Viral Growth and Techniques

Source: Tang, N., et al. Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing. J. Vis. Exp. (2019)This video demonstrates the clonal isolation and PCR-based screening of recombinant herpesvirus following site-specific excision of a GFP marker. Fluorescence-activated cell sorting is used to isolate non-fluorescent infected cells, and junction PCR confirms retention of the target gene in GFP-negative clones.

Source: Cross, T., et al. An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates. J. Vis. Exp. (2015).This video demonstrates the isolation of Arthrobacter bacteriophages from a soil sample using enrichment and plaque assay techniques. It highlights the infection, amplification, and visualization of phages as clear lysis zones formed on a bacterial lawn.

Source: Morales Vasquez, D., et al. Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2. J. Vis. Exp. (2021)This video demonstrates the protocol for carrying out plaque assay–based viral titration for quantifying infectious virus levels in tissue sample collected from a virus–infected mouse model.

Source: Vijayraghavan, S., Kantor, B. A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J. Vis. Exp. (2017).This video demonstrates the procedure for generating recombinant viral particles by transfecting embryonic kidney cells with plasmids encoding viral proteins and a gene of interest.

Source: Rustad, M., et.al. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp. (2017)This video demonstrates the purification of bacteriophages from a suspension containing host bacterial debris using a sucrose density gradient. It shows how the density gradient separates phages by buoyant density, enabling recovery of a visible band for resuspension and further analysis.

Source: Brauer, R. et al., Influenza Virus Propagation in Embryonated Chicken Eggs. J. Vis. Exp. (2015)This video demonstrates the synthesis and harvest of the influenza virus in fertilized chicken eggs. The virus is inoculated into the allantoic cavity, where it replicates in epithelial cells and buds into the allantoic fluid, which is then collected and stored for downstream applications.

Source: Nguyen, H., et al. Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus. J. Vis. Exp. (2021).This video demonstrates the propagation and harvesting of oncolytic herpes simplex virus in mammalian cell cultures, highlighting viral entry, replication, and the recovery of virus-loaded cells. It further shows methods for preserving infected cells for downstream processing.

Source: Nguyen, H., et al. Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus. J. Vis. Exp. (2021)This video demonstrates the purification of an oncolytic virus from infected mammalian cell cultures using nuclease treatment, sequential centrifugation, membrane filtration, and sucrose cushion centrifugation.

Source: Huang, D., et al. Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes. J. Vis. Exp. (2022).This video demonstrates the step-by-step procedure for the in vitro isolation of mosquito-associated viruses from mosquito homogenates using fibroblast-like mammalian cells.

Source: Eggers, C. H. Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi. J. Vis. Exp. (2022).This video demonstrates the isolation of borrelial phage particles from a Borrelia burgdorferi suspension using polyethylene glycol-mediated phase separation. The sample is centrifuged, treated with a salt solution, and a molecular crowding agent to precipitate phages. Chloroform extraction is done to remove debris and concentrate phage particles. The purified phage...

Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024)This video demonstrates the purification of bacteriophages from bacterial cell lysates using sequential centrifugation, membrane filtration, chloroform extraction, centrifugal filtration, and buffer exchange to obtain a purified phage preparation.

Source: Van Lidth de Jeude, et al. A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015).This video demonstrates the process of producing viral particles through transfection of embryonic kidney cells with plasmid–polyethylenimine complexes, which leads to viral genome expression, particle assembly, and virus collection in the medium.

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp.(2018)This video demonstrates preparation of a bacteriophage lysate containing transducing particles. A donor strain is infected, plaques form, and the soft agar overlay is processed with chloroform to yield a clarified lysate for downstream analysis.

Source: Weger-Lucarelli, J., et al. Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone. J. Vis. Exp. (2017)This video demonstrates the recovery of infectious Zika virus using electroporation of in vitro transcribed RNA. The process shows how synthetic RNA initiates infection and leads to viral replication in cultured cells.

