Purification Of Virus Research Articles

Fabrication and characterization of a cellulose monolith-like particle for virus purification

Monolith-like particles (MLP) are a new base bead architecture optimized for industrial chromatographic downstream processing of very large targets such as whole viral particles (5–300 nm diameter). They have been designed with large pore size though-channels to facilitate access of large diameter targets to the intraparticle chromatographic surface. Herein we describe a novel MLP bead architecture that have been fabricated and characterized for virus purification. This is the first study to demonstrate cellulose-based MLP beads applied for bioseparation of viral particles. The MLP was produced from cellulose acetate by a combined concept of thermally induced phase separation and oil-in-water emulsification. Hydrolyzation, cross-linking, and surface modification with dextran sulfate were carried out for pseudo-affinity chromatographic applications. Scanning electric microscopy analysis showed the presence of large pores that penetrated from the outer surface to within the intraparticle structure. Mercury intrusion porosimetry analysis estimated the mode pore radius to be 1.5 µm. For influenza A virus purification, the MLP surface modified with dextran sulfate exhibited significant higher dynamic binding capacity compared with commercial resins, Cellufine Sulfate and Capto DeVirS. These results demonstrate that viral particles can access the intraparticle surface within these novel large through-pore MLP beads. The novel MLP base architecture described in this study will be a useful support matrix for giant biomolecule and whole viral particle purification.

Safe use of PHI6 IN the experimental studies

Surrogate viruses theoretically provide an opportunity to study the viral spread in an indoor environment, a highly needed understanding during the pandemic, in a safe manner to humans and the environment. However, the safety of surrogate viruses for humans as an aerosol at high concentrations has not been established. In this study, Phi6 surrogate was aerosolized at high concentration (Particulate matter2.5: ∼1018 μg m−3) in the studied indoor space. Participants were closely followed for any symptoms. We measured the bacterial endotoxin concentration of the virus solution used for aerosolization as well as the concentration in the room air containing the aerosolized viruses. In addition, we measured how the bacterial endotoxin concentration of the sample was affected by different traditional virus purification procedures. Despite the purification, bacterial endotoxin concentration of the Phi6 was high (350 EU/ml in solution used for aerosols) with both (two) purification protocols. Bacterial endotoxins were also detected in aerosolized form, but below the occupational exposure limit of 90 EU/m3. Despite these concerns, no symptoms were observed in exposed humans when they were using personal protective equipment. In the future, purification protocols should be developed to reduce associated bacterial endotoxin levels in enveloped bacterial virus specimens to ensure even safer research use of surrogate viruses.

Scaling Up of Steric Exclusion Membrane Chromatography for Lentiviral Vector Purification.

Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.

Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance.

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.

Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55Gag

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

Genomic analysis and development of infectious clone of a novel carlavirus infecting blueberry.

A new blueberry virus was discovered using high throughput sequencing (HTS). Using sequence identity values, phylogenetics, serological and biological properties, we propose the virus, putatively named blueberry virus S (BluVS) to be a distinct species within the genus Carlavirus (family Betaflexiviridae). The genome was analyzed in depth and an infectious clone was developed to initiate studies on virus pathogenicity. Agroinfiltration of the binary vector construct produced severe systemic symptoms in Nicotiana occidentalis. Back inoculation using sap from agroinfiltrated N. occidentalis produced identical symptoms to the recipient plants (N. occidentalis) and virus purification yielded flexuous carlavirus-like particles. However, unlike BlScV, BluVS caused symptomless infection in Chenopodium quinoa and reacted weakly to BlScV antibodies in enzyme-linked immunosorbent assay (ELISA). Collectively those results provided evidence for the distinct speciation of BluVS. The availability of an infectious clone provides tools for future studies on the biology of the virus.

Improvement of viral vector purification using Mustang®️ Q membrane chromatography

David McCall, Editor, Cell & Gene Therapy Insights, talks to (pictured from left to right) Saadia Zakai, Product Development Manager, Gene Therapy, Pall Corporation, Mark Schofield, Senior R&D Manager, Pall Corporation and Hélène Lebas, Process Development Manager, Yposkesi, an SK pharmteco company

An efficient capture strategy for the purification of human adenovirus type 5 from cell lysates

An efficient capture step for human adenovirus type 5 from cell lysate was developed as an initial virus purification step from cell debris supernatant. Organosilane-based polymer particles were synthesized and experimental monomer screening allowed the selection of appropriate functionalities for the development of particles for virus binding. After elution, virus recoveries of 83 % were obtained with significant reduction of matrix proteins and residual host cell DNA. Therefore, the implemented capture strategy for adenovirus via polymer particles provides a scalable and reproducible approach to reduce time and cost during virus purification processes.

