Name: highly sensitive assay for measurement of arenavirus-cell attachment
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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 Cells in 48-well Plates
<ol>
	<li>Prepare 500 ml of complete Dulbecco&#8217;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) buffer solution by adding 50 ml of heat inactivated fetal bovine serum and 5 ml each of a 100x Penicillin-Streptomycin and 100x HEPES buffer solution to a 500 ml bottle of DMEM. This reagent can be stored at 4 &#176;C for months.</li>
	<li>Seed 2.5 x 104 Vero E6 cells per well in a 48-well tissue culture plate in a final volume of 500 &#181;l of complete DMEM. Incubate the plate at 37 &#176;C in a humidified incubator containing 5% CO2 for 10 to 18 hr.
	<ol>
		<li>Seed the plate at the end of the workday. Test each virus sample in either duplicate or triplicate wells. 
		Note: While a 48-well-based platform for this assay is described, it should be possible to scale the assay down for use in 96- or 384-well plates.</li>
	</ol>
	</li>
</ol>
2. Carry Out Virus-cell Attachment
Note: The virus used in this protocol, JUNV strain Candid #1 (C#1), has been successfully used as a live attenuated vaccine for the prevention of JUNV disease29. JUNV C#1 was derived from the virulent JUNV strain XJ through serial passage both in vivo and in vitro and differs from the parental XJ strain by 12 amino acids30. Because JUNV C#1 is a biosafety level (BSL)-2-rated pathogen, the work described for this section must be carried out using appropriate BSL-2 practices31. This includes the use of personal protective equipment (e.g. eye protection, laboratory coat, double gloves), a class II biosafety cabinet, and ensuring that all personnel have received the institutional training and approvals required to safely handle this organism.
<ol>
	<li>Dilute the JUNV C#1 sample(s) to be tested to the desired PFUs or focus forming units in ice-cold complete DMEM and store on ice until ready to add to each well. 
	Note: As an alternative to conducting the binding studies with virus diluted in complete DMEM, use a minimal medium containing PBS with a reduced serum concentration (2%) and an appropriate buffer to establish whether serum may cause interference with virus attachment. Similar attachment protocols for alternative viruses have reported the use of RPMI 1640 medium without bicarbonate containing 0.2% bovine serum albumin, 10 mM (2-(morpholino)ethanesulfonic acid), and 10 mM HEPES, pH 6.832. This binding media may be superior to prevent pH changes that could occur when media is removed from the CO2-environment of the incubator.
	<ol>
		<li>Inoculate wells with PFUs ranging from 200 to 2,000 to limit nonproductive attachment events. As shown in Figure 2, this assay is capable of detecting attachment using as few as 20 PFU (or as many as 2 x 106 PFU).</li>
		<li>Create a master mix of virus for inoculation onto cells. Each virus sample will be inoculated onto duplicate wells in a volume of 50 &#181;l per well. Therefore, if the addition of 2,000 PFUs of virus per well is desired, dilute 4,800 PFUs into a total volume of 120 &#181;l of ice cold complete DMEM.
		<ol>
			<li>To control for the specificity of the assay, use a matched pair of Chinese hamster ovary cell lines that either do not express TfR1 (TRVb) or express hTfR1 (TRVb-1) as JUNV attachment to these cells should either be reduced or not, respectively13,33. Alternatively, treat cells with a protease such as proteinase K to prevent arenavirus attachment34 or entry35. Soluble hTfR1 or the monocolonal antibody ch128.1, which is specific for hTfR1, could also be considered as they are known to block entry of JUNV into cells13,36. Additionally, pretreatment of cells with free mannose14 or incubation of particles with JUNV-specific neutralizing antibodies37 can inhibit virus entry.</li>
			<li>Please note, however, that these latter reagents and approaches, despite blocking JUNV entry, have not been confirmed to block attachment, although this is a likely explanation. The protocol described here could be used to formally determine whether these various approaches impact virion attachment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Remove plates from the 37 &#176;C incubator and place either in a 4 &#176;C refrigerator or on ice for 15 min to inhibit the ability of the plated cells to carry out endocytosis.</li>
	<li>Next, aspirate the complete DMEM from each well and rapidly wash each well twice using 100 &#181;l of ice cold PBS (phosphate buffered saline) pH 7.4. Then add 50 &#181;l of the pre-diluted virus samples (prepared in step 2.1) to the appropriate wells.</li>
	<li>Wrap the plate in plastic wrap and immediately place either back on ice or in a 4 &#176;C refrigerator for 1 to 1.5 hr to allow virus attachment to occur. Keep the plate, wash buffer, and virus samples cold at 4 &#176;C throughout this step.</li>
	<li>After the completion of the 4 &#176;C incubation, aspirate the virus inoculum and wash each well 3 times with 200 &#181;l of ice-cold PBS.</li>
</ol>
3. Viral RNA Extraction
<ol>
	<li>Purify viral RNA from JUNV C#1 particles attached to the monolayers by using the commercial kit (e.g., RNeasy Mini kit) according to manufacturer&#39;s protocol. Specifically, follow the &#34;purification of total RNA from animal cells using spin technology protocol&#34; on pages 23-28 in the RNeasy Mini Handbook (4th Edition, April 2006) with the modifications listed below.
	<ol>
		<li>Lyse the cells as directed in step 1B by adding 350 &#181;l of lysis buffer RLT directly to each well. Mix the buffer several times to ensure cell lysis. Note that cells easily lyse in this buffer and complete lysis can be verified by viewing wells under an inverted microscope.</li>
		<li>Transfer the resulting cell lysate to the kit column (step 3a) and follow the rest of the protocol as described. At this point, the virus has been neutralized by the lysis buffer so the remaining steps of the protocol can be done outside of the class II biosafety cabinet provided the kit tubes were not contaminated with infectious virus.</li>
		<li>Include the optional step 9 from the manufacturer&#39;s protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of Buffer RPE.</li>