Source: Mackler, R. M., et al. Assembly and Purification of Prototype Foamy Virus Intasomes. J. Vis. Exp. (2018)This video demonstrates the purification of prototype foamy virus (PFV) intasomes from a mixed protein-DNA suspension using salt extraction and size exclusion chromatography, yielding active complexes for downstream analysis.

Source: Suzuki, Y., et al. Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction. J. Vis. Exp. (2018).This video demonstrates the procedure for preparing viral samples for quantitative PCR (Polymerase chain reaction) analysis. Viral samples and RNA standards are serially diluted, treated with a proteinase K buffer, and incubated under controlled temperatures to release RNA. The processed RNA is then stabilized and stored for...

Source: Dembowski, J. A., et al. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017).This video demonstrates the isolation of protein–viral DNA complexes from cell nuclei using click chemistry and magnetic bead purification. The protocol involves labeling viral genomes with alkyne-modified nucleotides, biotinylation via a click reaction, and binding to streptavidin-coated magnetic beads. Following washing and heat elution, the purified...

Source: Ding, J. et. al., Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)This video shows the purification of recombinant virus particles from transfected human kidney cells. The virus is released by freeze–thaw lysis and purified using iodixanol gradients with block copolymer-assisted filtration.

Source: Rghei, A. D., et al. Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models. J. Vis. Exp. (2021)This video demonstrates the recovery of crude adeno-associated virus from transfected mammalian cells using chemical lysis and centrifugation, enabling high-yield AAV production for downstream purification and preclinical gene therapy applications.

Source: Lopez Jr., M. A., et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)This video demonstrates the purification of polyhistidine-tagged viral integrase using nickel affinity chromatography. The protocol uses imidazole gradient elution to selectively isolate the integrase based on its affinity for nickel-charged resin.

Source: Vijayraghavan, S. and Kantor, B. A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J. Vis. Exp., (2017)This video demonstrates a two-step ultracentrifugation method using sucrose gradients and cushions to purify and concentrate viral particles from cell culture supernatant. The procedure enables separation based on buoyant density, allowing recovery of intact virus suitable for downstream applications.

Source: Lopez Jr., M. et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)This video demonstrates the procedure for purifying viral integrase from bacterial nuclease contamination using heparin Sepharose column chromatography followed by SDS-PAGE analysis to confirm protein purity.

Source: Biesbrock, A. M. et al. Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture. J. Vis. Exp. (2014)This video demonstrates the in vitro propagation of Homalodisca coagulata Virus-01 using insect-derived host cells. It outlines the steps for virus purification, inoculation, and replication within cultured insect cells.

Source: Sahmi-Bounsiar, D. et al. Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation. J. Vis. Exp. (2019)This video demonstrates single-cell micro-aspiration to isolate and expand a single giant virus population from a mixed viral population using amoebae as host cells.

Source: Heidbuechel, J. P., et al. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J. Vis. Exp. (2019).This video demonstrates the process of infecting kidney epithelial cells with genetically engineered viruses for their production and harvesting to study viral replication.

Source: Bou Khalil, J. Y.,et al. A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis. J. Vis. Exp. (2016)This video demonstrates the enrichment of giant viruses from an environmental water sample using amoeba-based co-culture to enable downstream detection and analysis.

Source: Chen, Z., et al. Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry. J. Vis. Exp. (2018)This video demonstrates the method for the controlled expression of the bacteriophage Qβ coat protein in engineered E. coli. The resulting proteins self-assemble into virus-like particles, which are harvested for downstream bioconjugation.

Source: Gao, Y., et al. Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus. J. Vis. Exp. (2021)The video demonstrates the in vitro assembly of virus-specific nucleocapsids from a purified RNA-free nucleoprotein–phosphoprotein complex, their purification by size exclusion chromatography, and validation of full-length viral RNA within the assembled particles.

