Purification of viruses by centrifugation

Has PDF. Publication Type. More Filters. Extraction and purification of nucleic acids from viruses. View 4 excerpts, cites background and methods. Comparison of ultracentrifugation methods for concentration of recombinant alphaviruses: sucrose and iodixanol cushions. Sequencing and characterization of virus genomes. PloS one. Evaluation of methods to purify virus-like particles for metagenomic sequencing of intestinal viromes. BMC Genomics.

View 1 excerpt. Viruses in the Oceanic Basement. Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut. Benchmarking protocols for the metagenomic analysis of stream biofilm viromes. Viruses drive microbial diversity, function and evolution and influence important biogeochemical cycles in aquatic ecosystems.

Despite their relevance, we currently lack an understanding of their … Expand. Separation of subcellular components and viruses by combined rate- and isopycnic-zonal centrifugation. National Cancer Institute monograph. View 2 excerpts, references background.

Serum used as part of cell culture medium can act as a carrier, potentially increasing virus yield, but can also lead to clumping and can add to the background of the sample.

If purification in the presence of serum fails despite adequate starting virus titer, a serum-free preparation might be advantageous. For this reason an alternative serum-free purification protocol has been provided below. After concentration, but before further proteomics analysis, electron microscopy to look for virion density and spike coverage can be a useful quality control tool.

The reliability of results from virion purification will depend on the percentage of cell-derived exosomal vesicles that are co-purified with the virions, although the contributions of exosome-derived proteins can be estimated by performing the same purification in parallel on uninfected cell culture supernatant.

It is hoped that these techniques will facilitate further examination of coronavirus, torovirus, arterivirus, mesonivirus, and ronivirus virion proteomics. HEPES-saline: 0.

All other reagents and equipment as in Subheading 2. Since the quality of the virus preparation is the most important component of proteomics studies, two purification protocols are listed below. Either can yield high-quality coronavirus, but Subheading 3.

Serum-free purification Subheading 3. For best results, the purification process should be completed in 1 day, and the virus should be used immediately. This method is suitable for most coronaviruses that grow well in cultured cells, and has been used successfully with severe acute respiratory syndrome-coronavirus SARS-CoV , feline coronavirus FCoV and mouse hepatitis virus MHV , coronavirus virus-like particles, and torovirus in addition to several types of influenzavirus, arenavirus, and retrovirus-like particles [ 3 , 4 ].

In the case of infectious bronchitis virus IBV , this method has been used successfully for the purification of virus from embryonated chicken eggs. For the purpose of this protocol, it is assumed that IBV is being prepared on Vero cells. Remove and discard the culture medium 24 h after inoculation. Collect the cell culture supernatant 48 h after inoculation. Store a small sample for plaque assay titration see Note 4. Refill the pipette with 1 ml more sucrose solution than you will need, tilt the tube as much as possible without spilling the sample and place the pipette tip just above the bottom of the tube.

There will be visible lines at the border between steps if this is done correctly see Note 5. Transfer the supernatant to the largest available screw-cap centrifuge bottles that will fit your rotor, noting the total volume. It is best to use a high-capacity rotor at this stage Sorvall GSA, for example to minimize preparation time. During the centrifugation, prepare fresh screw-cap centrifuge bottles containing 10 g of dry PEG and 2. After centrifugation step 7 , an off-white or yellow pellet of cell debris will be visible.

Quickly decant the supernatant into the centrifuge bottles or conical flask prepared earlier with PEG and NaCl see Note 7. Transfer the solution to centrifuge bottles, if necessary. Decant and discard supernatants immediately to minimize the amount of virus that is lost by resuspending.

A large opaque white pellet should be present in each of the flasks following centrifugation, and may run from the bottom to top of one side if you used a fixed-angle rotor. Avoid passing the sample through a pipette at this step, if possible.

It is critical that the PEG pellets be completely resuspended before proceeding to the next step. The resuspended pellet will be viscous if this step is done correctly see Note 8. Optionally, add one-tenth the volume of each pellet of Tracking dye.

This can be left for 10 min with the resuspended PEG pellets, and will penetrate virions to fluorescently label the RNA inside without any additional permeabilization. This will make it possible to locate the virus-containing fraction or section of the pellet in any subsequent step, simply by resting the tube in a bottomless tube rack or clear beaker and illuminating the sample briefly with a UV transilluminator.

This step is useful for troubleshooting the purification procedure. It is important not to disturb the gradient layers at this stage. In this manner, overlay the resuspended PEG pellet carefully onto the sucrose gradients. Since the pellet will be compact, it is not important to brake the centrifuge slowly.

After centrifugation, decant and discard supernatants immediately. Invert the empty tubes on an absorbent surface for 5 min, and tap gently to wick away any remaining sucrose solution that may have gathered near the rim of the centrifuge tube. Do not use a pipette to resuspend the virus, as this may shear spikes and damage fragile viral envelopes see Note 9.

Use a P pipette tip from which the pointed end has been cut off to gently transfer the virus suspension to a cryovial with minimal shearing. Discard any insoluble material that remains, as most of the authentic virus will resuspend quickly and easily in comparison. At this stage, the virus should be monodisperse, and can be formalin inactivated if desired.

Purified virus for mass spectrometry should not be formalin fixed, but can instead be inactivated using the solvent that will be used for mass spectrometry, provided this has been validated for your virus see Note This alternative method is suitable for purification of viruses that grow to lower titers. Serum-free culture and preparation can also be used to remedy solutions that fail for cryo-EM or proteolysis due to high viscosity, non-viral protein contamination, or large amounts of insoluble material.

Percentage recovery will typically be lower than that with Subheading 3. Perform steps 2—15 of Subheading 3. The PEG-protein pellets should be white, and may be quite small and susceptible to resuspending quickly upon standing for even a few minutes. Decant and discard the supernatants immediately. Perform steps 16—22 of Subheading 3. The final translucent pellet may be small and quite difficult to see, but the presence of the virus can be confirmed using the Tracking dye and a transilluminator after removing the supernatant, if the dye was added at step 16 of Subheading 3.

The number of infectious virus particles in the final preparation should be directly assessed by plaque assay or similar means as a retrospective measure of quality.

Photographs of lysates and resin post-separation show that the resin was capable of removing contaminants such as phenol red pH indicator introduced from cell culture media. Furthermore, the lysate progressively clarified to almost-clear by the second sequential extraction. A Photographs depict steps of Capto Core in-slurry reovirus purification as described in text.

B—D Reovirus particles were purified by CsCl gradient ultracentrifugation or by 1—3 rounds of in-slurry purification with Capto Core resin in the absence or presence of microcentrifuge filtration to remove residual resin. E Several wild isolates of reovirus obtained from primary effluent by Dr. The integrity and purity of reovirus purified by the Capto Core slurry approach was examined by negative stain transmission electron microscopy EM.

It was evident that after 1, 2, or 3 rounds of slurry purification, reovirus particles remained intact similar to CsCl gradient ultracentrifugation Fig. However, with successive addition of Capto Core resin, there appeared to be an increase of large debris perhaps attributed to the resin itself. Virions were effectively clarified through simple MicroSpin columns with a 10 micron frit for bed support Fig.

The residual small particulates in virus preparations following MicroSpin column filtration may represent remnants of the resin rather than proteinaceous contaminants since SDS-PAGE gel electrophoresis showed that 2—3 rounds of Capto Core slurry purification produced similar virus purity and capture efficiency relative to CsCl gradient purification Fig. Furthermore, MicroSpin column filtration did not lower virus yields.

Importantly, virus titers were equivalent between CsCl gradient and Capto Core slurry approaches Fig. The quality of several independent preparations of natural isolates of reovirus Wild, W and our laboratory isolate of wild-type reovirus serotype 3 Dearing Wt seems comparable between CsCl gradients versus Capto Core Slurry approaches Fig. Altogether, results suggest that optimal large-scale slurry-based purification of reovirus should include 2—3 clarifications with Capto Core slurry, followed by an optional pass through polyethylene filter columns to remove large particulates.

Having successfully purified reovirus in suspension using the Capto Core resin in a medium scale preparation, we next optimized the application of this approach for small-scale but high-throughput applications Fig. To reduce processing time and complexity, samples were not sonicated but rather extracted by addition of Vertrel, vortexing, and centrifugation.

The resin was removed by brief centrifugation, and samples were subjected to protein electrophoresis and Imperial TM Coomassie blue staining Fig. Vertrel extraction followed by in-slurry purification with Capto Core resin was effective at maintaining high yields of reovirus proteins with minimal contamination by cellular proteins. We did note, however, that a non-specific NS protein persisted in preparations unless large volumes of resin were used.

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.