Purification of viruses ppt

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Additional treatment with RNase and DNase prior to in-slurry purification did not visibly improve virus purity. C L cells were exposed to purified reovirus samples from B at indicated dilutions.

Results from two independent preparations are provided. F Infectivity was assessed by plaque titration. G Samples from E were subjected to western blot analysis.

A separate membrane was blotted with rabbit anti-STAT1 and Cy5-conjugated anti-rabbit secondary antibodies. The integrity of reovirus preparations was then tested by two methods. The in-slurry Capto Core purification method was then further validated and optimized. To test applicability to alternative cell lines, we used reovirus-infected NIH3T3 fibroblasts transformed with constitutively active Ras oncogene.

Ras-transformed NIH3T3 cells are highly susceptible to reovirus 7 , 8 , 9 and can reach high cell density, supporting efficient reovirus production. Starting with four times more cells then L cells i. Virus purity was monitored by Imperial TM coomassie- Fig. Relative to unpurified lysates or Vertrel extraction alone, 2—3 sequential in-slurry purifications successfully removed most cellular contaminants including the aforementioned non-specific NS protein. Silver staining showed some contaminating bands and smears in virus preparations purified by both CsCl gradient ultracentrifugation and Capto core in-slurry approaches.

Plaque titration demonstrated retained infectivity of purified reovirus Fig. For optimal time-savings versus purity and infectivity, two rounds of in-slurry purification seemed optimal.

Western blot analysis was used to assess levels of specific viral and cellular proteins at higher sensitivity. To determine if the Capto Core in-slurry method could extend to other viruses, we evaluated the method for purification of adenovirus. Adenovirus is a common cause of respiratory illnesses though generally causing only mild symptoms. Adenoviruses are frequently used as vectors for gene expression and are under investigation as oncolytic virotherapies.

A high-throughput purification strategy for adenoviruses would facilitate rapid comparison of natural and engineered adenovirus variants. In contrast to reoviruses, adenoviruses have a DNA genome and replicate in the cell nucleus. Coomassie blue staining showed effective removal of cellular proteins from mock- and infected- cell lysates especially in the absence of sonication.

Similar persistence of dominant adenovirus structural proteins such as hexon and protein V was found between conditions 1—5 Fig. Note that as with reovirus, only a portion of viral proteins produced in infected cells are expected to be assembled into whole virions and therefore recovered from total cell lysates.

Mock- and adenovirus infected lysates were frozen and thawed three times, then either left unpurified lysate or subjected to 4 alternative purification strategies as summarized in A. Locations of major adenovirus proteins are indicated. Densitometric analysis for quantities of hexon proteins is provided. D Coomassie blue staining shows relative adenovirus hexon proteins in purified samples.

E Similar to D but all samples were diluted further by one-fifth and virus-infected cells were quantified flow cytometry. Percent of cells positive for GFP expression are indicated. The best two conditions freeze-thaw with or without Vertrel extraction prior to Capto Core in-slurry purification were then applied to two additional first generation E1 and E3- deleted adenovirus constructs Fig. Addl70—3 contains no transgene insert, while AdGFP encodes green fluorescent protein.

Virion purity was assessed by Imperial TM coomassie- or silver staining. Results suggest that inclusion of Vertrex extraction provides added virion purity, as indicated by further removal of proteins absent from CsCl purified virions.

Vertrel extraction did however slightly reduce recovery, as indicated by a 2—3 fold reduction in hexon protein band intensity. Silver staining suggests that adenovirus purified by Capto Core in-slurry is less pure than adenovirions purified by CsCl density ultracentrifugation, and therefore the in-slurry approach is most valuable for adenovirus screening applications where ultracentrifugation or commercial adenovirus purification columns are not feasible.

Adenovirus titers were similar pre- versus post-purification, indicating reproducible recovery of infectious adenovirus virions regardless of the specific construct used for analysis. The GFP-expressing adenovirus construct AdGFP was also used as a complementary approach to evaluate the infectivity of recovered virions.

Method 1 freeze-thaw plus Capto Core produced equal infectivity to lysates alone, while method 3 inclusion of Vertrel extraction resulted in approximately 4-fold lower levels of hexon and infectious virions. These results demonstrate that infectivity-per-particle is preserved following extraction with Vertrel and purification with Capto Core slurry; albeit Vertrel extraction seems to reduce the number of particles by 2—4 times Fig.

Furthermore, method 1-purified adenovirus had 5-fold lower hexon levels than unpurified samples Fig. Here we present a new method for purifying either reovirus or adenovirus samples in high-throughput format. The capto Core resin can be removed by centrifugation, or completely clarified through MicroSpin columns. The in-slurry method further eliminates the need for chromatography equipment, reduces contamination between samples, and is easily scaled for small, medium, or large preparations.

It should be noted that for cost saving purposes, the resin is advertised as being reusable. Although we did successfully reuse the Capto Core chromatography columns 2—3 times initially data not shown , we opted for single-use applications to avoid cross-contamination between virus mutants.

The main foreseeable drawback to purification through Capto Core columns or in-slurry, is that genome-devoid empty virions are not eliminated. Conversely, since mutations might affect the efficiency of genome packaging, the inclusion of empty virions might help uncover previously overlooked phenotypes.

Our study focused on reoviruses and adenoviruses. The in-slurry adaptation for high-throughput purification is therefore likely to work for these and other viruses as well. Once optimized for individual virus families, Capto Core chromatography or in-slurry methods would enable large numbers of virus samples to be rapidly compared for phenotypes such as infectivity.

For example, the in-slurry method could easily be applied to 24—30 samples in a standard centrifuge or even multiples of 96 samples in well plates. We envision this approach being applied similar to routine high-throughput purification of plasmids and RNA.

The 0. Finally, an increasing number of laboratories use viruses as molecular tools, for example to deliver genes or interfering RNAs using adenovirus, adeno-associated virus, or lentivirus vectors. The rapid in-slurry virus purification strategy may accelerate these routine procedures. The development of the Capto Core chromatography and in-slurry methodology is expected to contribute significantly to the efforts of those having interest in reovirus or adenovirus biology.

For example, many reovirus protein functions have been characterized by comparing phenotypes among natural reovirus variants, reovirus genome reassortants, and laboratory derived reovirus mutants 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , In conjunction with reverse genetics approaches for these viruses 20 , 21 , 22 , 23 , 24 , 25 , it is now possible to design copious virus mutants, purify them in-slurry, and make rapid comparisons of virus proteins, virion structure, and virus replication.

Furthermore, both reovirus and adenovirus are heavily pursued as oncolytic therapies. We and others have demonstrated that reovirus mutants exhibit differences in their oncolytic activities, and that second-generation reovirus variants may possess improved oncolytic potency 17 , 19 , 26 , We have found the Capto Core slurry method to be extremely useful for purifying and comparing various natural isolates Fig.

For transgene-containing adenovirus vectors, this method of purification should yield vector preparations free of transgene product which can confound analysis of vector activity, in little more time than required to prepare a crude lysate. Importantly, this approach should provide a quick method to prepare immune-modulating viruses that are free of cellular proteins such as cytokines, calreticulin and HMGB1 that are known to alter immunologic responses.

Protocols for virus purification by CsCl, by Capto Core chromatography columns, and by Capto Core in-slurry approach are described in the Supplementary Materials and Methods. Natural wild isolates of reovirus were obtained from Edmonton primary effluent collected and identified by Xiao-Li Pang Alberta Provincial Laboratory, Canada.

Adl was generated by previously described methods AdGFP was generated by recombining an dE1-deleted, E3-deleted adenovirus vector backbone with an expression plasmid containing the murine CMV immediate early promoter controlling the green fluorescent protein gene using the AdMax system Microbix The adML-4 expressing IL-2 was previously described Remaining cells were scraped into media and subjected to centrifugation to pellet cells and debris. For western blot analysis, Reovirus proteins were detected using polyclonal rabbit anti-reovirus antibodies generously provided by Dr.

Patrick Lee, Dalhousie University, followed by Cy2-conjugated anti-rabbit secondary antibodies Jackson Immunoresearch , A separate blot was incubated with rabbit anti-STAT1 Cell Signalling , followed by Cy5-conjugated anti-rabbit secondary antibodies. For negative staining, virus preparations were applied to carbon-coated formvar films and stained with 0.

How to cite this article : James, K. Chahal, P. Validation of a high-performance liquid chromatographic assay for the quantification of Reovirus particles type 3.

Transfiguracion, J. Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles. B Biomed. Segura, M. Overview of current scalable methods for purification of viral vectors. Methods Mol. Blom, H. Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification.

Vaccine 32, — Zhao, D. Enterovirus71 virus-like particles produced from insect cells and purified by multistep chromatography elicit strong humoral immune responses in mice. Mendez, I. A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.

J Virol. Methods 90, 59—67 Marcato, P. Ras transformation mediates reovirus oncolysis by enhancing virus uncoating, particle infectivity, and apoptosis-dependent release. Mol Ther. Shmulevitz, M. Cancer Res 70, — Yin, P. First, the mixture may contain some substance that should be isolated from the rest of the mixture: this process of isolating and thus removing substances considered to be contaminants is called purification.

RNA Viruses. Which is the first step in the purification process? Table of Contents. Post navigation Previous post. Next post. We use cookies on our website to give you the most relevant experience by remembering your preferences and repeat visits. Do not sell my personal information.

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