Source: Fricano-Kugler, C. J., et al. Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection. J. Vis. Exp. (2016).This video demonstrates the step-by-step process for concentrating and purifying lentiviral particles from a mixed suspension containing host cell debris. The procedure utilizes centrifugation, filtration, and PEG-salt precipitation to isolate viral particles while removing soluble impurities. The final product is resuspended in a...

Source: Rustad, M. et al. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp. (2017)This video demonstrates the amplification and recovery of infectious bacteriophages from E. coli cultures. It outlines the steps for plaque isolation, phage replication, controlled lysis, and purification.

Source: Wang, M., et al, Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice. J. Vis. Exp. (2020).This video demonstrates intranasal infection of a mouse with a pathogenic influenza virus to establish a respiratory tract infection. The procedure outlines the administration of the virus into the nasal cavity. The virus enters host cells, replicates, and activates the immune system.

Source: Asad, S., et al. Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells. J. Vis. Exp. (2019)This video demonstrates antibody-dependent enhancement of Zika virus infection in human primary macrophages, using dengue antibody–virus complexes to model how cross-reactive antibodies facilitate Fcγ receptor–mediated viral entry and replication for studying infection mechanisms and immune interactions.

Source: Klaus, J. P., et al Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (2016).This video demonstrates how to measure arenavirus attachment to monkey kidney cells. The assay uses low temperatures to block endocytosis and isolate the virus binding step. The approach enables precise quantification of surface-bound virus particles under conditions that restrict internalization.

Source: Fernández-Fernández, R., et. al. Bacteriophage Removal from Infected Salmonella Cultures. J. Vis. Exp. (2024)This video demonstrates the use of exogenous LPS (Lipopolysaccharide) as a receptor mimic to inactivate lytic bacteriophages in Salmonella cultures. Through sequential incubation, centrifugation, and washing, phages are eliminated, resulting in a viable, phage-free bacterial suspension for downstream applications.

Source: Zhao, J., et al. Potato Virus X-Based microRNA Silencing (VbMS) In Potato. J. Vis. Exp. (2020).This video demonstrates the use of Agrobacterium tumefaciens to deliver a Potato Virus X (PVX)-based construct into in vitro-grown potato plants for virus-based microRNA silencing (VbMS). The method enables systemic suppression of a target microRNA, allowing researchers to study its role in plant development through observable phenotypic changes.

Source: Esposito, A. M.,et al. High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion. J. Vis. Exp. (2018)This video demonstrates a co-culture fluorescent assay to monitor cell-to-cell viral transmission. The assay uses a red-to-green reporter cassette to visualize viral membrane fusion and evaluate the efficacy of test compounds in blocking viral entry.

Source: Yates, J. G. E., et al. Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid. J. Vis. Exp. (2022)The video demonstrates the purification and concentration of virus particles from clarified allantoic fluid collected from virus-infected embryonated chicken eggs. It showcases a series of filtration and separation steps, including depth filtration, tangential flow filtration, and density gradient ultracentrifugation to isolate intact viruses. The virus-rich...

Source: Otter, C. J., et al. Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions. J. Vis. Exp. (2023)This video demonstrates the growth of coronavirus in human nasal epithelial cells cultured at an air-liquid interface, mimicking infection and viral replication in the human nasal cavity.

Source: Krishnamurthi, R., et al. Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. J. Vis. Exp. (2024)This video demonstrates the induction of lytic replication in a prophage integrated into the bacterial genome and the capture of gene expression changes through timed sampling for transcriptomic analysis.

Source: Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)This video demonstrates the antibody-mediated neutralization of pseudoviruses carrying coronavirus spike proteins using patient serum, studied through high-throughput fluorescent imaging of infected epithelial cell monolayers.

Source: Ruiz, A. J. and Russell, S. J. MicroRNA-based Regulation of Picornavirus Tropism. J. Vis. Exp. (2017)This video demonstrates the use of synthetic microRNA mimics to inhibit viral replication in cultured epithelial-like cells selectively. It highlights how microRNA-directed genome silencing can block virus spread and confirm targeting specificity through downstream infectivity analysis.

