Name: highly sensitive assay for measurement of arenavirus-cell attachment
Uploaded: 2016-03-02T14:00:00
Description: Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

We thank Markus Thali, Nathan Roy, Christopher Ziegler, Emily Bruce, and Benjamin King for helpful discussions and the National Institutes of Health for support of these studies through grants T32 AI055402 (JK), R21 AI088059 (JB), and P20RR021905 (JB).

Note: It is critical that the described protocol be carried out using strict pre-PCR technique (see 28 for an excellent overview on how to set up a pre-PCR laboratory and how to conduct experiments using good pre-PCR technique). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. An overview of the protocol is shown in Figure 1.
1. Prepare Media and Seed Cell...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+heterogeneity+in+the+termini+of+lymphocytic+choriomeningitis+virus+genomic+and+antigenomic+RNAs.">Sequence heterogeneity in the termini of lymphocytic choriomeningitis virus genomic and antigenomic RNAs.</a> J. Virol. 68, 7659-7664 (1994).</li><li>Haist, K., Ziegler, C., Botten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strand-Specific+Quantitative+Reverse+Transcription-Polymerase+Chain+Reaction+Assay+for+Measurement+of+Arenavirus+Genomic+and+Antigenomic+RNAs.">Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.</a> PLoS One. 10, e0120043 (2015).</li><li>Radoshitzky, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+receptor+1+is+a+cellular+receptor+for+New+World+haemorrhagic+fever+arenaviruses.">Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses.</a> Nature. 446, 92-96 (2007).</li><li>Martinez, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+human+DC-SIGN+and+L-SIGN+for+entry+and+infection+of+host+cells+by+the+New+World+arenavirus,+Junin+virus.">Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus, Junin virus.</a> Biochem. Biophys. Res. Commun. 441, 612-617 (2013).</li><li>Martinez, M. G., Cordo, S. M., Candurra, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Junin+arenavirus+cell+entry.">Characterization of Junin arenavirus cell entry.</a> J. Gen. Virol. 88, 1776-1784 (2007).</li><li>Martinez, M. G., Forlenza, M. B., Candurra, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+cellular+proteins+in+Junin+arenavirus+entry.">Involvement of cellular proteins in Junin arenavirus entry.</a> Biotechnol J. 4, 866-870 (2009).</li><li>Nunberg, J. H., York, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Curious+Case+of+Arenavirus+Entry,+and+Its+Inhibition.">The Curious Case of Arenavirus Entry, and Its Inhibition.</a> Viruses. 4, 83-101 (2012).</li><li>Botten, J., King, B., Klaus, J., Zielger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Viral Hemorrhagic Fevers. , 233-260 (2013).</li><li>Klaus, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+cargo+receptor+ERGIC-53+is+required+for+the+production+of+infectious+arenavirus,+coronavirus,+and+filovirus+particles.">The intracellular cargo receptor ERGIC-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles.</a> Cell Host Microbe. 14, 522-534 (2013).</li><li>Rojek, J. M., Perez, M., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+entry+of+lymphocytic+choriomeningitis+virus.">Cellular entry of lymphocytic choriomeningitis virus.</a> J. Virol. 82, 1505-1517 (2008).</li><li>Lavanya, M., Cuevas, C. D., Thomas, M., Cherry, S., Ross, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+screen+for+genes+that+affect+Junin+virus+entry+uncovers+voltage-gated+calcium+channels+as+a+therapeutic+target.">siRNA screen for genes that affect Junin virus entry uncovers voltage-gated calcium channels as a therapeutic target.</a> Sci Transl Med. 5, 204ra131 (2013).</li><li>Ellenberg, P., Edreira, M., Scolaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+superinfection+of+Vero+cells+persistently+infected+with+Junin+virus.">Resistance to superinfection of Vero cells persistently infected with Junin virus.</a> Arch. Virol. 149, 507-522 (2004).</li><li>Brandenburg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+poliovirus+entry+in+live+cells.">Imaging poliovirus entry in live cells.</a> PLoS Biol. 5, e183 (2007).</li><li>Petersen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Major+Cellular+Sterol+Regulatory+Pathway+Is+Required+for+Andes+Virus+Infection.">The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection.</a> PLoS pathogens. 10, e1003911 (2014).</li><li>Jonsson, N., Gullberg, M., Israelsson, S., Lindberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+studies+of+virus+interaction+at+the+host+cell+surface+using+enteroviruses+and+real-time+PCR.">A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR.</a> Virol J. 6, 217 (2009).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+attachment+mediated+by+apolipoprotein+E+binding+to+cell+surface+heparan+sulfate.">Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate.</a> J. Virol. 86, 7256-7267 (2012).</li><li>Shannon-Lowe, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-induced+B-cell+transformation:+quantitating+events+from+virus+binding+to+cell+outgrowth.">Epstein-Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth.</a> J. Gen. Virol. 86, 3009-3019 (2005).</li><li>Mifflin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+up+a+PCR+laboratory.">Setting up a PCR laboratory.</a> CSH Protoc. 2007, pdb top14 (2007).</li><li>Maiztegui, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+a+live+attenuated+vaccine+against+Argentine+hemorrhagic+fever.+AHF+Study+Group.">Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.</a> J. Infect. Dis. 177, 277-283 (1998).</li><li>Goni, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+features+of+attenuated+Junin+virus+vaccine+strain+candidate.">Genomic features of attenuated Junin virus vaccine strain candidate.</a> Virus Genes. 32, 37-41 (2006).</li><li>Chosewood, L. C., Wilson, D. 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> Biosafety in microbiological and biomedical laboratories. , (2009).</li><li>White, J., Matlin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fusion+by+Semliki+Forest,+influenza,+and+vesicular+stomatitis+viruses.">Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses.</a> J. Cell Biol. 89, 674-679 (1981).</li><li>McGraw, T. E., Greenfield, L., Maxfield, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+the+human+transferrin+receptor+cDNA+in+Chinese+hamster+ovary+cells+deficient+in+endogenous+transferrin+receptor.">Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.</a> J. Cell Biol. 105, 207-214 (1987).</li><li>Borrow, P., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+lymphocytic+choriomeningitis+virus-binding+protein(s):+a+candidate+cellular+receptor+for+the+virus.">Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.</a> J. Virol. 66, 7270-7281 (1992).</li><li>Rojek, J. M., Spiropoulou, C. F., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+receptors+for+the+South+American+hemorrhagic+fever+viruses+Junin,+Guanarito,+and+Machupo.">Characterization of the cellular receptors for the South American hemorrhagic fever viruses Junin, Guanarito, and Machupo.</a> 346. , 476-491 (2006).</li><li>Helguera, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antibody+recognizing+the+apical+domain+of+human+transferrin+receptor+1+efficiently+inhibits+the+entry+of+all+new+world+hemorrhagic+Fever+arenaviruses.">An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.</a> J. Virol. 86, 4024-4028 (2012).</li><li>Sanchez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+monoclonal+antibodies:+characterization+and+cross-reactivity+with+other+arenaviruses.">Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses.</a> J. Gen. Virol. 70, 1125-1132 (1989).</li><li>Trombley, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+panel+of+real-time+TaqMan+polymerase+chain+reaction+assays+for+detection+and+absolute+quantification+of+filoviruses,+arenaviruses,+and+New+World+hantaviruses.">Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.</a> Am. J. Trop. Med. 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The protocol we describe is straightforward and robust provided the following critical considerations are observed. First, strict pre-PCR technique must be employed (see 28 for an excellent review). It is imperative that all reagents and equipment are free of amplified DNA containing the qPCR target sequence and that appropriate controls are in place to detect such contamination. Second, for the virus-cell attachment portion of the protocol, the virus, cells, and media must be kept at 4&#160;&#176;C to prevent...