Recent Advances in Methods Detecting the Dengue Virus

Dengue fever, caused by the Dengue virus (DENV), has emerged as a mild to lethal human infection. Globally, an estimated 400 million patients have been infected with DENV over the past 10 years, and this number is expected to increase in the coming years. The DENV, possessing a single positive- stranded RNA with five serotypes, is transmitted by mosquitos of the Flaviviridae family—particularly, the Flavivirus genus and Aedes species. The DENV genome encodes three structural and seven non- structural proteins. In the Kingdom of Saudi Arabia (KSA), serotype 2 of the dengue virus (DENV-2) emerged in 1994 and caused a major epidemic in Jeddah, KSA. Dengue outbreaks first appeared in Makkah in 2004, according to the characteristics studied in hospitalized patients. Major factors causing the wide and rapid distribution of the virus include increased urbanization, migration, global commerce, weather variation, inadequate mosquito control, the development of pesticide resistance in mosquitos, irregular use of insecticides, and shifting climatic circumstances. Detection methods currently used for DENV include the detection of viral antigens (Ag) (virus extraction and purification, immunofluorescence test, and NS1 detection assay), serological assays (plaque reduction neutralization titers (PRNT), IgM/IgG immunological assays), and RNA detection using RT-PCR. Low sensitivity, specificity, and accessibility of the detection protocols represent major challenges necessitating the advent of more amenable protocols. The Aedes mosquito is the primary vector for horizontal transmission of DENV. DENV-infected mosquitos infect people, and DENV passes from one human to another through this vector. Once acquired, the virus requires 5–7 days of incubation before the patient exhibits various symptoms of dengue fever; subsequently, uninfected mosquitoes that come in contact with infected patients feed on their blood and become infected. The DENV may also be spread through the mating of male and female Aedes mosquitoes. The reverse transcription loop-mediated isothermal application (RT-LAMP) has emerged as one of the most adaptable viral detection procedures. This method could prove to be an excellent pathogen detection tool because it is cheap, simple, sensitive, cost-effective, accessible, and fast. The method relies on the use of 4–6 primers to recognize eight different loci in the target sequence contained in the DENV clinical isolates with a 100% success rate and a sensitivity of about 93%. We strongly recommend the use of LAMP in detecting spots of virus spread, especially in urban regions where accessibility to detection methods is scarce.

Viral Coinfections.

In nature, viral coinfection is as widespread as viral infection alone. Viral coinfections often cause altered viral pathogenicity, disrupted host defense, and mixed-up clinical symptoms, all of which result in more difficult diagnosis and treatment of a disease. There are three major virus-virus interactions in coinfection cases: viral interference, viral synergy, and viral noninterference. We analyzed virus-virus interactions in both aspects of viruses and hosts and elucidated their possible mechanisms. Finally, we summarized the protocol of viral coinfection studies and key points in the process of virus separation and purification.

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly.

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Roadmap to success in AAV purification. In-process control, high throughput & novel column modalities as necessary means for control over scalable AAV process

Manufacture and purification of recombinant adeno-associated viruses (rAAV) require development and optimization of processes to ensure the best possible quality of the final rAAV product. To do so, different strategies in upstream can be used to achieve the highest possible viral titer and lowest amount of impurities, both of which further influence downstream. Second challenge involves removal of cell debris where different pre-treatments can be utilized. In the next step, optimized capture of rAAV on a cation-exchange chromatography should be developed to remove impurities and achieve a high recovery of rAAV. In the end, several chromatographic options are available to remove empty and defected capsids, so only functional viruses can be isolated. Here, the process of manufacturing and purification of rAAV has been designed using monolithic columns to achieve this important goal of preparing rAAV for the use in gene therapy. 

Bioinformatics Predicted Linear Epitopes of the Major Coat Protein of the Beet Yellows Virus for Detection of the Virus in the Cell Extract of the Infected Plant.

Beet yellows virus, which belongs to the genus Closterovirus, family Closteroviridae and has a significant negative economic impact, has proven to be challenging to detect and diagnose. To obtain antibodies against BYV, we propose an easier bioinformatics approach than the isolation and purification of the wild virus as an antigen. We used the SWISS-MODEL Workspace (Biozentrum Basel) protein 3D prediction program to discover epitopes of major coat protein p22 lying on the surface of the BYV capsid. Sequences coding these epitopes were cloned into plasmid pQE-40 (Qiagen) in frame with mouse dihydrofolate reductase gene. Fused epitopes were expressed in Escherichia coli and isolated by the Ni-NTA affinity chromatography. Murine antibodies were raised against each epitope and in a combination of both and characterized by dot-ELISA and indirect ELISA. We successively used these antibodies for diagnosis of virus disease in systemically infected Tetragonia tetragonioides. We believe the approach described above can be used for diagnostics of difficult-to-obtain and hazardous-to-health viral infections.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP.