Source: Lucas-Hourani, M. et.al., High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses. J. Vis. Exp. (2014)This video shows a luciferase-based antiviral assay in human embryonic kidney cells infected with recombinant measles virus. It shows that serially diluted test compounds inhibit viral replication, leading to lower luminescence in a dose-dependent manner while maintaining host cell viability.

Source: Heidbuechel, J. P., et al. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J. Vis. Exp. (2019).This video demonstrates a cell-based assay to evaluate the target-specific immunomodulatory activity of a virally encoded bispecific T-cell engager (BTE). The BTE links tumor cells expressing a target receptor with T cells, triggering tumor lysis and enzyme release. A colorimetric readout, based on substrate conversion, indicates T-cell activation. Higher...

Source: Kalveram, B., et al. Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis. J. Vis. Exp. (2013)This video demonstrates the use of click chemistry and flow cytometry to measure virus-induced suppression of host-cell transcription. Mammalian cells are infected with a virus that expresses a nuclear virulence protein disrupting the host transcription factor complex, while control cells remain transcriptionally active. Incorporation of 5-ethynyl uridine...

Source: Chiarella, A. M. et. al., Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers. J. Vis. Exp. (2018)This video demonstrates the generation of genetically modified mouse embryonic stem cells using lentiviral transduction. Lentiviral particles deliver an antibiotic resistance marker, allowing stable genomic integration and selection of transduced cells.

Source: Fuchs, M. et al. Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments. J. Vis. Exp. (2016)This video demonstrates adenovirus-mediated delivery of a fluorescent transgene combined with siRNA–calcium phosphate transfection in cervical cancer cells. The adenovirus expresses a fluorescent replacement protein, while the siRNA induces targeted mRNA degradation through RISC binding. This sequential co-transfection...

Source: Zhang, J., et al. P. Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production. J. Vis. Exp. (2014)This video demonstrates intranasal delivery of murine herpesvirus in mice to induce a lung-specific immune response. Lung tissue is subsequently processed to obtain cytokine-containing supernatants for downstream assessment of host immune responses.

Source: Zhang, J., et al. Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production. J. Vis. Exp. (2014)This video demonstrates the process of infecting murine fibroblast cells with herpesvirus and monitoring the resulting antiviral cytokine release and gene expression. It outlines key steps, including viral infection, media collection for cytokine quantification, and cell harvesting for gene expression analysis.

Source: Zhang, L., et al. Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts. J. Vis. Exp. (2021)The video demonstrates the preparation of gut tissue from a plant virus–infected insect vector for fluorescence imaging. Fixed guts were washed, permeabilized, and incubated with two fluorescent antibodies in a blocking solution—one targeting a viral antigen in vesicles, the other a vesicle-associated membrane protein. The tissue was counterstained for actin...

Source: Schaeffer, J. W., Chandler, et al. Detection of Viruses from Bioaerosols Using Anion Exchange Resin. J. Vis. Exp. (2018).This video demonstrates the detection of viruses from bioaerosols using an anion exchange resin. A liquid impinger containing the resin is used to capture viruses from the air. The resin is collected and treated with a virus lysis buffer containing carrier RNA. The lysate is transferred to a microcentrifuge tube for viral RNA isolation, followed by qRT-PCR to amplify...

Source: Yuan, M., et al. A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors. J. Vis. Exp. (2016)This video demonstrates the process of performing a fluorescent plaque assay using recombinant vaccinia virus encoding red fluorescent protein (RFP) in fibroblast cells, followed by plaque isolation and purification for further analysis.

Source: Williams, S. M. et al. Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis. J. Vis. Exp. (2015)This video demonstrates tapping-mode AFM to visualize phage–antigen interactions on a mica surface. It outlines the steps for antigen immobilization, phage binding, and high-resolution nanoscale imaging.