Arenaviruses are a family of enveloped, single-stranded RNA viruses that are maintained primarily in rodents in nature1-3. While these viruses typically establish an asymptomatic, persistent infection in rodent reservoirs1,2, they can cause severe disease in humans3. Important pathogenic species include the New World Junin virus (JUNV)4 and the Old World Lassa virus5, which are the etiologic agents of Argentine hemorrhagic fever in South America and Lassa fever in Africa, respectively. Lymphocytic choriomeningitis virus (LCMV), the prototypic virus of the family6, has a worldwide distribution and is responsible for a variety of disease states including aseptic meningitis in immunocompetent individuals6, severe birth defects in the developing fetus7, or high lethality in immunosuppressed individuals following solid-organ transplantation8,9. The lack of United States Food and Drug Administration (FDA)-approved vaccines or effective antivirals to combat these viruses highlights the critical need to develop novel strategies to therapeutically target these emerging/re-emerging pathogens.
The arenavirus genome consists of two single-stranded RNA segments, the large (L) (~7.2 kb) and small (S) (~3.6 kb) segments10. The S segment encodes the viral nucleoprotein (NP) and envelope glycoprotein (GP) while the L segment encodes the viral polymerase (L) and matrix protein (Z). The L and S genomic RNA segments (vRNAs) are packaged into virions10-12. The initial step of arenavirus infection is attachment of viral particles to host cells through an interaction between the viral GP and its corresponding host cell receptors which, for JUNV, includes the human transferrin receptor 1 (hTfR1)13,14. Following attachment, JUNV particles are taken into the cell via clathrin-mediated endocytosis15. Particles then traffic to the late endosome where the low pH of this compartment triggers the activation of GP16,17. Once activated, the GP-encoded fusion peptide inserts into the endosomal membrane, initiating fusion between the viral and endosomal membranes. Successful fusion results in the release of the virion-packaged vRNAs into the cytoplasm, where they serve as templates for genomic replication and transcription. When sufficient quantities of viral structural proteins and vRNAs are generated, new particles assemble and bud from the infected cell to complete the life cycle18.
To facilitate the discovery of new strategies to therapeutically target the pathogenic arenaviruses, our group has focused on the identification of host factors that are critical for virus propagation, but dispensable for the host. In this context, defining the precise stage(s) of the viral life cycle controlled by such host factors is necessary to guide the development of antiviral strategies. Herein, we describe a quantitative (q)RT-PCR-based assay that can be used to measure arenavirus-cell attachment for the purpose of identifying whether selected host proteins and/or antiviral molecules influence this initial stage of viral entry19. Alternative approaches to measure arenavirus-cell attachment have featured virions labeled with biotin20, fluorescent dyes21, or radioactive isotopes22. Drawbacks to these methods can include the requirement for large quantities of input virus (e.g. high multiplicities of infection (MOI)), the use of radioactivity, and/or additional handling steps to prepare input virus (e.g. concentration and/or labeling of virus). In particular, the use of high MOI makes it difficult to distinguish productive versus nonproductive attachment events15,23. The qRT-PCR-based assay described here can detect attachment events using as few as 20 plaque forming units (PFUs) of virus, which translates into an MOI of 0.0004 for a 48-well plate. Therefore, the strong sensitivity of the assay overcomes the requirement for high MOI as well as any virus labeling or concentration steps. Further, the assay can be i) adapted to measure virion endocytosis as well as release of virus genome into the cytoplasm following fusion and ii) tailored for use with other viruses through the development of virus-specific qRT-PCR reagents (for examples, see 24-27).

<table border="0" cellpadding="0" cellspacing="0" width="730">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMEM</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">11965-118</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15140-163</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HEPES Buffer Solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15630-130</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PBS pH 7.4</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">10010-023</td>
			<td style="width:160px;">1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Vero E6 cells</td>
			<td style="width:131px;">American Type Culture Collection</td>
			<td style="width:141px;">CRL-1586</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Costar 48-well plate</td>
			<td style="width:131px;">Corning</td>
			<td style="width:141px;">3548</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RNeasy mini kit</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">74106</td>
			<td style="width:160px;">do not mix buffers RLT or&#160; RW1 with bleach</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">QIAshredder homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">79654</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Taqman Universal PCR Master Mix</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4326614</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Multiscribe Reverse Transcriptase (50U/&#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4311235</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">dNTP Mixture (10 mM; 2.5 mM each dNTP)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0260</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0010</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">RNase Inhibitor (5000U/100 &#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0119</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RT primer 5&#8217;-AAGGGTTTAAAAATGGTAGCAGAC-3&#8217;</td>
			<td style="width:131px;">Integrated DNA Technologies</td>
			<td style="width:141px;">250 nmol DNA oligo</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Forward qPCR primer&#160; 5&#8217;-CATGGAGGTCAAACAACTTCCT-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Reverse qPCR primer 5&#8217;-GCCTCCAGACATGGTTGTGA-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">TaqMan Probe 5&#8217;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4316033</td>
			<td style="width:160px;">HPLC purified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">MicroAmp Fast Optical 96-well reaction plate (0.1 mL)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4346906</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4376598</td>
			<td style="width:160px;">or other qPCR instrument</td>
		</tr>
	</tbody>
</table>

highly sensitive assay for measurement of arenavirus-cell attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

To demonstrate the sensitivity and dynamic range of the qPCR assay, known quantities of a plasmid encoding the NP gene of JUNV C#1 (range 5-5 x 107 copies) were subjected to qPCR as described in Section 4 of the protocol. The assay is sensitive and can reliably detect as few as 5 copies of the target nucleic acid (Figure 2). Further, the dynamic range of the assay is large and, as shown in Figure 2, can cover at least 7 logs of template. While ...

Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

The overall goal of this protocol is to quantitatively measure arenavirus particle attachment to host cells. The main advantage to this technique is its high sensitivity. It can also be adapted to measure virion endocytosis, as well as the release of genome into the cytoplasm following fusion.To begin, to make complete DMEM, to a 500 milliliter bottle of DMEM, add 50 milliliters of heat inactivated fetal bovine serum, and five milliliters each of 100X penicillin-streptomycin, and 100X HEPES buffer. At the end of the day, seed 2.5 times 10 to the 4th Vero E6 cells per well, in triplicate for each virus, in a 48-well tissue culture plate in a final volume of 500 microliters of complete DMEM. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 10 to 18 hours.Under a Class 2 biosafety cabinet, using ice-cold complete DMEM, dilute the Junin Candid 1 virus samples to be tested to 200 to 2, 000 focus forming units or plaque forming units. Plaque forming units will be referred to as PFUs in the remainder of the protocol. For example, to inoculate with 50 microliters of 2, 000 PFUs in duplicate, add 4, 800 PFUs into a total volume of 120 microliters of medium.Store on ice until ready to add to the Vero cultures. Remove the plates from the 37 degree Celsius incubator and place on ice or at 4 degrees Celsius for 15 minutes to inhibit the ability of the plated cells to carry out endocytosis. Next, aspirate the complete DMEM from each well, and use 100 microliters of ice-cold PBS to rapidly wash each well twice.Then add 50 microliters of the pre-diluted virus samples to the appropriate wells. Wrap the plate in plastic and immediately place it back on ice or at 4 degrees Celsius for 60 to 90 minutes to allow virus attachment to occur. Keep the wash buffer and virus samples at 4 degrees Celsius throughout this step.After the incubation, aspirate the virus inoculum and use 200 microliters of ice-cold PBS to wash each well three times. To extract viral RNA from Junin Candid 1 particles, use a commercial RNA isolation kit according to the manufacturer's protocol for purification of RNA from animal cells using spin technology as follows. Lyse the cells as directed by adding 350 microliters of lysis buffer, RLT, directly to each well.Mix the buffer several times to ensure cell lysis. Then use an inverted microscope to ensure lysis by the absence of cells. At this point, the virus has been neutralized, so the remaining steps can be performed outside of the Class 2 biosafety cabinet, provided kit tubes were not contaminated.Transfer the lysate to the kit column, and follow the rest of the protocol as described. Include the optional Step 9 from the manufacturer's protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of buffer RPE. For the final step, use 30 microliters of nuclease-free double-distilled water to elute the purified RNA from the spin column.Store the RNA at 80 degrees Celsius, or use directly in the qRT-PCR assay. To convert Junin Candid 1 S-segment RNA into cDNA for qPCR, set up a 50 microliter RT Reaction Master Mix using the reagents listed in the text protocol. Gently mix the RT Master Mix, and then aliquot 40 microliters into individual 0.2 milliliter PCR tubes.Next, add 10 microliters of viral RNA to each tube. Include a no template control tube and a no RT enzyme control tube to screen for contamination of RT reagents. Include RNA extracted from uninfected cells with a full RT Master Mix to control for assay specificity in the presence of cellular RNA.Also include a known positive viral RNA sample with a full Master Mix to verify the quality of the RT reagents. Use the cycling conditions listed below for RT.When the program is complete, leave the samples in the thermocycler at 4 degrees Celsius or store them at 20 degrees Celsius. Next, prepare 25 microliter qPCR reactions by combining the qPCR Master Mix, PCR primers, and the qPCR probe as listed in the text protocol.Gently mix the solution, and then add 20 microliters to each qPCR well. Add five microliters of each RT reaction containing plasmin numbers that fall within the standard curve to the appropriate qPCR wells in triplicate. Load the qPCR plate into the thermocycler and run the reaction conditions shown below.Finally, collect and analyze the data to determine the quantity of viral RNA present in each sample, according the manufacturer's protocol. As shown here, known quantities of a plasmid encoding the NP gene from Junin virus Candid 1 were subjected to qPCR to demonstrate the sensitivity and dynamic range of the assay. In this experiment, various quantities of Junin virus Candid 1 were prebound to Vero E6 cells and quantified by qRT-PCR.As illustrated in this figure, the assay can detect viral attachment events using as few as 20, or as many as 2 times 10 to the 6th PFU, which translates to multiplicities of infection of 0.0004 and 40, respectively. In this example, the protocol was modified to also measure the rate of amplification of the viral genome inside of cells over time. Interestingly, the quantity of viral genome appears to decrease during the first six hours following attachment.This suggests that a portion of the viral RNA is degraded. This could happen to particles that fail to be taken into the cell, and degrade in the extracellular space, or perhaps due to degradation within the endolysosomal network. The data shown demonstrates that 12 or 18 hours post-infection would be optimal times to screen for active genome replication.Once mastered, this technique can be done in a single day. After watching this video, you should have a very good idea of how to accurately quantitate arenavirus attachment to host cells.

Research

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