The authors would like to thank several members of the lab for their valuable suggestions in the development of agroinfiltration and in vitro assembly assays. This work was funded by a grant from University of California. This study was supported in parts by a grant from the National Science Foundation (CBET-1144237).

1. Plant material
<ol>
	<li>Nicotiana benthamiana plants to be used in the encapsidation assay should be at 4 leaves stage (approximately 3-4 week old plants).</li>
</ol>
2. Delivery and expression of functional viral genome components to plant cells by agroinfiltration
<ol>
	<li>Day 1: The Agrobacterium strain (eg. EH 105 or GV 3101) harboring the pCass- BMV RNA 1, BMV RNA 2 and BMV RNA 31,2 are grown on LB agar plates supplemented with 50 mg/ml of kanamycin (selects for the pCASS vector) and 100mg/ml of rifampicin (selects for the Agrobacterium). Incubation is being carried out at 28 &deg;C for two days.</li>
	<li>Day 3: Inoculate single colony from the LB agar plate into 2 ml of LB broth containing 50 mg/ml of kanamycin and 100 mg/ml of rifampicin for 1 day at 28 &deg;C in orbital shaker set at 250 RPM.</li>
	<li>Day 4: Inoculate 50 ml of the LB broth supplemented with 50 mg/ml of kanamycin and 100 mg/ml of rifampicin, 10 mM MES pH 5.6 and 100 mM acetosyringone with one ml of culture in a 250 ml of Erlenmeyer flask for 16 hours at 28 &deg;C on orbital shaker at 250 RPM.</li>
	<li>Day 5: Transfer the culture (when OD600 reached 1.0) to an oak ridge tube or sterile Falcon tube and centrifuge for 10 minutes at 5000 rpm at 4 &deg;C.</li>
	<li>Dissolve the pellet in 10 ml of 10 mM MgCl2 and centrifuge for 10 minutes at 5000 RPM at 4 &deg;C, Repeat this step one more time to ensure complete removal of the antibiotic.</li>
	<li>Suspend the pellet in 10 ml buffer containing 10 mM MgCl2 and 10 mM MES (pH 5.6).</li>
	<li>Measure the OD600 for each culture of BMV RNA 1, RNA 2 and RNA 3 and adjust to 0.1 OD600 using 10 mM MgCl2 and 10mM MES (pH 5.6).</li>
	<li>Mix 1 ml each all three 0.1 OD600 culture suspensions.</li>
	<li>Add 100 mM of acetosyringone, mix gently and keep the mixture undisturbed at room temperature for 3 hours.</li>
	<li>Draw the culture suspension into 1 ml. syringe without needle.</li>
	<li>Infiltrate the above culture suspension into abaxial side of 2-3 well-expanded leaves of N. benthamiana (5 leaves stage, 3-4 week old plants) by gently pressing the syringe to one half of the abaxial surface of leaf.</li>
	<li>Repeat this infiltration procedure to other leaves.</li>
	<li>Keep infiltrated plants in the green house with 24 &deg;C.</li>
	<li>Harvest infiltrated leaves 3 to 4 days post infiltration (dpi).</li>
</ol>
3. Purification of BMV virions
<ol>
	<li>Collect N. benthamiana leaves agroinfiltrated with a mixture containing all three wild type BMV agrotransformants.</li>
	<li>Grind leaves in a sterile pestle and mortar with equal volume (w/v) of BMV extraction buffer (0.5 M NaAc; 0.08 M MgAc, pH 4.5 and 1/100 volume of &beta;-mercaptoethanol which is to be added just before use)3. To maximize virus yield, it is recommended to add acid washed sand (~0.5-1 g) that facilitates easy grinding and efficient cell disruption.</li>
	<li>Filter the leaf extract through 2-3 layers of muslin cloth and collect the flurry.</li>
	<li>Again grind the retained portion on muslin cloth with equal volume of BMV extraction buffer to the initial weight of leaf material.</li>
	<li>Repeat the filtration procedure using muslin cloth. These steps should be carried out at 4 &deg;C.</li>
	<li>Transfer the filtrate solution to a centrifuge tube and add equal volume of pre-chilled chloroform and vortex (or shake) for 5 minutes at the room temperature till the color of the suspension turns light green.</li>
	<li>Centrifuge the emulsified solution for 15 minutes at 12,000 RPM at 4 &deg;C.</li>
	<li>Collect the aqueous phase and stir for 30 minutes on magnetic stirrer to remove any residual chloroform.</li>
	<li>Transfer the suspension to a sterile ultracentrifuge tube.</li>
	<li>Underlay 1 ml. 10% sucrose (prepared in virus extraction buffer) as a cushion3.</li>
	<li>Centrifuge at 30,000 rpm for 3 hours in a Beckman ultracentrifuge at 4 &deg;C.</li>
	<li>Discard the supernatant and suspend pellet in desired volume of BMV suspension buffer (to prepare suspension buffer dilute BMV extraction buffer to 1/10 with sterile distilled water).</li>
	<li>The partially purified virion preparation from above step is subjected to 10-40% sucrose density gradient centrifugation for 150 min at 28000 rpm at 4 &deg;C.</li>
	<li>Collect the virus band either manually or by a fractionator. The virus band will appear blue under white light illumination.</li>
	<li>Dilute the sucrose solution containing virus sample at least 50% with virus suspension buffer.</li>
	<li>Centrifuge diluted virus containing sucrose solution for 3 hours at 30,000 rpm in a Beckman ultracentrifuge at 4 &deg;C.</li>
	<li>Finally suspend the highly purified virus pellet in a desired volume of virus suspension buffer.</li>
	<li>Measure the concentration of the virion (mg/ml) using spectrophotometer at OD260.</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/3645/3645eq1.jpg" />
Note: Based on RNA content, the extinction coefficient for BMV is 54.
<ol start="19">
	<li>Verify the purity of virions by viewing under TEM (Fig. A)</li>
</ol>
4. Preparation of capsid protein subunits for in vitro assembly
<ol>
	<li>Prepare 1 Liter of 1x virus dissociation buffer (0.5 M CaCl2; 50 mM Tris HCl, pH 7.5; 1.0 mM EDTA; 1.0 mM DTT and 0.5 mM PMSF)5.</li>
	<li>Prepare dialysis membrane according to Sambrook et al 6.</li>
	<li>Dispense required concentration of purified virus into a dialysis bag.</li>
	<li>Place the virus containing dialysis bag into a beaker containing the 1x virus dissociation buffer.</li>
	<li>Dialyze 24 hours at 4&deg;C while stirring.</li>
	<li>Collect the solution from the dialysis bag after 24 hours and centrifuge at 12,000x g for 15 min at 4 &deg;C. This will separate the BMV RNA from the dissociated virions.</li>
	<li>Collect the supernatant and centrifuge at 35,000 rpm in Beckman centrifuge for 3 hours at 4&deg;C to pellet any un-dissociated virus particle.</li>
	<li>Collect the supernatant.</li>
	<li>At this stage it is imperative to remove any contaminating virion RNA from dissociated capsid protein subunits. To do this, the supernatant from step 8 should be subjected to another round of over night reassembly of capsid protein subunits without adding any RNA (in RNA 1x assembly buffer, see below) followed by an high speed centrifugation (30,000 rpm for 3 hrs at 4 &deg;C) to pellet any assembled virions. After this reassembly step, the supernatant containing coat protein subunits would be free of any residual contaminating RNA. However, to ensure this, it is advisable to perform an additional in vitro reassembly with only coat protein subunits using RNA 1x assembly buffer. After over night reassembly, the preparation must be free of assembled virions (verify using TEM). </li>
	<li>Determine the concentration of capsid protein subunits by measuring at OD 254 and 280 nm or by other methods such as Bradford assay.</li>
	<li>Perform 12-15% SDS-PAGE followed by western blot to determine the integrity of the dissociated capsid protein subunits.</li>
</ol>
5. In vitro assembly of RNA containing virions
<ol>
	<li>Prepare RNA transcripts to be re-assembled into virions and calculate the concentration7.</li>
	<li>Mix capsid protein subunits and RNA transcript at a ratio of 1:5 (wt/wt)5.</li>
	<li>Dispense the above mixture to a dialysis bag and properly secure to avoid any leaks.</li>
	<li>Prepare 1,000 ml. of RNA 1x assembly buffer (50 mM NaCl; 50 mM Tris-HCl, pH 7.2; 10 mM KCl; 5 mM MgCl2, 1.0 mM DTT)5.</li>
	<li>Place the dialysis bag containing the reaction mixture against 1x assembly buffer into the beaker and stir.</li>
	<li>Dialyze the re-assembly reaction at 4 &deg;C for 24 hours by gentle stirring.</li>
	<li>Collect the mixture from the dialysis bag after 24 hours and add 1.5 ml of RNA assembly buffer.</li>
	<li>Pass this mixture through a Centricon-100 column by centrifuging at low speed (2,000x g) for 30 minutes.</li>
	<li>Wash the column once with 1.5 ml of assembly buffer at 4 &deg;C for 30 min.</li>
	<li>Repeat the above washing step twice.</li>
	<li>Finally, elute the re-assembled virions by centrifugation at 10,000g at 4 &deg;C for 5 min.</li>
	<li>Estimate the virion concentration at OD 260 nm.</li>
</ol>
6. Fabrication of Optical Viral Ghosts (OVGs)
<ol>
	<li>Add Indocyanine green (ICG)8 to the purified capsid protein at desired concentration ratio(the weight ratio of Indocyanine green and capsid protein used in this report was 1:10) and mix thoroughly by pipetting.</li>
	<li>Dialyze the capsid protein and ICG solution against the OVG assembly buffer (1 M NaCl; 50 mM NaAc; 1 mM EDTA; and 1 mM DTT, pH4.8) at 4 &deg;C for 24 hours.</li>
	<li>After 24 hours, collect the solution from the dialysis bag (12 kDa pore size) and centrifuge at 90,000 rpm for 1 hour at 4 &deg;C (Fig. B)</li>
	<li>Remove the supernatant and suspend the pellet in BMV suspension buffer by vortexing or allowing it to suspend by itself in BMV suspension buffer overnight at 4 &deg;C.</li>
	<li>Verify the morphology of OVGs by TEM (Fig. C).</li>
	<li>Measure the absorbance from 240 nm to 950 nm using spectrophotometer (Cary 50, Varian Inc.); the presence of ICG is confirmed by signature absorbance peaks around 700 nm and 790 nm8. (Fig. D)</li>
	<li>The typical ICG fluorescence peaks at ~700 nm and ~800 nm can be seen upon 620 nm excitation of OVG solution (Fig. D) using spectrofluorometer (Fluorolog 3, Jobin-Yvon).</li>
</ol>
7. Representative Results
<img alt="Figure A" src="/files/ftp_upload/3645/3645figA.jpg" /> 
Figure A. TEM image of negatively stained BMV virions purified from N. benthamiana plants agroinfiltrated with a mixture of all three wild type BMV agroconstructs (scale bar = 100 nm).
<img alt="Figure B" src="/files/ftp_upload/3645/3645figB.jpg" /> 
Figure B. Pellet of in vitro assembled ICG containing OVGs following high-speed centrifugation.
<img alt="Figure C" src="/files/ftp_upload/3645/3645figC.jpg" /> 
Figure C. TEM image of negatively stained ICG containing OVGs (scale bar = 100 nm).
<img alt="Figure D" src="/files/ftp_upload/3645/3645figD.jpg" /> 
Figure D. Absorbance (Top) and fluorescence (bottom) spectra of OVGs. The excitation wavelength used for OVG emission was 620 nm.

<ol>
	<li>Annamalai, P., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-independent+expression+of+genome+components+and+capsid+protein+of+brome+mosaic+virus+in+planta:+a+functional+role+for+viral+replicase+in+RNA+packaging.">Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.</a> Virology. 338, 96-111 (2005).</li><li>Annamalai, P., Rofail, F., Demason, D. A., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+packaging+mechanism+in+positive-strand+RNA+viruses:+synchronized+coexpression+of+functional+multigenome+RNA+components+of+an+animal+and+a+plant+virus+in+Nicotiana+benthamiana+cells+by+agroinfiltration.">Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.</a> J. Virol. 82, 1484-1495 (2008).</li><li>Annamalai, P., Rao, A. L., Foster, N., Johansen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Virology Protocols. 451, 251-264 (2008).</li><li>Lane, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bromoviruses.">The bromoviruses.</a> Adv. Virus Res. 19, 151-220 (1974).</li><li>Choi, Y. G., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+studies+on+bromovirus+capsid+protein.+VII.+Selective+packaging+on+BMV+RNA4+by+specific+N-terminal+arginine+residuals.">Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.</a> Virology. 275, 207-217 (2000).</li><li>Sambrrok, J., Russel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (2001).</li><li>Dreher, T. W., Rao, A. L., Hall, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+in+vivo+of+mutant+brome+mosaic+virus+RNAs+defective+in+aminoacylation.">Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation.</a> J. Mol. Biol. 206, 425-438 (1989).</li><li>Jung, B., Rao, A. L., Anvari, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+nano-constructs+composed+of+genome-depleted+brome+mosaic+virus+doped+with+a+near+infrared+chromophore+for+potential+biomedical+applications.">Optical nano-constructs composed of genome-depleted brome mosaic virus doped with a near infrared chromophore for potential biomedical applications.</a> A.C.S. Nano. 5, 1243-1252 (2011).</li><li>Voinnet, O., Lederer, C., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+movement+protein+prevents+spread+of+the+gene+silencing+signal+in+Nicotiana+benthamiana.">A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.</a> Cell. 103, 157-167 (2000).</li><li>Wroblewski, T., Tomczak, A., Michelmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Agrobacterium-mediated+transient+assays+of+gene+expression+in+lettuce,+tomato+and+Arabidopsis.">Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis.</a> Plant Biotechnol. J. 3, 259-273 (2005).</li></ol>

We have nothing to disclose.

Agroinfiltration approach presented here can widely be applicable to a wide range of plant viruses. A hallmark feature of this approach is synchronized delivery of multiple agroconstruct to the same cell-a major drawback commonly associated with routinely used mechanical inoculation of plant viruses. In vivo and in vitro assembly studies using brome mosaic virus as a model can be conducted efficiently by following few tips.(i)For successful infiltration of N. benthamiana leaves, do not water th...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>MES Sodium salts</td>
			<td>Sigma-aldrich</td>
			<td>M2993</td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>Sigma-aldrich</td>
			<td>I2633</td>
		</tr>
		<tr>
			<td>Beckman ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Model: L8-70M</td>
		</tr>
		<tr>
			<td>Centricon-100 column</td>
			<td>Millipore</td>
			<td>YM-100</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Cary 50, Varian Inc.</td>
			<td>Part number 
			10068900</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>Fluorolog 3, Jobin-Yvon.</td>
			<td>Part number FL3-21</td>
		</tr>
	</tbody>
</table>

simple and robust in vivo and in vitro approach for studying virus assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Watch this Scientific Journal Video about Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly at JoVE.com

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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Immunology and Infection

12.1K Views.  University of California, Riverside. A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Video: Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...