Purification of viruses, especially for therapeutic purposes, is a tedious and challenging task. The challenges arise due to the size and surface complexity of the virus particles. VSV-GP is a promising oncolytic virus, which has been approved for phase I clinical trials by the Food and Drug Administration (FDA) of United States and Paul Ehrlich Institute (PEI) of Germany. The virus particles of VSV-GP are larger in size than vectors commonly used for gene therapy (e.g., adenovirus, adeno-associated virus, etc.). The current established proprietary clinical-grade manufacturing process for the purification of VSV-GP encompasses several chromatographic and non-chromatographic steps. In this study, we describe a new single-step purification process for the purification of VSV-GP virus, using cation exchange convective flow column with relatively higher yields. The purified virus was characterized for its quality attributes using TCID50 assay (for viral infectivity), host cell protein contaminant ELISA, SDS-PAGE, size exclusion chromatography (SEC), and cryo-electron microscopy. Furthermore, the purified viral therapeutic material was tested in vivo for its efficacy and safety. All these characterization methods demonstrated a therapeutic virus preparation of high purity and yield, which can be readily used for various studies.

Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes

The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 1013 vp·mL−1) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 1013 capsids·mL−1·min−1) and logarithmic reduction value of host cell proteins (HCP LRV ~ 1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.

Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles

The determination of the electrostatic charge of biological nanoparticles requires a purified, mono-disperse, and concentrated sample. Previous studies proofed an impact of the preparation protocol on the stability and electro-hydrodynamics of viruses, whereas commonly used methods are often complex and do not allow the required sample throughput. In the present study, the application of the (I) steric exclusion chromatography (SXC) for the Orf virus (ORFV) purification and subsequent physicochemical characterization was evaluated and compared to (II) SXC followed by centrifugal diafiltration and (III) sucrose cushion ultracentrifugation. The three methods were characterized in terms of protein removal, size distribution, infectious virus recovery, visual appearance, and electrophoretic mobility as a function of pH. All preparation techniques achieved a protein removal of more than 99 %, and (I) an infectious ORFV recovery of more than 85 %. Monodisperse samples were realized by (I) and (III). In summary, ORFV samples prepared by (I) and (III) displayed comparable quality. Additionally, (I) offered the shortest operation time and easy application. Based on the obtained data, the three procedures were ranked according to eight criteria of possible practical relevance, which delineate the potential of SXC as virus preparation method for physicochemical analysis.

Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions.

Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captured along with the capsids and incorporated into the virions as host cell signatures. In this report, we reviewed the existing information on host cell signatures in human cytomegalovirus (HCMV) virions. We analyzed the published proteomes of the HCMV virion preparations that identified a large number of host cell proteins. Virion purification methods are not yet advanced enough to separate all of the components of the rich extracellular material, including the large amounts of non-vesicular extracellular particles (NVEPs). Therefore, we used the proteomic data from large and small extracellular vesicles (lEVs and sEVs) and NVEPs to filter out the host cell proteins identified in the viral proteomes. Using these filters, we were able to narrow down the analysis of the host cell signatures within the virions and determine that envelopment likely occurs at the membranes derived from the tubular recycling endosomes. Many of these signatures were also found at the autophagosomes, suggesting that the CMV-infected cell forms membrane organelles with phagophore growth properties using early endosomal host cell machinery that coordinates endosomal recycling.

Reducing the Effectiveness of Ward Particulate Matter, Bacteria and Influenza Virus by Combining Two Complementary Air Purifiers.

Air purifiers should pay much attention to hospital-associated infections, but the role of a single air purifier is limited. The goal of this study was to evaluate the effectiveness of the combined application of the nonequilibrium positive and negative oxygen ion purifier (PNOI) and the high-efficiency particulate air filter (HEPA) on a complex, polluted environment. Two of the better performing purifiers were selected before the study. The efficacy of their use alone and in combination for purification of cigarette particulate matter (PM), Staphylococcus albicans, and influenza virus were then evaluated under a simulated contaminated ward. PNAI and HEPA alone are deficient. However, when they were combined, they achieved 98.44%, 99.75%, and 100% 30 min purification rates for cigarette PM, S. albus, and influenza virus, respectively. The purification of pollution of various particle sizes and positions was optimized and reduced differentials, and a subset of airborne influenza viruses is inactivated. Furthermore, they were superior to ultraviolet disinfection for microbial purification in air. This work demonstrates the strong purification capability of the combined application of these two air purifiers for complex air pollution, which provides a new idea for infection control in medical institutions.

POROSTM CaptureSelectTM AAVX elution optimization study for optimized recovery of AAV6 capsids

Watch the video or view the poster to learn about:Optimum elution conditions to maximize recovery of AAV6 capsidsThe impact of pH (2.0, 2.5, and 3.0) and excipient effect (arginine, MgCl2, and propylene glycol) on AAV recoveryHigh-throughput screening and RoboColumn approachesDr Jenny England is an R&D Manager in the Application group in Purification and Pharma Analytics at Thermo Fisher Scientific. Jenny is a biophysicist by training and earned her PhD from Georgetown University. After graduate school, she did a post doc at the National Cancer Institute that focused on structure-based drug design for protein kinase complexes. Jenny currently leads the application group for process development of the POROS resin products for antibody, mRNA, plasmid, and viral vector purification. Additionally, Jenny evaluates new and emerging technologies that can be applied to solve unmet customer needs in the bioproduction workflow.