		<li>In the final step the manufacturer&#39;s protocol (step 10), elute the purified RNA from the spin column using 30 &#181;l of nuclease-free ddH2O. Store RNA at -80 &#176;C at this point or use directly in the qRT-PCR assay. To prevent degradation of purified viral RNA samples by nucleases, use nuclease-free reagents and equipment and keep thawed RNA samples on ice. 
		Note: Buffers RLT and RW1 contain guanidine salt and should not be mixed with bleach. Buffer RW1 contains ethanol.</li>
	</ol>
	</li>
</ol>
4. qRT-PCR Measurement of Viral Genome
<ol>
	<li>To convert JUNV C#1 S segment RNA into cDNA for subsequent qPCR, subject the viral RNA (generated in step 3.1.4) to RT in a total volume of 50 &#181;l.
	<ol>
		<li>To set up the RT reaction, first create a master mix containing each of the following reagents per reaction: 6.75 &#181;l of nuclease-free ddH2O, 5 &#181;l of a 2 &#181;M stock of the RT primer S 2098- to 2075- (5&#39;-AAGGGTTTAAAAATGGTAGCAGAC-3&#39;), which is complementary to the NP region of the S segment genomic RNA, 5 &#181;l of 10X PCR buffer II, 11 &#181;l of a 25 mM MgCl2 solution, 1.25 &#181;l (62.5 Units) of RT enzyme, 1 &#181;l (0.4 Units) of RNase inhibitor, and 10 &#181;l of a 500 &#181;M stock of dNTPs. 
		Note: During the course of arenavirus replication, 4 viral S segment RNA species are generated: a full length genomic RNA (vRNA), a full length antigenomic RNA (vcRNA) (that is the reverse complement of the vRNA), and two subgenomic mRNAs encoding the NP and GP genes, respectively. The RT primer listed here is specific for only the S segment vRNA, but not the vcRNA, NP mRNA, or GP mRNA.</li>
		<li>Gently mix the RT master mix and then aliquot 40 &#181;l into individual 0.2 ml PCR tubes. Add 10 &#181;l of viral RNA to each tube. Include i) a no template control tube (e.g. add 10 &#181;l of nuclease-free ddH2O to 40 &#181;l of the master mix) and ii) a no RT enzyme control (e.g. add 10 &#181;l of a known-positive viral RNA sample to 40 &#181;l of master mix that did not receive any RT enzyme) to screen for contamination of the RT reagents with amplified DNA containing the viral target sequence. Include RNA extracted from uninfected cells with the full RT master mix to control for assay specificity in the presence of cellular RNA.</li>
		<li>Additionally, include a known-positive viral RNA sample to the full master mix to verify the quality of the RT reagents. Note that the volume of the RT reaction can be reduced to 25 &#181;l if necessary.</li>
	</ol>
	</li>
	<li>Subject the RT tubes to the following cycling conditions: 25 &#176;C for 10 min, 48 &#176;C for 30 min, and 95 &#176;C for 5 min. Note that samples can be stored at -20 &#176;C at this point or left overnight in the thermocycler by adding a 4 &#176;C step.</li>
	<li>Perform qPCR in a total volume of 25 &#181;l.
	<ol>
		<li>Make a qPCR master mix by combining the following: 2.5 &#181;l (of a 9 &#181;M stock) of each the forward PCR primer S1937+ to 1958+ (5&#39;-CATGGAGGTCAAACAACTTCCT-3&#39;) and reverse PCR primer S 2001- to 1982- (5&#39;-GCCTCCAGACATGGTTGTGA-3&#39;), 2.5 &#181;l (of a 2 &#181;M stock) of the qPCR probe S 1960+ to 1975+ (5&#39;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#39;), and 12.5 &#181;l of the 2X universal qPCR master mix. Note that the forward primer reported here, which is specific for JUNV C#1, differs by 1 nt from the originally reported primer sequence38, which targets the virulent JUNV strain Romero.</li>
		<li>Gently mix the solution and then add 20 &#181;l to each qPCR well. Add 5 &#181;l of each RT reaction to the appropriate qPCR wells. Conduct qPCR in triplicate for each sample tested. Note that the PCR reaction volume can be reduced to as little as 12.5 &#181;l if needed. 
		Note: The software associated with the qPCR machine will generate absolute copy numbers of JUNV C#1 S segment genomic RNA by comparing the value of each unknown sample to a series of standard dilutions of plasmid encoding the JUNV C#1 NP gene, which is the target of the qPCR primer and probe set. The qPCR assay can only provide quantitation for values that fall within the range of the standard curve. For this reason, include the following quantities of plasmid (in triplicate wells): 5, 50, 5 x 103, 5 x 105, and 5 x 107 copies per well.</li>
	</ol>
	</li>
	<li>Load the qPCR plate into the thermocycler and run the following reaction conditions: 95 &#176;C for 10 min and 40 cycles of 95 &#176;C for 15 sec and 60 &#176;C for 1 min.
	<ol>
		<li>Collect and analyze the data to determine the quantity of viral RNA present in each sample according to manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>To establish a standard curve using a newly purified sample of plasmid containing the qPCR target sequence, first determine the plasmid copy number in this solution.
	<ol>
		<li>Calculate the molecular weight of the plasmid. If the sequence of the entire plasmid is known, calculate this using the following formula: MW = ([A * 312.2] + [G * 328.2] + [C * 288.2] + [T * 303.2] - 61.13). Remember to include the total number of a particular nucleotide found on each strand of the double stranded plasmid.</li>
		<li>If the size of the plasmid is known but its exact sequence is not, calculate the MW under the assumption that each dsDNA base pair has a MW of 600. 
		Note: The pCAGGS-JUNV C#1 NP plasmid used here has a MW of 4.2 x 106.</li>
		<li>Once the MW of the plasmid has been determined, calculate how many copies per &#181;l are present in the stock plasmid solution.</li>
		<li>First calculate the total &#181;g of plasmid in the solution, then convert this to moles of plasmid (e.g. if 69 &#181;g of plasmid are contained in 300 &#181;l of water, then 6.9 x 10-5 g of plasmid * 1 mole/4.2 x 106 g of plasmid = 1.6 x 10-11 moles of plasmid), which can be converted to copy number (e.g. 1.6 x 10-11 moles of plasmid * 6.022 x 1023 molecules/mole = 9.8 x 1012 molecules (or copies)).</li>
		<li>Divide the total copies of plasmid in the plasmid solution by the volume of that solution to get copies per &#181;l (e.g. 9.8 x 1012 copies/300 &#181;l = 3.28 x 1010 copies).</li>
		<li>Dilute this solution appropriately so that 5 &#181;l volumes can be loaded onto the PCR plate to deliver the range of copy numbers needed to establish the standard curve.</li>
	</ol>
	</li>
</ol>

