We wish to thank Ms. Laura M. Cooper and Dr. Lijun Rong from the University of Illinois at Chicago for providing the pseudovirus samples used in this protocol (SARS-CoV-2, SARS-CoV-1, H5N1), as well as Dr. Alvaro Hernandez and Dr. Chris Wright of the DNA Services facility of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign for their assistance with high-throughput sequencing, and many members of the Lu group who have helped us with in vitro selection and aptamer characterization techniques. This work was supported by a RAPID grant from the National Science Foundation (CBET 20-29215) and a seed grant from the Institute for Sustainability, Energy, and Environment at the University of Illinois at Urbana-Champaign and Illinois-JITRI Institute (JITRI 23965). A.S.P. thanks the PEW Latin American Fellowship for financial support. We also thank the Robert A. Welch Foundation (Grant F-0020) for support of the Lu group research program at the University of Texas at Austin.

The authors have nothing to disclose.

SELEX allows not only the identification of aptamers with high affinity, in the pM-nM range22,43,44,45, but also with high and tunable selectivity. By taking advantage of counter selection, aptamers with challenging selectivity can be obtained. For instance, the Li group has demonstrated the ability to obtain sequences that can differentiate pathogenic bacterial strains from non-pathogenic stra...

Virus infections have enormous economic and societal impacts around the world, as became increasingly apparent from the recent COVID-19 pandemic. Timely and accurate diagnosis is paramount in treating viral infections while preventing the spread of viruses to healthy people. While many virus detection methods have been developed, such as PCR tests1,2 and inmunoassays3, most of the currently used methods are not capable of determining whether the detected virus is actually infectious or not. This is because the presence of components of the virus alone, such as viral nucleic acid or proteins, does not indicate that the intact, infectious virus is present, and levels of these biomarkers have shown poor correlation with infectivity4,5,6. For example, viral RNA, commonly used for the current PCR-based COVID-19 tests, has very low levels in the early stages of infection when the patient is contagious, while the RNA level is often still very high when patients have recovered from the infection and are no longer contagious7,8. The viral protein or antigen biomarkers follow a similar trend, but typically appear even later than the viral RNA and thus are even less predictive of infectability6,9. To address this limitation, some methods that can inform on the infectivity status of the virus have been developed, but are based on cell culture microbiology techniques that require a long time (days or weeks) to obtain results4,10. Thus, developing new sensors that can inform on the infectability of clinical or environmental samples can avoid delays in treatment and further spread of the virus. However, very few methods can obtain sensing molecules that can recognize an intact infectious virion and differentiate it from the same virus that has been rendered non-infectious.
In this context, aptamers are particularly well-suited as a unique biomolecular tool11,12,13,14. Aptamers are short, single-stranded DNA or RNA molecules with a specific nucleotide sequence that allows them to form a specific 3D conformation to recognize a target with high affinity and selectivity15,16. They are obtained by a combinatorial selection process called systematic evolution of ligands by exponential enrichment (SELEX), also known as in vitro selection, that is carried out in test tubes with a large random DNA sampling library of 1014-1015 sequences17,18,19. In each round of this iterative process, the DNA pool is first subjected to a selection pressure through incubation with the target under the desired conditions. Any sequences that are not bound to the target are then removed, leaving behind only those few sequences that are able to bind under the given conditions. Finally, the sequences that have been selected in the previous step are amplified by PCR, enriching the population of the pool with the desired functional sequences for the next round of selection, and the process is repeated. When the activity of the selection pool reaches a plateau (typically after 8-15 rounds), the library is analyzed by DNA sequencing to identify the winning sequences exhibiting the highest affinity.
SELEX has unique advantages that can be exploited to gain increased selectivity against other similar targets20,21, such as for infectivity status of the virus22. First, a wide variety of different types of targets can be used for the selection, from small molecules and proteins to whole pathogens and cells16. Thus, to obtain an aptamer that binds to an infectious virus, an intact virus can be used as the target, instead of a viral surface protein19. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need for disruption of the virus. Second, SELEX can be tailor-made to remove competing targets21,23, such as other similar viruses or non-infectious inactivated viruses, using counter selection steps in each round of selection22. During the counter selection steps, the DNA pool is exposed to targets for which binding is not desired, and any sequences that bind are discarded.
In this work, we provide a protocol that can be generally applied for selecting aptamers that bind to an infectious virus but not to the same virus that has been rendered non-infectious by a particular disinfection method or to another related viruses. This method allows recognition agents to be obtained based on functional differences of the virus surface, which do not need to be known in advance, and so offers an additional advantage for the detection of newly emerged pathogens or for understudied diseases.

