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 endocytosis of surface-bound virions. Third, the quantity of cells collected for RNA extraction must not exceed the RNA binding capacity of the RNA purification filter. Fourth, purified viral RNA samples must be protected from ribonuclease-mediated degradation through the use of nuclease-free reagents and equipment and by keeping them on ice when thawed. Fifth, the range of the standard curve included in the qPCR assay must be sufficiently large to capture all values observed from the unknown samples. Sixth, technical replicates should be included for both the virus-cell attachment portion of the assay as well as the qPCR of individual RNA samples. Seventh, controls (e.g. cell lines that express hTfR1 or not) can be included to control for the specificity of the assay. Finally, all personnel must have appropriate biosafety training and institutional approval to handle infectious JUNV C#1 or other pathogenic arenaviruses.
The qPCR portion of the protocol calls for a qPCR probe primarily to ensure the specificity of the product being amplified during each stage of the PCR reaction. The cost of the assay could be reduced, however, by eliminating this qPCR probe in favor of another qPCR chemistry that does not require a probe (e.g. the use of the asymmetrical dye SYBR Green I25) and/or by reducing the volume of the qPCR reaction mixture used. If a probe-independent qPCR approach is used, it would be important to verify that the reaction conditions are stringent enough to ensure specificity of the qPCR primers. It is also important to be aware that arenavirus RNA, whether extracted from cells or virions, can be nonspecifically primed to form cDNA during the RT in the absence of a virus-specific RT primer12. Therefore, copy numbers detected using a vRNA-specific RT primer do not strictly reflect just the vRNA species targeted by the RT primer. If specificity for genome or antigenome is required, we have reported a means to circumvent this nonspecific priming phenomenon through the use of biotinylated RT primers and streptavidin beads12.
An important strength of this assay is its extreme sensitivity. Other arenavirus-cell attachment assays featuring radioactively-labeled22, biotinylated20 or fluorescently-labeled21 particles have required MOIs of 0.2, 100, or 1000, respectively. While the use of radioactively-labeled particles results in good sensitivity, it necessitates the use of radioactivity, which increases the logistical complexity and safety concerns associated with this particular assay. The qPCR-based method described here requires as little as 20 PFU (MOI of ~0.0004 in a 48-well plate) and can reliably detect attachment over a large dynamic range of MOIs (e.g. MOIs from 0.0004 to at least 40) (Figure 3). The strong sensitivity of this assay makes it possible to use low MOIs for more accurate measurement of productive binding events by standard infectious virus. The high sensitivity also significantly reduces the quantity of virus needed for the assay and, in doing so, limits the additional handling steps that may impact the quality of virions being used in the assay (e.g. polyethylene glycol-based precipitation of virions, labeling virions with fluorophores or radioactive isotopes and/or sucrose-banding of virions). Additionally, the qPCR assay itself is straightforward to perform as well as highly accurate and reproducible.
The cell-attachment assay described can be modified to measure additional stages of viral entry including viral particle endocytosis as well as the final step of virus fusion, which is release of viral genome into the cytoplasm. To measure particle endocytosis, the same steps of the attachment protocol would be followed through step 2.5, which is attachment of particles to cells at 4 &#176;C. Cells would then be warmed to allow viral particles to be endocytosed, followed by treatment with a protease to strip any externally-bound virions that were not endocytosed as described22,24. Sections 3 and 4 of this protocol would then be followed to extract viral RNA and quantitate copies of viral genomic RNA as a measure of endocytosis. To measure genome uncoating following fusion of viral and endosomal membranes, cells would be prebound with virus at 4 &#176;C, warmed (to permit endocytosis and fusion) and stripped of external particles, and then collected for analysis. In one approach, cells could be lysed in an isotonic buffer to preserve intracellular vesicles, and then split into 2 fractions: one that would be treated with a cocktail of RNases (uncoated viral RNA is susceptible to degradation) and the other that would not39. Alternatively, cells could be separated into cytosolic or endosomal fractions40. In both cases, qRT-PCR would be used to quantitate viral genome in the cytoplasm as a measure of viral genome uncoating. Last, the qRT-PCR assay can be easily modified to study virus attachment, endocytosis, or fusion for other viruses provided the RT-PCR reagents are tailored to the new virus of interest (for examples see 24-27).