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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. Hyg. 82, 954-960 (2010).</li><li>Helenius, A., Marsh, M., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Semliki+forest+virus+penetration+by+lysosomotropic+weak+bases.">Inhibition of Semliki forest virus penetration by lysosomotropic weak bases.</a> J. Gen. Virol. 58 (Pt 1), 47-61 (1982).</li><li>Nour, A. M., Li, Y., Wolenski, J., Modis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+membrane+fusion+and+nucleocapsid+delivery+into+the+cytoplasm+are+distinct+events+in+some+flaviviruses.">Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses.</a> PLoS pathogens. 9, e1003585 (2013).</li></ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>11965-118</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-163</td><td>100x</td></tr><tr><td>HEPES Buffer Solution</td><td>Life Technologies</td><td>15630-130</td><td>100x</td></tr><tr><td>PBS pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td>1x</td></tr><tr><td>Vero E6 cells</td><td>American Type Culture Collection</td><td>CRL-1586</td><td></td></tr><tr><td>Costar 48-well plate</td><td>Corning</td><td>3548</td><td></td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74106</td><td>do not mix buffers RLT or&amp;nbsp; RW1 with bleach</td></tr><tr><td>QIAshredder homogenizer</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>Taqman Universal PCR Master Mix</td><td>Life Technologies</td><td>4326614</td><td></td></tr><tr><td>Multiscribe Reverse Transcriptase (50U/&amp;micro;l)</td><td>Life Technologies</td><td>4311235</td><td></td></tr><tr><td>dNTP Mixture (10 mM; 2.5 mM each dNTP)</td><td>Life Technologies</td><td>N808-0260</td><td></td></tr><tr><td>GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution</td><td>Life Technologies</td><td>N808-0010</td><td></td></tr><tr><td>RNase Inhibitor (5000U/100 &amp;micro;l)</td><td>Life Technologies</td><td>N808-0119</td><td></td></tr><tr><td>RT primer 5&amp;rsquo;-AAGGGTTTAAAAAT&lt;br/&gt;GGTAGCAGAC-3&amp;rsquo;</td><td>Integrated DNA Technologies</td><td>250 nmol DNA oligo</td><td>standard desalting</td></tr><tr><td>Forward qPCR primer&amp;nbsp; 5&amp;rsquo;-CATGGAGGTCAAACAACTTCCT-3&amp;rsquo;</td><td>Life Technologies</td><td>4304971</td><td>standard desalting</td></tr><tr><td>Reverse qPCR primer 5&amp;rsquo;-GCCTCCAGACATGGTTGTGA-3&amp;rsquo;</td><td>Life Technologies</td><td>4304971</td><td>standard desalting</td></tr><tr><td>TaqMan Probe 5&amp;rsquo;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&amp;rsquo;</td><td>Life Technologies</td><td>4316033</td><td>HPLC purified</td></tr><tr><td>MicroAmp Fast Optical 96-well reaction plate (0.1 mL)</td><td>Life Technologies</td><td>4346906</td><td></td></tr><tr><td>StepOnePlus Real-Time PCR System</td><td>Life Technologies</td><td>4376598</td><td>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 ...

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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 ...

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JoVE Journal

Immunology and Infection

9.5K Views.  University of Vermont. 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.

Video: Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Biology

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

Source: Tai, C., et al. <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015).
This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows assessment of the impact of the test compound on viral envelope fusion with endosomes.

Test do oceny wpływu badanych związków na wnikanie wirusa i fuzję w komórkach gospodarza

Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum

Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 105 cells/mL in pre-warmed complete DMEM media.
Then, seed 100 microliters of cells, per well, into clear 96-well flat-bottomed plates to the concentration of 1 x 104 cells per well. Incubate the plate at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 22 hours.
On day two, generate serial dilutions of the serum samples of interest in 1.5-milliliter microcentrifuge tubes using pre-warmed complete DMEM.
To each tube with diluted serum samples, add 66 microliters of the 7.5 x 106 viral genome per microliter virus working solution. Mix the virus/serum dilutions by pipetting, and then, place the tubes containing the virus/serum mixtures in an incubator at 37 degrees Celsius with 5% carbon dioxide for 30 minutes, to allow potential neutralization to occur.
After 30 minutes, pipette 100 microliters of the virus/serum mixture to each well on the 96-well plate containing 1 x 104 cells per well, then, wrap the plate in a foil to place in an incubator at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 24 hours.
On day three, aspirate the media from the wells of the 96-well plate using a fume hood vacuum without disrupting the adhered cells, and add 50 microliters of 4% paraformaldehyde to each well. After wrapping the plate in foil, leave it for 10 minutes at room temperature.
Later, wash and aspirate the cells twice with 200 microliters of PBS at room temperature. After the second wash, pipette 200 microliters of pre-warmed PBS into each well, and wrap the plate in foil followed by incubation at 65 degrees Celsius for 90 minutes to denature endogenous alkaline phosphatase activity.
Following incubation, add 50 microliters of the freshly prepared, dissolved BCIP/NBT into each well, and incubate the wrapped plates at room temperature for 2 to 24 hours. Later, take photos of each well using a 4x objective lens in a light microscope camera, ensuring the consistent use of the same exposure, white balancing, and light settings for all assays.

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