<table><tbody><tr><td>10% Ammonium persulfate (APS)</td><td>BioRad</td><td>1610700</td><td></td></tr><tr><td>100% Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>1x PBS without calcium &amp;amp; magnesium</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>40% acrylamide/bisacrylamide (29:1) solution</td><td>BioRad</td><td>1610146</td><td></td></tr><tr><td>Agencourt AMPure XP Beads</td><td>Beckman Coulter</td><td>A63880</td><td>DNA clean-up beads - Section 7.2.2</td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Merck</td><td>UFC501024</td><td>cut-off 10 kDa</td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Merck</td><td>UFC510024</td><td>cut-off 100 kDa</td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>100165</td><td></td></tr><tr><td>C1000 Touch Thermal Cycler with Dual 48/48 Fast Reaction Module</td><td>BioRad</td><td>1851148</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>CFX Connect Real-Time PCR Detection System</td><td>BioRad</td><td>1855201</td><td></td></tr><tr><td>Digital Dry Baths/Block Heaters</td><td>Thermo Scientific</td><td>88870001</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin C1</td><td>Thermo Fisher</td><td>65001</td><td>streptavidin-modified magnetic beads - Section 4.9</td></tr><tr><td>EDTA disodium salt</td><td>Sigma-Aldrich</td><td>324503</td><td></td></tr><tr><td>Eppendorf Safe-Lock microcentrifuge tubes</td><td>Sigma-Aldrich</td><td>T9661</td><td>1.5 mL</td></tr><tr><td>Lenti-X p24 Rapid Titer Kit</td><td>Takara Bio USA, Inc.</td><td>632200</td><td>Lentivirus quantification kit - Section 3.3.2.1</td></tr><tr><td>MagJET Separation Rack, 12 x 1.5 mL tube</td><td>Thermo Scientific</td><td>MR02</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Microseal &#039;B&#039; PCR Plate Sealing Film, adhesive, optical</td><td>BioRad</td><td>MSB1001</td><td>non-UV absorbing</td></tr><tr><td>Mini-PROTEAN Tetra Cell for Ready Gel Precast Gels</td><td>BioRad</td><td>1658004EDU</td><td></td></tr><tr><td>Mini-PROTEAN Short Plates</td><td>BioRad</td><td>1653308</td><td></td></tr><tr><td>Mini-PROTEAN Spacer Plates with 0.75 mm Integrated Spacers</td><td>BioRad</td><td>1653310</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Lonza</td><td>51200</td><td></td></tr><tr><td>Multiplate 96-Well PCR Plates, high profile, unskirted, clear</td><td>BioRad</td><td>MLP9611</td><td></td></tr><tr><td>Nanodrop One</td><td>Thermo Scientific</td><td>ND-ONE-W</td><td></td></tr><tr><td>OneTaq DNA Polymerase</td><td>New England BioLab</td><td>M0480S</td><td></td></tr><tr><td>Ovation Ultralow v2 + UDI</td><td>Tecan</td><td>0344NB-A01</td><td>High-troughput sequencing library preparation kit - Section 7.2.</td></tr><tr><td>PIPETMAN G (100-1000 &amp;micro;L, 20-200 &amp;micro;L, 2-20 &amp;micro;L and 0.2-2 &amp;micro;L)</td><td>Gilson</td><td>F144059M, F144058M, F144056M, F144054M</td><td></td></tr><tr><td>Purifier Logic+ Class II, Type A2 Biosafety Cabinets</td><td>Labconco</td><td>4261</td><td></td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>Invitrogen</td><td>Q32850</td><td>fluorescence-based&amp;nbsp; dsDNA quantification&amp;nbsp; kit - Section 7.2.3</td></tr><tr><td>SHARP Classic Low Retention Pipet Tips (10 uL, 200 uL, 1000 uL)</td><td>Thomas Scientific</td><td>1158U43, 1159M44, 1158U40</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sorvall Legend Micro 17R Microcentrifuge</td><td>Thermo Scientific</td><td>75002440</td><td></td></tr><tr><td>SsoFast EvaGreen Supermix</td><td>BioRad</td><td>1725201</td><td>qPCR mastermix - Section 6.2.</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tubes and Ultra Clear Caps, strips of 8</td><td>USA scientific</td><td>AB1183</td><td>PCR tubes</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5128</td><td></td></tr></tbody></table>

in vitro selection of aptamers to differentiate infectious from non-infectious viruses

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

Since DNA aptamers can be obtained using SELEX in a test tube15, this SELEX strategy was carefully designed to include both positive selection steps toward the intact, whole infectious virus (i.e., retain the DNA molecules that bind to the infectious virus), as well as counter selection steps for the same virus that has been rendered non-infectious by a particular disinfection method, specifically UV-treatment, by discarding the DNA sequences that can bind to the non-infectious virus. A schematic ...

Watch this Scientific Journal Video about In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses at JoVE.com

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, ...

in-vitro-selection-aptamers-to-differentiate-infectious-from-non

Research

JoVE Journal

Bioengineering

1.6K Views.  Universidad de Buenos Aires. We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

Video: In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Immunology and Infection

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Cancer Research

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biology

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Summary

Explore More Videos

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

Assays for the Identification of Novel Antivirals against Bluetongue Virus

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Primer-Free Aptamer Selection Using A Random DNA Library

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy