The authors appreciate the contributions of Greg Smith, Lisa Pomeranz, Matt Lyman, Marlies Eldridge, Halina Staniszewska Goraczniak, and members of the Enquist lab in fine-tuning this protocol.

1. First Day: Viral Infection and Preparation of Buffers
<ol>
	<li>Prepare 5-10 dishes (15 cm diameter) of tissue culture cells for infection, e.g. PK-15 cells for pseudorabies virus (PRV) or Vero cells for herpes simplex virus (HSV).</li>
	<li>When cells are 95 - 100% confluent, infect them at a (MOI) of 5-10. To do this, inoculate each plate using virus stock in a total volume of 4 ml per plate, then incubate the plates for 1 hour at 37&deg;C. Rock plates gently every 15 minutes to ensure that the cell monolayer remains fully coated by the virus inoculum. Meanwhile, warm the medium for the next step.</li>
	<li>After one hour of infection, aspirate viral inoculum from the plates, add 15 ml of warm medium, and incubate at 37&deg;C for 12-20 hours in a humidified incubator.</li>
	<li>Prepare LCM buffers (see Table 1) and rock overnight at 4&deg;C for thorough mixing. Prepare TNE for resuspension of viral DNA (see Table 2).</li>
</ol>
2. Second Day, Phase 1: Cell Lysis and Ultracentrifugation
<ol>
	<li>Visually confirm that infection of cells has caused uniform cytopathic effect (CPE). Allow infection to progress until all cells have rounded up, but have not lifted off the plate.</li>
	<li>Scrape the infected cells into the medium, and combine cells and medium into 50 ml conical tubes. The number of conical tubes needed for this step will depend on the number of dishes used for infection in Step 1.2 above, e.g. one 50 ml conical tube can be used to pellet a maximum of 3 dishes of 15 ml medium each. Optional: Rinse the plates with 10 ml PBS and combine with the cells and medium.</li>
	<li>Centrifuge cells and medium for 10 minutes at 2000 relative centifugal force (rcf) at room temperature, then aspirate supernatant and resuspend each pellet in 10 ml of PBS. If the pellet from one viral infection was spread into multiple conical tubes in Step 2.2, these can be recombined into one conical tube at this step. Repeat centrifugation and aspiration. Optional: This pellet may be stored at -20&deg;C and the protocol continued at a later time.</li>
</ol>
3. Second Day, Phase 2: Ultracentrifugation
Keep the cell pellet and LCM buffers on ice whenever possible during the following steps.
<ol>
	<li>Add &beta;-mercaptoethanol to 0% glycerol - LCM buffer. Begin cooling the ultracentrifuge (for step 3.9 below) by setting it to 4&deg;C and turning on the vacuum.</li>
	<li>Resuspend the cell pellet in 5 ml of 0% glycerol - LCM buffer until it is no longer clumpy. If necessary, vortex the tube to break up any undissolved cell pellet.</li>
	<li>Extract by adding 1.5 ml Freon (1,1,2-trichloro-1,2,2-trifluoroethane) and vortexing vigorously. Immediately centrifuge for 10 min at 2000 rcf at a temperature of 4&deg;C. Collect the top layer with a wide-bore pipette tip (avoiding the interface) and transfer to a new tube. Optional: If the pellet is a gelatinous solid, you can quickly pour the top layer into a new tube.</li>
	<li>Repeat the Freon extraction and centrifuge again.</li>
	<li>Collect the top layer (~5 ml) and transfer to a new polyallomer ultracentrifuge tube. Add &beta;-mercaptoethanol to the two remaining glycerol - LCM buffers.</li>
	<li>Prepare a gradient in the polyallomer centrifuge tube by underlaying the Freon extract (top layer) with 3 ml of 5% glycerol - LCM (middle layer), and then underlaying again with 2.5 ml of 45% glycerol - LCM (bottom layer). Use a thin pipette to avoid overflow of the tube contents.</li>
	<li>If needed, prepare an equivalent balance tube using the same proportions of LCM buffers.</li>
	<li>Transfer tubes into ultracentrifuge buckets. Balance buckets to within 0.1 g of each other, by gently adding extra 0% glycerol - LCM buffer to the top layer (~50-100 &mu;l at a time).</li>
	<li>Using a cold ultracentrifuge with a horizontal or swinging bucket rotor, spin the balanced samples for 1 hour at 77,000 rcf (e.g. 25,000 rpm for a SW41Ti rotor) at a temperature of 4&deg;C.</li>
	<li>During ultracentrifugation, make a glass hook to capture floating DNA at the ethanol precipitation step (4.6 below). Hold both ends of a glass Pasteur pipet and suspend the middle section over a flame. When a section of glass is almost molten, pull gently to stretch the glass, bend it gently to create a tightly angled hook (&lt;90 degrees), and pull sharply to seal off the end. If the tip of the glass hook is open, hold the tip over the flame so that the glass melts enough to close it.</li>
	<li>After the ultracentrifugation, you should see a thin opaque pellet with a dark spot in the middle. Carefully aspirate the liquid from the tube, including drippings along the sides, but avoiding the pellet at the bottom of the tube.</li>
	<li>Add 0.5 ml TNE at room temperature.</li>
	<li>Let the pellet sit for at least 10 minutes to allow hydration of the pellet. Optional: The pellet may also resuspend in TNE overnight, if kept at 4&deg;C.</li>
</ol>
4. Third Day: "Ghost" DNA Precipitation
<ol>
	<li>Break up the pellet by pipetting with a small-bore pipette tip (e.g. a p200 pipette tip). Do not worry about shearing at this step, because the viral DNA is still contained in capsids at this point. Add the following reagents to the tube containing the viral pellet, at room temperature, to destroy capsids:
	<ul>
		<li>4.25 ml TNE</li>
		<li>0.25 ml 10% SDS</li>
		<li>2 mg Proteinase K</li>
	</ul>
	Gently rock to mix these reagents. There should be a slight increase in viscosity.</li>
</ol>
Beware of shearing from here on, e.g. use large-bore pipettes and do not vortex the viral material
<ol start="2">
	<li>Extract viral proteins by adding 5 ml of phenol-chloroform and immediately begin inverting or vortexing to maintain an emulsion. Mix for 10 seconds, then centrifuge the emulsion for 5 minutes at 2000 rcf at room temperature. Collect the top layer with a wide-bore pipette tip, avoiding the interface, and transfer to a new tube. Optional: Use phase lock gel (PLG) tubes in this step to avoid contamination from the interface.</li>
	<li>Repeat the phenol-chloroform extraction and centrifuge again.</li>
	<li>After the second phenol-chloroform extraction, collect the top layer into a 30 ml glass Corex tube and chill the tube at -20&deg;C for 10-15 minutes, taking care that it does not freeze.</li>
	<li>Add 10 ml of ice-cold ethanol, cover the tube (e.g. by stretching a piece of Parafilm M over the top), and invert to mix immediately. Watch for a DNA "ghost" to appear, which consists of a thin ropy precipitate. Invert the tube gently to mix further and cause the sticky DNA precipitates to cluster together.</li>
	<li>Use a glass hook to capture the DNA ghost(s). Carefully blot the DNA to remove excess liquid, then place the hook (tip down) into a 1.5 ml tube and allow to dry. Add 0.25 - 0.5 ml of TE to dissolve the DNA ghost. Allow resuspension of DNA to proceed for at least one hour. Store DNA at 4&deg;C. Optional: To maximize DNA yield, break off the glass hook into the 1.5 ml tube, so that resuspension off the glass fragments can continue over time.</li>
</ol>
5. Representative Results
With a sufficiently high MOI, cells should achieve full CPE within ~18 hours post infection (hpi) for wild-type PRV strains, and ~24 hpi for wild-type HSV-1 strains (Figure 1). Once the infected cell pellet is harvested, the remaining protocol steps can be complete in one long day or carried out over two days (Figure 2).
When preparing glass hooks to capture the DNA ghost, it is important to make the hook angle be less than 90 degrees, so that later it can fit into the base of an 1.5 ml tube (Figure 3). Sealing the tip of this hook prevents DNA loss inside the pipet.
DNA ghosts form during the precipitation step and will appear as white, ropy threads (Figure 4). These aggregate and stick to each other and/or the glass hook used to capture the DNA. Thus the same glass hook can be used to gather up multiple threads of DNA from within the precipitation solution.
<table border="1">
	<tbody>
		<tr>
			<td>Choose column based on desired volume:</td>
			<td>10 ml</td>
			<td>15 ml</td>
			<td>20 ml</td>
			<td>40 ml</td>
		</tr>
		<tr>
			<td>1M KCL</td>
			<td>1.3 ml</td>
			<td>1.9 ml</td>
			<td>2.5 ml</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>1M Tris (pH 7.4)</td>
			<td>300 &mu;l</td>
			<td>450 &mu;l</td>
			<td>600 &mu;l</td>
			<td>1.2 ml</td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>50 &mu;l</td>
			<td>75 &mu;l</td>
			<td>100 &mu;l</td>
			<td>200 &mu;l</td>
		</tr>
		<tr>
			<td>0.5 EDTA</td>
			<td>10 &mu;l</td>
			<td>15 &mu;l</td>
			<td>20 &mu;l</td>
			<td>40 &mu;l</td>
		</tr>
		<tr>
			<td>NP40/IGEPAL</td>
			<td>50 &mu;l</td>
			<td>75 &mu;l</td>
			<td>100 &mu;l</td>
			<td>200 &mu;l</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol (add immediately before use)</td>
			<td>4.3 &mu;l</td>
			<td>6.5 &mu;l</td>
			<td>8.6 &mu;l</td>
			<td>17.2 &mu;l</td>
		</tr>
<tr>
<td colspan="5">&nbsp;</td>
</tr>
		<tr>
			<td colspan="5">Use proportions for desired percent glycerol (0, 5, or 45%)</td>
		</tr>
		<tr>
			<td>0% glycerol</td>
			<td>None</td>
			<td>None</td>
			<td>None</td>
			<td>None</td>
		</tr>
		<tr>
			<td>water</td>
			<td>8.3 ml</td>
			<td>12.5 ml</td>
			<td>16.7 ml</td>
			<td>33.4 ml</td>
		</tr>
		<tr>
			<td>5% glycerol</td>
			<td>0.5 ml</td>
			<td>0.8 ml</td>
			<td>1.0 ml</td>
			<td>2.0 ml</td>
		</tr>
		<tr>
			<td>water</td>
			<td>7.8 ml</td>
			<td>11.7 ml</td>
			<td>15.7 ml</td>
			<td>31.4 ml</td>
		</tr>
		<tr>
			<td>45% glycerol</td>
			<td>4.5 ml</td>
			<td>6.8 ml</td>
			<td>9.0 ml</td>
			<td>18.0 ml</td>
		</tr>
		<tr>
			<td>water</td>
			<td>3.8 ml</td>
			<td>5.7 ml</td>
			<td>7.7 ml</td>
			<td>15.4 ml</td>
		</tr>
	</tbody>
</table>
Table 1. Recipes for preparation of LCM buffers
<table border="1">
	<tbody>
		<tr>
			<td>For 50 ml volume</td>
		</tr>
		<tr>
			<td>5 ml 1M NaCl</td>
		</tr>
		<tr>
			<td>2.5 ml 1M Tris, pH 7.5</td>
		</tr>
		<tr>
			<td>1 ml 0.5 EDTA</td>
		</tr>
		<tr>
			<td>41.5 ml H2O</td>
		</tr>
	</tbody>
</table>
Table 2. Preparation of TNE (0.1 M NaCl; 50 mM Tris, pH 7.5; 10 mM EDTA)
<table border="1">
	<tbody>
		<tr>
			<td>For 1 L volume</td>
		</tr>
		<tr>
			<td>0.2 g KCl</td>
		</tr>
		<tr>
			<td>0.2 g KH2PO4</td>
		</tr>
		<tr>
			<td>8 g NaCl</td>
		</tr>
		<tr>
			<td>1.15g Na2HPO4</td>
		</tr>
		<tr>
			<td>H2O to 1L</td>
		</tr>
	</tbody>
</table>
Table 3. Preparation of PBS (2.7 mM KCl; 1.5 mM KH2PO4; 137 mM NaCl; 8.1 mM Na2HPO4; pH 7.0)
<img alt="Figure 1" src="/files/ftp_upload/3151/3151fig1.jpg" /> 
Figure 1. PK15 cells (A) prior to infection, (B) midway through infection, and (C) at cytopathic effect (CPE), after a high multiplicity of infection.
<img alt="Figure 2" src="/files/ftp_upload/3151/3151fig2.jpg" /> 
Figure 2. Overview of the protocol.
<img alt="Figure 3" src="/files/ftp_upload/3151/3151fig3.jpg" /> 
Figure 3. Three representative examples of glass hooks used to collect the viral DNA ghosts.
<img alt="Figure 4" src="/files/ftp_upload/3151/3151fig4.jpg" /> 
Figure 4. Representative example of a well-formed and abundant DNA "ghost".

<ol>
	<li>Szpara, M. L., Parsons, L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variability+in+clinical+and+laboratory+isolates+of+herpes+simplex+virus+1+reveals+new+mutations.">Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.</a> J Virol. 84, 5303-5313 (2010).</li><li>Banfield, B. W., Kaufman, J. D., Randall, J. A., Pickard, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pseudorabies+virus+strains+expressing+red+fluorescent+proteins:+new+tools+for+multisynaptic+labeling+applications.">Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications.</a> J Virol. 77, 10106-10112 (2003).</li><li>Kobiler, O., Lipman, Y., Therkelsen, K., Daubechies, I., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+carrying+a+Brainbow+cassette+reveal+replication+and+expression+of+limited+numbers+of+incoming+genomes.">Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes.</a> Nat Commun. 1, 146-146 (2010).</li><li>Enquist, L. W., Madden, M. J., Schiop-Stanley, P., Vande Woude, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+herpes+simplex+type+1+DNA+fragments+in+a+bacteriophage+lambda+vector.">Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.</a> Science. 203, 541-544 (1979).</li><li>Smith, G. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+transposon+mutagenesis+in+Escherichia+coli+of+a+full-length+infectious+clone+of+pseudorabies+virus,+an+alphaherpesvirus.">Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.</a> J Virol. 73, 6405-6414 (1999).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiol Mol Biol Rev. 69, 462-500 (2005).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. , 2381-2397 (2001).</li><li>Mendez, I. I., Hermann, L. L., Hazelton, P. R., Coombs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+freon+substitutes+in+the+purification+of+reovirus+and+calicivirus.">A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.</a> J Virol Methods. 90, 59-67 (2000).</li><li>Gharabaghi, F., Aymard, M., Trotemann, P., Gerdil, 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+rapid+and+simplified+micromethod+for+subtyping+varicella-zoster+virus.">A rapid and simplified micromethod for subtyping varicella-zoster virus.</a> J Med Virol. 31, 129-134 (1990).</li><li>Granstedt, A. E., Szpara, M. L., Kuhn, B., Wang, S. S., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+monitoring+of+in+vivo+neural+activity+using+a+circuit-tracing+pseudorabies+virus.">Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.</a> PLoS One. 4, e6923-e6923 (2009).</li></ol>

No conflicts of interest declared.

Portions of this protocol were originally developed for viral DNA isolation in BSL4 conditions, but it adapts equally well to non-BSL conditions.4 We commonly use this protocol to isolate DNA from the alpha-herpesviruses PRV and HSV-1, which have DNA genomes enclosed in a proteinaceous capsid and surrounded by a lipid envelope.7,8 However it is likely directly adaptable to other large DNA viruses, including beta- and gamma-herpesviruses and adenoviruses. Similar extractions are commonly used for RNA...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent	</td>
			<td>Company	</td>
			<td>Catalogue number	</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Freon (1,1,2-trichloro-1,2,2-trifluoroethane)</td>
			<td>Fisher</td>
			<td>T178-4</td>
			<td>Check with your institution for guidelines on appropriate disposal of Freon-containing waste, or see Mendez et al. for potential Freon alternatives.9</td>
		</tr>
		<tr>
			<td>Phase lock gel tubes, Heavy 15ml capacity</td>
			<td>5 PRIME	</td>
			<td>2302850</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Polyallomer ultracentrifuge tubes</td>
			<td>Beckman*</td>
			<td>331372*</td>
			<td>*Select tubes appropriate for your own ultracentrifuge; these are included as an example only</td>
		</tr>
		<tr>
			<td>NP-40 / IGEPAL</td>
			<td>Sigma</td>
			<td>I-3021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PK-15 cells</td>
			<td>ATCC</td>
			<td>CCL-33</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS	</td>
			<td>HyClone</td>
			<td>SH30028.03</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of viral dna from nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

Preparation of Viral DNA from Nucleocapsids

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

preparation-of-viral-dna-from-nucleocapsids

Research

JoVE Journal

Immunology and Infection

20.9K Aufrufe.  Princeton University. Wir beschreiben den Prozess der Isolierung hochreiner Herpesvirus Nukleokapsid DNA aus infizierten Zellen. Die endgültige DNA aus der Lösung erfasst ist von hoher Konzentration und Reinheit und ist damit ideal für Hochdurchsatz-Sequenzierung, high fidelity PCR-Reaktionen, und Transfektionen neue virale Rekombinanten produzieren geeignet.

Serie: Preparation of Viral DNA from Nucleocapsids

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...