The authors would like to acknowledge the technical advice and support from Ren Sun (University of California, Los Angeles) and Seungmin Hwang (Washington University). This work was funded by the Baxter Foundation, National Institutes of Health grants (R01 CA140964 and R21 AI083841 to C. Liang).

The following protocol will describe the study of virus titers and viral genome loads in the lytic and latent infection cycle of &gamma;HV68 in mice, which can theoretically be used to evaluate infection by other viruses sharing similar lifestyle to that of &gamma;HV68.
1. Amplification of &gamma;HV68
<ol>
	<li>Thaw a frozen vial of &gamma;HV68 in 37 &deg;C water bath.</li>
	<li>Add viral inoculums to NIH3T12 or 3T3 cell culture (~50% confluency) in 10 cm dishes and culture the infected cell...

<ol>
	<li>Damania, B., Choi, J. K., Jung, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+activities+of+gammaherpesvirus+membrane+proteins.">Signaling activities of gammaherpesvirus membrane proteins.</a> J. Virol. 74, 1593-1601 (2000).</li><li>Stevenson, P. G., Efstathiou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+mechanisms+in+murine+gammaherpesvirus-68+infection.">Immune mechanisms in murine gammaherpesvirus-68 infection.</a> Viral. Immunol. 18, 445-456 (2005).</li><li>Flano, E., Husain, S. M., Sample, J. T., Woodland, D. L., Blackman, 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=Latent+murine+gamma-herpesvirus+infection+is+established+in+activated+B+cells,+dendritic+cells,+and+macrophages.">Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages.</a> J. Immunol. 165, 1074-1081 (2000).</li><li>Sunil-Chandra, N. P., Efstathiou, S., Nash, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gammaherpesvirus+68+establishes+a+latent+infection+in+mouse+B+lymphocytes+in+vivo.">Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo.</a> J. Gen. Virol. 73, 3275-3279 (1992).</li><li>Simas, J. P., Swann, D., Bowden, R., Efstathiou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+murine+gammaherpesvirus-68+transcription+during+lytic+and+latent+infection.">Analysis of murine gammaherpesvirus-68 transcription during lytic and latent infection.</a> J. Gen. Virol. 80, 75-82 (1999).</li><li>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=KSHV+and+the+pathogenesis+of+Kaposi+sarcoma:+listening+to+human+biology+and+medicine.">KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine.</a> J. Clin. Invest. 120, 939-949 (2010).</li><li>Wen, K. W., Damania, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi+sarcoma-associated+herpesvirus+(KSHV):+molecular+biology+and+oncogenesis.">Kaposi sarcoma-associated herpesvirus (KSHV): molecular biology and oncogenesis.</a> Cancer. Lett. 289, 140-150 (2009).</li><li>E, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Bcl-2-mediated+evasion+of+autophagy+aids+chronic+infection+of+gammaherpesvirus+68.">Viral Bcl-2-mediated evasion of autophagy aids chronic infection of gammaherpesvirus 68.</a> PLoS. Pathog. 5, e1000609-e1000609 (2009).</li><li>Marques, S., Efstathiou, S., Smith, K. G., Haury, M., Simas, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+gene+expression+of+latent+murine+gammaherpesvirus+68+in+B+lymphocytes.">Selective gene expression of latent murine gammaherpesvirus 68 in B lymphocytes.</a> J. Virol. 77, 7308-7318 (2003).</li><li>McCausland, M. M., Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+PCR+technique+for+detecting+lymphocytic+choriomeningitis+virus+in+vivo.">Quantitative PCR technique for detecting lymphocytic choriomeningitis virus in vivo.</a> J. Virol. Methods. 147, 167-176 (2008).</li><li>Weck, K. E., Kim, S. S., Virgin, H. I., Speck, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+regulate+murine+gammaherpesvirus+68+latency.">B cells regulate murine gammaherpesvirus 68 latency.</a> J. Virol. 73, 4651-4661 (1999).</li><li>Weck, K. E., Kim, S. S., Virgin, H. I., Speck, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+are+the+major+reservoir+of+latent+murine+gammaherpesvirus+68+in+peritoneal+cells.">Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells.</a> J. Virol. 73, 3273-3283 (1999).</li></ol>

No conflicts of interest declared.

&gamma;HV68 has been widely used as a model to understand the pathogenesis of human &gamma;-HVs2,4,5. In this protocol, we described three routinely used methods, including plaque assay for infectious virus titer, IC assay for viral latent load, and qPCR for viral genome load, to evaluate the acute and latent infection of &gamma;HV68 after intranasal inoculation in mice.
The plaque assay has been used extensively to determine the virus titer in infected cells or tissues, but the op...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Preparation Procedure</td>
		</tr>
		<tr>
			<td>Methylcellulose (MC) overlay medium for viral plaque assay</td>
			<td><ol>
			<li>Heat ~ 250 ml of distilled water in TC bottle to &gt; 80&deg;C (boiling for 3 min in microwave)</li>
			<li>Add gradually 2.5 g MC powder into heated water, stirring over low heat until dissolved.</li>
			<li>Autoclave immediately for 15 min at liquid cycle.</li>
			<li>Stirring the autoclaved MC media at 4&deg;C overnight.</li>
			<li>Combine 250 ml MC media with 200ml 2 x MEM (Gibco-BRL) + 50 ml FBS to give 500 ml overlay media. </li>
			</ol></td>
		</tr>
		<tr>
			<td>Fix/stain medium for plaque assay</td>
			<td>0.2% (w/v) crystal violet in 20% ethanol.</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>NH4Cl 0.15M, KHCO3 10mM, EDTA 0.1mM</td>
		</tr>
		<tr>
			<td>Complete DMEM</td>
			<td>Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine, and 1% penicillin-streptomycin (Gibco-BRL).</td>
		</tr>
	</tbody>
</table>
Table 1. Specific media used in this protocol
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>K2753</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0512-250g</td>
		</tr>
		<tr>
			<td>2 x MEM</td>
			<td>Life Technologies</td>
			<td>11935</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Faclon</td>
			<td>352340</td>
		</tr>
		<tr>
			<td>Omni tissue homogenizer</td>
			<td>OMNI International</td>
			<td>TH115</td>
		</tr>
		<tr>
			<td>DNeasy Blood & Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
		</tr>
		<tr>
			<td>iQTM SYBRH Green Supermix</td>
			<td>BioRad</td>
			<td>170-8882</td>
		</tr>
		<tr>
			<td>CFX96 real-time PCR system</td>
			<td>Bio-Rad</td>
			<td>184-5072</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0001</td>
		</tr>
		<tr>
			<td>Sorvall SA-600</td>
			<td>Thermo Scientific</td>
			<td>096-124022</td>
		</tr>
	</tbody>
</table>
Table 2. Specific reagents and equipment

measurement of &#947;hv68 infection in mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Watch this Scientific Journal Video about Measurement of Î³HV68 Infection in Mice at JoVE.com

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Research

JoVE Journal

Immunology and Infection

13.5K Görüntüleme.  University of Southern California, Los Angeles. γ-herpesvirüslerinin (γ-HVS) ev sahibi ömür boyu kalıcılığı kurmak. Enfeksiyon γ-HV68 ile farelerin genetik çözülebilir sağlar In vivo ΓHVs ömrü / patogenezinde karakterizasyonu modeli. Bu protokol, plak oluşturan, bulaşıcı merkezi, ve qPCR deneyleri enfeksiyonu takip eden akut ve latent aşamasında γHV68 enfeksiyon tespit ve kantitatif açıklamaktadır.