Methods of Virus Purification

As the COVID-19 pandemic continues to disrupt the quality of life, investigators are gaining more insight into the SARS-CoV-2 virus behind this disease. Continued research in elucidating the dynamics of the SARS-CoV-2 life cycle is essential to facilitate the design and development of novel diagnostics and antiviral therapies. Epigenetic modifications are known to play important roles in the life cycles of RNA viruses like human coronavirus. The SARS-CoV-2 epigenome thus presents a suitable target of remedial treatment. Modified adenosines, for instance, such as m⁶A, m⁶A_m, and 2'-O-me are reported to affect the viability of specific RNA viruses by modulating viral cap structures, viral replication, innate sensing pathways, and the innate immune response. The SARS-CoV-2 RNA genome contains more than 50 m6A modification sites.

The relatively low abundance of viral genomic material within the nucleic acid milieu of clinical samples places constraints on the utility of epigenetics-related applications, like m⁶A RNA methylation ELISAs, to specifically study the virus epigenome. Such assays require highly pure input material, free from host-derived impurities whose epigenetic modifications can also be detected and interfere with results. Described below are some common nucleic acid purification methods, with a focus on viral RNA isolation.

Filtration

Membrane filtration is a physical separation technique whereby the contents of a liquid either pass through or are excluded by the membrane, depending on the properties of the membrane (e.g., porosity) and the liquid contents (e.g., particle size). By choosing a membrane with the proper pore size (e.g., 0.45 μm; PMID: 30619900), a liquid virus sample can be filtered to exclude cells and other large non-viral contaminants.

Nuclease treatment

Capsids and envelopes afford viral genomes with an added layer of protection against nuclease degradation (PMID:21413309). DNAse and RNAse are commonly exploited to eliminate exogenous nucleic acid while the protected viral genome remains intact.

Host-derived genomic DNA and small RNA can be separated from the viral RNA genome using the phenol/chloroform extraction method, which involves the differential partitioning of DNA and RNA into organic and aqueous phases, respectively (PMID:29984193). First, a monophasic solution is created by adding acidic phenol and acidic buffer to the nucleic acid-containing sample. The addition of chloroform creates a biphasic system in which DNA and protein partition into the lower organic phase while the upper aqueous phase retains the RNA. The aqueous phase can be additionally treated with isopropanol and processed by silica column-based purification to extract either total RNA or individual small (miRNA, tRNA, 5 S rRNA, 5.8 S rRNA) and large (mRNA, 18 S rRNA, 28 S rRNA, snRNA) RNA fractions, depending on the added volume of alcohol.

Centrifugation

Low-speed centrifugation (e.g., 6000 × g for 10 min at 4 °C; PMID: 24036074) is a simple and convenient way to purify viruses. Cells and large cellular debris are pelleted, and the suspended virions in the supernatant can be subjected to more stringent purification.

The size and density of viruses are distinct from those of cell organelles (e.g., mitochondria, ribosomes, nuclei), biological macromolecules (e.g., DNA, RNA, protein), and other host constituents sullying viral samples. Ultracentrifugation utilizes these physicochemical properties to separate viruses from non-viral elements. This separation is typically achieved through a combination of sucrose and cesium chloride density gradients (PMID: 15217618). Prior to ultracentrifugation, cellular debris is eliminated during an initial centrifugation step. Remaining impurities are further removed by first applying sucrose density gradient centrifugation, which segregates particles based on their S value, or sedimentation coefficient. Cesium chloride density gradient equilibrium centrifugation, which segregates particles based on their buoyant density, is then applied to the resulting virus crude fraction to obtain purified virus fragments.

Precipitation

Concentrated virus preps are often required for storage or downstream applications like infection and viral genome isolation. Precipitation methods have been developed to rapidly, easily, and cheaply concentrate viruses and remove impurities on the basis of solubility in solutions of inorganic salts or polyether compounds (e.g., ammonium sulfate, PEG-6000). For example, at 40% saturation levels, ammonium sulfate has been reported to induce virus precipitation from cell culture medium supplemented with 10% FCS, with the majority (85%) of protein remaining solubilized (PMID: 11021595).

Column chromatography

A variety of chromatographic resins (e.g., Protein A, anion exchange, cation exchange) are available that, depending on the chromatography operating parameters (e.g., resin type; buffer composition and pH; etc.), can be used to separate viruses from host nucleic acid, protein, and the like (PMID:12641291; PMID: 19575414, PMID: 24504234). However, such methods require expensive instrumentation, elaborate protocols, specialized technical expertise, and trained personnel for operation.

VIDISCA

A method for virus discovery based on the cDNA-amplified restriction fragment–length polymorphism technique, or VIDISCA for short (PMID:15034574), allows for the preferential amplification of viral nucleic acid. Blood plasma/serum and culture supernatant input samples have been successfully tested with this method. In a typical VIDISCA protocol, viral nucleic acid is first selectively enriched via centrifugation and DNase treatment to eliminate cells, mitochondria, and their associated genetic content. The encapsulated viral genome is then extracted. For single-stranded RNA viruses like coronaviruses, a reverse-transcription step with random hexamer primers is required, followed by second-strand synthesis of the complementary DNA. The resulting double-stranded DNA (dsDNA) is purified with phenol/chloroform extraction and ethanol precipitation. Purified dsDNA is subsequently processed with restriction enzymes that digest short recognition sequences present in virtually every virus. After ligation of anchors to the ends of the digested fragments, the target undergoes two rounds of PCR amplification, an initial preamplification followed by a second “nested” selective fragment amplification, using primers specific for the anchor sequences.

Approximately 80% of total cellular RNA is ribosomal (rRNA), and host-derived RNA constitutes the majority of RNA found in human plasma samples (PMID:28646219). Therefore, modifications to the VIDISCA protocol have been made that incorporate selective rRNA depletion for improved assay sensitivity and specificity. The use of rRNA reverse transcription-blocking oligonucleotides, as well as non-random hexamers that cannot anneal to rRNA, have been shown to inhibit non-viral cDNA synthesis and reduce background amplification.

Selective rRNA depletion with rRNA-specific DNA “scissor probes”

Ribosomal RNA contamination can also be selectively depleted with rRNA-hybridizing oligonucleotide probes and concomitant nuclease-mediated hybrid digestion (PMID: 22900061). These synthetic probes are designed to complement particular rRNA sequences. RNase H treatment leads to targeted cleavage of rRNA in the rRNA/DNA duplexes, while DNase I is employed to remove residual probe. The rRNA-depleted sample can then be subjected to downstream purification. Given the proper reaction conditions, rRNA is specifically cleaved while the integrity of non-hybridized RNA is preserved.

Virus amplification using in vitro cell cultures

Given the low titers at which viruses are usually produced, in vitro cell cultures are utilized for amplifying virions to analytically relevant amounts. Inoculated Vero and HEK293T cells have been effectively used for virus expansion purposes PMID: 27773536). The method involves a media change 24 hours post-inoculation, and virions released by infected cells are then harvested from the supernatant after another 24-hour incubation.

Commercial viral RNA extraction kits

Column- and magnetic bead-based methods provide a quick and convenient means to extract the viral RNA genome from clinical specimens. EpiGentek offers a variety of magnetic bead and spin column kits for isolating RNA from diverse sample sources (cells, tissues, whole blood, saliva, nasopharyngeal swabs) and species (viral, mammalian, bacterial, fungal, plant). Detergents and chaotropic salt are used to lyse cells and inactivate RNases. A specialized high salt buffering system allows RNA bases to bind to the magnetic beads or to the glass fiber matrix of the spin column while contaminants pass through. Impurities are efficiently washed away, and the pure nucleic acid is eluted with an aqueous buffer, without the need for phenol extraction or alcohol precipitation. These kits are ideal for downstream PCR analysis; primers targeting the viral genome can be used in conjunction with those that amplify the RNase P gene of host DNA, which also binds to the columns/beads and serves as an internal or extraction control.

Combining methods

The methods included above are generally not sufficient, when performed alone, for adequate purification of viruses. Studies focused on the virus epigenome require highly pure input material, without interference from the epigenetic modifications of host DNA, RNA, or protein. Combinations of the aforementioned methods can increase viral recovery, yield, and quality. To illustrate, low titer RNA viruses can be amplified and subsequently retrieved from the supernatant of inoculated cell cultures. Following nuclease treatment and centrifugation to remove host contaminants, the viral genome can be extracted with a commercial isolation kit for downstream m6A RNA methylation analysis.