See Figure 1 for an illustration summarizing the following protocol.
Safety Note: All material that has been in contact with assembled viral particles needs to be disinfected with Virkon solution or other suitable disinfectant.
1. Preparation of plasmid DNA stocks (~ 2 days)
<ol>
	<li>The following plasmids are required 1:
	<ul>
		<li>pRV1 - Containing the AAV2 Rep and Cap sequences</li>
		<li>pH21 - Containing the AAV1 Rep and Cap sequences</li>
		<li>pFdelta6 - Adenovirus-helper plasmid</li>
		<li>AAV plasmid containing the recombinant expression cassette flanked by AAV2 packaging signals (inverted terminal repeats, ITRs)</li>
	</ul>
	</li>
	<li>While pFdelta6 should be grown in Stbl2 competent cells to prevent partial deletion, pRV1 and pH21 can be grown in DH5alpha competent cells. AAV plasmids can be grown in Stbl2 cells if partial deletions occur in DH5alpha cells.</li>
	<li>Plasmid DNA should be of high quality and free of RNA contaminants. Plasmids can be screened for integrity using the following digests:
	<ul>
		<li>pRV1 - digest with XbaI to give bands of 7.5 kb and 3.8 kb</li>
		<li>pH21 - digest with EcoRI to give bands of 4.5 kb, 2.8 kb and 0.2 kb</li>
		<li>pFdelta6 - digest with HindIII to give bands of 5.5 kb, 3 kb, 3 kb, 2.3 kb and 1.5 kb</li>
	</ul>
	</li>
</ol>
2. Preparation of Human Embryonic Kidney 293 (Hek293) cells for transfection (2 - 3 days)
<ol>
	<li>Plate two 80% confluent 150 cm2 flasks of Hek293 cells into five 15 cm diameter Nunc tissue culture dishes. Cells should be 70 - 80% confluent before transfection (approximately 48 hours after plating). Culture cells in standard Dulbecco's modified Eagle medium (DMEM) with low glucose containing 10% foetal calf serum and 100 U/ml penicillin/ 100 &mu;g/ml streptomycin.</li>
	<li>3 hours before transfection remove DMEM and replace with Iscove's modified Dulbecco's medium (IMDM) containing 5% foetal calf serum.</li>
</ol>
3. Transfection of viral plasmids (~1 hour for transfection, 3 days for incubation)
<ol>
	<li>Prepare the following for a single batch (5 x 15 cm tissue culture plates) of virus:
	<ul>
		<li>62.5 &mu;g AAV plasmid</li>
		<li>125 &mu;g pFdelta6</li>
		<li>31.25 &mu;g pRV1</li>
		<li>31.25 &mu;g pH21</li>
		<li>1650 &mu;l 2.5 M CaCl2</li>
		<li>12 ml dH2O</li>
	</ul>
	</li>
	<li>In a class 2 tissue culture hood sterile filter the transfection mixture into a 50 ml tube.</li>
	<li>Whilst vortexing the solution, quickly add 13 ml of 2 x HEPES buffered saline (pH 7.05). Replace lid of 50 ml tube and continue to vortex for 15 seconds. Leave to stand for 1 minute 45 seconds, a fine white precipitate should form.</li>
	<li>Gently add 5 ml of the transfection solution to each 15 cm tissue culture dish. Swirl plates to mix and return to incubator.</li>
	<li>16 hours after transfection remove IMDM medium and replace with DMEM.</li>
</ol>
4. Lysing of cells and harvesting of rAAVs (2 hours)
<ol>
	<li>72 hours after transfection, remove media from cell culture plates and discard. All waste should be treated with Virkon solution or other suitable disinfectant.</li>
	<li>Gently wash the cells in warm 1x phosphate buffered saline (PBS; pH 7.4).</li>
	<li>Add 25 ml warm PBS to each plate and gently remove cells with a cell scraper. Collect suspension in 50 ml tubes.</li>
	<li>Pellet cells at 800 x g for 10 minutes.</li>
	<li>Discard supernatant and resuspend pellet in 150 mM NaCl, 20 mM Tris pH 8.0, use 10 ml per tissue culture plate. Split into two 50 ml tubes.</li>
	<li>Prepare a fresh solution of 10% sodium deoxycholate in dH2O. Add 1.25 ml of this to each tube for a final concentration of 0.5%. Add benzonase nuclease to a final concentration of 50 units per ml. Mix tube thoroughly.</li>
	<li>Incubate at 37&deg;C for 1 hour.</li>
	<li>Remove cellular debris by centrifuging at 3000 x g for 15 mins. Transfer to fresh 50 ml tube, ensure all cell debris has been removed to prevent blocking of heparin columns (see step 5). At this stage, the samples can be stored at -20&deg;C before continuing. We have stored samples for several weeks at -20&deg;C without a reduction in infectivity.</li>
</ol>
5. Heparin column purification of rAAVs (2 - 3 hours)
<ol>
	<li>Setup HiTrap heparin columns using a peristaltic pump so that solutions flow through the column at 1 ml per minute for steps 5.2 to 5.4. It is important to ensure no air bubbles are introduced into the heparin column.</li>
	<li>Equilibrate the column with 10 ml 150 mM NaCl, 20 mM Tris, pH 8.0.</li>
	<li>Apply 50 ml virus solution to column and allow to flow through.</li>
	<li>Wash column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0.</li>
	<li>Using a 5 ml syringe continue to wash the column with 1 ml 200 mM NaCl, 20 mM Tris, pH 8.0, followed by 1 ml 300 mM NaCl, 20 mM Tris, pH 8.0. Discard the flow-through.</li>
	<li>Using 5 ml syringes and gentle pressure elute the virus from the column by applying:
	<ul>
		<li>1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0</li>
		<li>3.0 ml 450 mM NaCl, 20 mM Tris, pH 8.0</li>
		<li>1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0</li>
		<li>Collect these in a 15 ml centrifuge tube.</li>
	</ul>
	</li>
</ol>
6. Concentration and sterile filtration of rAAVs (1 hour)
<ol>
	<li>Concentrate vector using Amicon ultra-4 centrifugal filter units with a 100,000 molecular weight cutoff. Load 4 ml of column eluate into the concentrator and centrifuge at 2000 x g for 2 minutes (at room temperature). Discard flowthrough and reload concentrator with remaining virus solution and repeat centrifugation. The concentrated volume should be approximately 250 &mu;l. If concentrated volume is significantly more than this, discard the flow through and continue to centrifuge in one minute steps until volume is approximately 250 &mu;l.</li>
	<li>Add 250 &mu;l of PBS to virus for a final volume of 500 &mu;l and remove from concentrator.</li>
	<li>Filter vector through a 13 mm diameter 0.2 &mu;m syringe filter. Vector should be aliquoted and stored at -80&deg;C until required.</li>
</ol>
7. Titering of viral stocks (3 days)
This requires a promoter that is active in Hek293 cells driving a reporter gene or an immunocytochemically detectable gene product. When producing a vector that is not compatible with Hek293 cells other cell lines or primary cell cultures can be used.
<ol>
	<li>Prepare 18 poly-L-lysine coated glass coverslips or 18 wells of Nunc chamber slides by seeding Hek293 cells so that they are 40 - 50% confluent. For titration of Cre-dependent rAAVs use Hek293 cells that stably express Cre recombinase 2.</li>
	<li>Infect each well with serial dilutions of the AAV vector (Fig 2A). Use 3 wells per dilution. We routinely add virus directly to each well.</li>
	<li>After 72 hours fix Hek293 cells by adding an equal volume of 4% paraformaldehyde (PFA) to medium (giving a final concentration of 2% PFA) for ten minutes at room temperature.</li>
	<li>Wash cell three times in PBS, rinse in dH2O and coverslip.</li>
	<li>Count the number of transduced cells from the three wells that have the highest dilution factor, but still contain infected cells, find the average of these and multiply by the dilution factor to give the number of infectious units per microliter (Fig 2B).</li>
</ol>
8. Anticipated Results
Hek293 cells transduced with a virus encoding enhanced green fluorescent protein (eGFP) are shown in Figure 2B. We generally achieve consistent titers of around 6 x 106 infectious particles per micro litre. We routinely use these vectors for stereotaxic injection into the adult rodent brain. This provides a powerful technique for region-specific gene expression 2. To combine this region specificity with cell-type selective gene expression we inject Cre recombinase-dependent rAAVs into target regions of Cre-transgenic mice 2. Figure 2C-E shows examples of stereotaxic injections of Cre-dependent rAAVs encoding eGFP into different brain regions of adult parvalbumin-Cre transgenic mice 3.
<img alt="Figure 1" src="/files/ftp_upload/3348/3348fig1.jpg" /> 
Figure 1. Schematic illustration of the protocol for rAAV production. 1.) Hek293 cells are plated and grown to 70 - 80% confluence. 2.) AAV and helper plasmids are prepared and transfected into Hek293 cells. 3.) Medium is changed back to DMEM 16 hours after transfection and Hek293 cells are cultured for another 56 hours. 4.) Hek293 cells are harvested and lysed. rAAV vectors are separated from cell debris by centrifugation. 5.) Viral particles are purified through heparin columns before concentration and sterilisation. 6.) Hek293 cells are grown on 24 well plates and infect with serial dilutions of the AAV vector to determine the number of infectious units.
<img alt="Figure 2" src="/files/ftp_upload/3348/3348fig2.jpg" /> 
Figure 2. Titering and in vivo application of rAAV1/2. (A) Setup of Hek293 cells for measuring viral titer. Hek293 cells are transduced with serial dilutions of rAAVs. C, uninfected control; N, 1 &mu;l of undiluted viral stock. (B) Hek293 cells infected with rAAVs expressing eGFP. (C-E) Viral eGFP expression is restricted to Cre-expressing neurons in different target regions of parvalbumin-Cre transgenic mice after stereotaxic injection of Cre-activated rAAVs. Examples images show GFP immunoreactivity in (C) the reticular thalamus and globus pallidus, (D) the dentate gyrus and (E) the CA1 region of the hippocampus. DG, dentate gyrus; RT, reticular thalamic nucleus; GP, globus pallidus. Scale bars: B, 500 &mu;m; C, 100 &mu;m; D, 100 &mu;m; E, 500 &mu;m.

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+injection+of+rAAV+with+mannitol+enhances+gene+expression+in+the+rat+brain.">Combined injection of rAAV with mannitol enhances gene expression in the rat brain.</a> Molecular Therapy. 3, 225-232 (2001).</li><li>Fu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-complementary+adeno-associated+virus+serotype+2+vector:+global+distribution+and+broad+dispersion+of+AAV-mediated+transgene+expression+in+mouse+brain.">Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain.</a> Molecular Therapy. 8, 911-917 (2003).</li><li>Ghosh, A., Yue, Y., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Transgene+Reconstitution+with+Hybrid+Dual+AAV+Vectors+Carrying+the+Minimized+Bridging+Sequences.">Efficient Transgene Reconstitution with Hybrid Dual AAV Vectors Carrying the Minimized Bridging Sequences.</a> Human Gene Therapy. 22, 77-83 (2011).</li><li>McCarty, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+delivery+and+selective+transduction+of+glial+cells+in+the+mammalian+brain+by+AAV+serotypes+isolated+from+nonhuman+primates.">Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 17, 1692-1702 (2009).</li><li>Chtarto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-inducible+transgene+expression+mediated+by+a+single+AAV+vector.">Tetracycline-inducible transgene expression mediated by a single AAV vector.</a> Gene Therapy. 10, 84-94 (2003).</li><li>Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+humoral+immune+response+to+the+tetracycline+(Tet)+activator+in+rats+injected+intracranially+with+Tet-off+rAAV+vectors.">Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors.</a> Gene Therapy. 17, 616-625 (2010).</li></ol>

No conflicts of interest declared.

rAAV vectors are becoming increasingly valuable for in vivo studies in animals. Here, we have described a simple and inexpensive protocol for producing rAAVs, which may facilitate the widespread application of this useful vector by avoiding the costly outsourcing of virus production to companies. The protocol (based on reference 1) describes the production of chimeric rAAV1/2 vectors containing capsid proteins from both parental serotypes at equal ratios 4. The purification of the rAAV vectors by binding to heparin columns relies on the expression of AAV2 capsid proteins, but can be combined with serotypes other than AAV1 outlined here. Additionally, AAV6 has been reported to bind to heparin, although with a reduced affinity, and could potentially be purified with heparin columns using this procedure 5,6. It should be noted however, that other serotypes will require different concentrations of NaCl for heparin column elution 5,7.
Packaging rAAV genomes was shown to be optimal between 4.1 and 4.9 kb with a sharp reduction in packaging efficiency up to 5.2 kb 8. This packaging limit can be considered the largest disadvantage of the rAAV system, since cell type-specific regulation of transgene expression is usually achieved by large cis acting elements which can not be accommodated within the small AAV particles. To overcome low titer batches, such as those resulting from attempting to package genes that are close to the AAV packing limit we are combining separate viral batches concentrated to 250 &mu;l into one 500 &mu;l batch, rather than adding PBS as outlined in step 6.3. Reliable cell-type specific transduction can be achieved by combining Cre-driver mice and conditional rAAV cassettes (see below) to avoid stretching the packaging limit.
Of note, while this protocol attempts to provide easy-to-follow instructions on the production of high titer rAAV, it requires the presence of AAV2 capsid protein moieties for purification by heparin affinity chromatography. Serotypes other than AAV2 must be purified using alternate procedures 9. One advantage of heparin column purification is it is believed to produce AAV vectors that have greater infectivity rate than those produced using a caesium chloride gradient 10. However, there are also some drawbacks to this method. For example, other proteins that bind heparin may also be present in the purified viral stock. While the tropism of heparin-purified vectors can be influenced by the vector titer10, our approach to specifically target either excitatory20 or inhibitory neurons (Fig. 2) employs Cre-induced activation of AAV-mediated transgene expression and is titre-independent. An alternative to heparin column purification is the use of an iodixanol density gradient, which produces a higher viral titer and purer preparation than the use of a caesium chloride gradient 10. This method can also be combined with heparin column purification to produce a vector that is greater than 99% pure 11. Therefore, careful consideration needs to be taken by individual groups as to which viral purification method is best for their particular downstream application.
The most common problem associated with this protocol is low viral titer. In our experience this can usually be traced to low transfection efficiency, or the elution of virus from the heparin columns. All transfection materials should be at room temperature before transfection, and test transfections should be performed to find the optimal conditions in each laboratory. Other methods of transfection, such as lipofectamine, are highly efficient, but can be prohibitively expensive. Additionally, the concentration and pH of the heparin column elution solutions is critical for complete elution of virions from heparin columns without damage. Another important point is that the packaging cells must adhere well to the surface of the tissue culture dishes. If the cells detach at some stage during the procedure it is advised to terminate the experiment and defrost a new vial of cells.
Spatio-temporal control of transgene expression in rodents is easily achieved by accurate anatomical targeting and the developmental stage of the animal. Efficient AAV-mediated gene transfer has been shown in utero 12,13. In the adult brain, the area infected with rAAV particles after stereotaxic injection can be adjusted from very small targeted regions, to much larger areas not only by changes in the viral titer but also by altering the injection parameters. These include the volume and speed of injections and the inclusion of the polyol mannitol with the virus suspension 14. Mannitol can also be used to enhance infection of a variety of tissues after systemic rAAV injection 15.
The packaging capacity of rAAV (approximately 4.7 kb) is probably the most limiting factor in terms of the genes that can be expressed from these viral vectors. However, recent studies have shown that this can be partially overcome by splitting larger genes or expression cassettes into separate rAAV vectors and introducing a bridging sequence, initiating gene expression when virions infect the same cell 16. Expression levels of genes carried by rAAV vectors can also be greatly enhanced by using self-complimentary rAAV vectors 17. Stereotaxic injection of rAAV vectors provides a rapid, inexpensive and powerful method for inducing gene expression in a region-specific manner. In combination with 2nd generation RNA interference strategies 18 rAAVs can also be used for region-specific gene-knock down. rAAVs can be combined with Cre-transgenic mice or cell-type specific promoters to achieve circuit- and cell-type-specific gene expression, permitting genetic manipulations with high spatial resolution 2,19-21, while fitting rAAVs with e.g. the tet system adds temporal control over virus-mediated gene expression 22,23.These examples illustrate the enormous combinatorial potential of these vectors and predict a rapid increase in rAAV-based studies over the years to come.

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amicon Ultra centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC810024</td>
			<td>100,000 MWCO</td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbeco's modified Eagle medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>10567014</td>
			<td>Supplement with 10% FCS and 10 units/ml penicillin/100 &mu;g/ml streptomycin</td>
		</tr>
		<tr>
			<td>Hek293 cells</td>
			<td>American type culture collection</td>
			<td>CRL-1573</td>
			<td>Use at passage less than 30</td>
		</tr>
		<tr>
			<td>HEPES buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>51558</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HiTrap Heparin cartridge</td>
			<td>Sigma-Aldrich</td>
			<td>54836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iscove's modified Dulbecco's medium</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
			<td>Supplement with 5% FCS </td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D5670</td>
			<td>Make fresh solution for each batch</td>
		</tr>
		<tr>
			<td>15 cm Tissue culture dishes</td>
			<td>Fisher Scientific</td>
			<td>157150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stbl2 competent cells</td>
			<td>Invitrogen</td>
			<td>10268-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Virkon solution</td>
			<td>Fisher Scientific</td>
			<td>NC9821357</td>
			<td>Leave to disinfect overnight before discarding to drain</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents required for protocol

production and titering of recombinant adeno-associated viral vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Production and Titering of Recombinant Adeno-associated Viral Vectors

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

production-and-titering-of-recombinant-adeno-associated-viral-vectors

Research

JoVE Journal

Immunology and Infection

61.2K ビュー.  University of Aberdeen. 組換えアデノ随伴ウイルス（rAAVs）ベクトルはのためにますます貴重になってきています in vivoで動物での試験。我々はrAAVsが実験室で製造することができるとどのようにこれらのベクトルが生成される感染性粒子の数の正確な読みを与えるために滴定することができる方法について説明します。

ビデオ: Production and Titering of Recombinant Adeno-associated Viral Vectors

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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 ...

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.

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. ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...