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 not shown, we have detected as many as 5 x 109 copies of nucleic acid.
To illustrate the utility of the assay for measurement of JUNV C#1 particle attachment, various quantities of JUNV C#1 were prebound to Vero E6 cells as described in the protocol. After washing away unbound particles, cells were lysed for RNA purification and the resulting RNA samples were subjected to qRT-PCR. As shown in Figure 3, the assay can detect viral attachment events using as few as 20 or as many as 2 x 106 PFU, which translates to MOIs of 0.0004 and 40, respectively.
As shown in Figure 4, it is possible to modify the protocol to not only measure viral attachment, but also the rate of amplification of the genome contained in the incoming viral particles over time. This modified approach can be used as a surrogate to determine whether further defects in the remaining steps of viral entry (e.g. particle endocytosis and/or fusion and release of viral genome into the cytoplasm) might be occurring. Interestingly, the quantity of viral genome appears to decrease during the first 6 hr following attachment, which suggests that a portion of the incoming viral genome 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 hr post-infection would be optimal times to screen for active genome replication.
<img alt="Figure 1" src="/files/ftp_upload/53682/53682fig1.jpg" /> 
Figure 1: Depiction of protocol workflow. The primary steps of the virus-cell attachment assay are 1) incubation of virus particles with cells at 4 &#176;C to allow virus-cell attachment to occur followed by washes to remove unbound virus, 2) purification of viral RNA from the cells, 3) reverse transcription (RT) to convert JUNV S segment genomic RNA (vRNA) into cDNA, and 4) qPCR to enumerate copies of JUNV S vRNA as a measure of virus-cell attachment. <a href="https://www.jove.com/files/ftp_upload/53682/53682fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53682/53682fig2.jpg" /> 
Figure 2: qPCR assay for detection of JUNV S segment genome is sensitive and can be used over a large dynamic range. The JUNV S segment qPCR primer-probe set was tested for its ability to accurately measure known quantities (5, 16.6, 50, 5 x 103, 5 x 105, or 5 x 107 copies) of a DNA standard control plasmid encoding the target sequence. Depicted are the amplification plots (A) and standard curve fit lines (B). R2, correlation coefficient of primer-probe set. <a href="https://www.jove.com/files/ftp_upload/53682/53682fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53682/53682fig3.jpg" /> 
Figure 3: JUNV-cell attachment assay reliably measures particle attachment using as few as 20 PFU. Vero E6 cells were prebound with known quantities of JUNV C#1 (2 x 101, 2 x 102, 2 x 103, 2 x 104, 2 x 105, or 2 x 106 PFU) for 1.5 hr at 4 &#176;C. Unbound particles were removed by washing, cells were lysed for RNA extraction, and the resulting RNA samples were subjected to qRT-PCR to detect JUNV C#1 S segment vRNA as a measure of particle attachment to Vero E6 cells. Data represent the mean copy number per well from duplicate wells &#177; SEM.
<img alt="Figure 4" src="/files/ftp_upload/53682/53682fig4.jpg" /> 
Figure 4: Kinetics of JUNV C#1 genome replication following infection. Vero E6 cells were incubated with 2 x 103 PFU of JUNV C#1 (MOI of 0.04) for 1.5 hr at 4 &#176;C. Following several washes in ice-cold PBS, cells were either harvested immediately (0 hr time point) or warmed to 37 &#176;C for the indicated times before collection. In each case, total RNA was extracted from cells and subjected to qRT-PCR to detect JUNV C#1 S segment vRNA. Values are listed as mean copies per well from duplicate wells &#177; SEM.

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 ...

highly-sensitive-assay-for-measurement-of-arenavirus-cell-attachment

Nanyang Technological University

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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

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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 ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

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Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

Plaque Assay for Murine Norovirus

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

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

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM