001001 (2010).</li><li>Strauss, W. M., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+genomic+DNA+from+mammalian+tissues.">Preparation of genomic DNA from mammalian tissues.</a> Current Protocols in Molecular Biology. , 2-2 (1998).</li><li>Song, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+viral+genes+essential+for+replication+of+murine+gamma-herpesvirus+68+using+signature-tagged+mutagenesis.">Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 3805-3810 (2005).</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J. Mol. Biol. 26, 365-369 (1967).</li><li>Eva-Maria Borst, E., Crnkovic-Mertens, I., Messerle, M., Zhao, S., Stodolsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+&#946;-herpesvirus+genomes+as+bacterial+artificial+chromosomes.">Cloning of &#946;-herpesvirus genomes as bacterial artificial chromosomes.</a> Methods in Molecular Biology. , 256-256 (2004).</li><li>Sun, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Meylan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Xu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li></ol>

No conflicts of interest declared.

In response to viral infection, the MAVS-dependent innate immune signaling pathways are activated to promote the production of antiviral inflammatory cytokines10-14. Using murine &gamma;HV68 as a model virus for human oncogenic Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus3,4, we discovered that &gamma;HV68 usurps the MAVS-IKK&beta; pathway to promote viral lytic replication via transcriptional activation5. Employing genetically modified MEFs and techniques in molecular v...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Electro-MAX DH10B competent cells</td>
			<td>Invitrogen</td>
			<td>18290-015</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>M0512</td>
		</tr>
		<tr>
			<td>POWERPREP HP Plasmid Miniprep System</td>
			<td>OriGene</td>
			<td>NP100004</td>
		</tr>
		<tr>
			<td>POWERPREP HP Plasmid Midiprep System</td>
			<td>OriGene</td>
			<td>NP100006</td>
		</tr>
	</tbody>
</table>

dissecting host-virus interaction in lytic replication of a model herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

dissecting-host-virus-interaction-lytic-replication-model

Research

JoVE Journal

Immunology and Infection

10.8K 조회수.  UT Southwestern Medical Center. 우리는 모델 herpesvirus, 감마 herpesvirus 68 (γHV68)의 lytic 복제에 호스트 신호 분자의 핵심 역할을 식별 할 수있는 프로토콜을 설명합니다. 유전자 변형 마우스 긴장과 γHV68 lytic 복제 배아 섬유 아세포를 활용, 프로토콜 phenotypic 특성화 및 바이러스 lytic 복제에서 바이러스 호스트 상호 작용의 분자 심문을 모두 할 수 있습니다.

비디오: Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.