Source: DeMarino, C., et al. Purification of High Yield Extracellular Vesicle Preparations Away from Virus. J. Vis. Exp. (2019)The video demonstrates the separation of EVs from virions and other contaminants by density gradient centrifugation, followed by capture and concentration with hydrogel nanoparticles. Washed fractions are then ready for downstream identification of those enriched in EVs.

Source: Van Lidth de Jeude, J. F., et al. A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015).This video demonstrates the processing of lentivirus-transduced mouse intestinal organoids for paraffin embedding, to enable their downstream analysis for assessing gene expression.

Source: Chen, H. et. al. Live Cell Imaging of the TGF-β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System. J. Vis. Exp. (2018)This video demonstrates the preparation of luciferase-expressing breast cancer cells using recombinant adenoviruses for in vivo imaging.

Source: Ren, S., et al. A Protocol for Analyzing Hepatitis C Virus Replication. J. Vis. Exp. (2014).This video demonstrates an immunofluorescence-based method to detect Hepatitis C virus replication in infected hepatoma cells. The protocol involves cell fixation, antibody staining for viral protein and double-stranded RNA, and visualization using fluorophore-tagged secondary antibodies. Dual signal detection under a fluorescence microscope confirms active viral replication within the host cells.

Source: Stauffer, S. et al. In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation. J. Vis. Exp. (2016)This video demonstrates the pH-dependent disassembly of influenza A virus (IAV) cores using gradient ultracentrifugation. It outlines glycerol gradient preparation, ultracentrifugation, and how different pH conditions affect IAV core disassembly.

Source: Philip, A. A., et al., Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)This video demonstrates the isolation of rotavirus double-stranded RNA from clarified cell lysates. It further illustrates the separation and visualization of genome segments through polyacrylamide gel electrophoresis.

Source: Philip, A. A., et. al., Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)This video demonstrates a plaque assay technique to monitor rotavirus infection in epithelial cells. Trypsin activation enhances viral entry, and staining highlights viable cells, enabling visualization of infection-induced plaques.

Source: Stauffer, S. et al., In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation. J. Vis. Exp. (2016)This video demonstrates the effect of pH on the solubilization of influenza A viral cores. Gel electrophoresis and protein staining reveal the progressive disassembly of the core structure under acidic conditions.

Source: Moore, M. D., et al. Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity. J. Vis. Exp. (2017)The video demonstrates a protocol for monitoring the heat-induced aggregation of virus-like particles using dynamic light scattering (DLS). It begins with preparing a monodisperse suspension of purified particles. The sample is then loaded into a preheated DLS instrument. As the capsid proteins denature with heat, exposed hydrophobic regions cause particle...

Source: Tran, B. N., et. al. High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells. J. Vis. Exp. (2022)This video demonstrates the use of quantum dot–conjugated spike protein (QD-Spike) to visualize dose-dependent viral entry in ACE2-GFP–expressing cells. Confocal imaging captures the internalization of QD-Spike via receptor-mediated endocytosis, modeling early coronavirus entry.

Source: Mateer, E., et al., Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection. J. Vis. Exp. (2019)This video demonstrates the confocal imaging of viral double-stranded RNA (dsRNA) and cytoplasmic pattern recognition receptors (PRRs) in virus-infected epithelial cells. It highlights how antibody-based fluorescent labeling reveals the interaction between viral replication intermediates and host immune sensors.

Source: Xu, C., et al. An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection. J. Vis. Exp. (2018).This video demonstrates the phenotyping of EBV-infected T and NK cell lines using flow cytometry, highlighting the preparation, antibody staining, and analysis steps to identify lineage-specific surface markers. It shows how marker profiles confirm distinct T-cell and NK-cell populations.

Source: Cross, T., et al. An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates. J. Vis. Exp.(2015)This video demonstrates the isolation of bacteriophages from a soil sample. A soil sample is collected and suspended in a phage buffer and allowed to settle the sediment. The extract is filtered, and nutrients are added. A vacuum filtration is done to remove microbial contaminants. The filtrate with phages is aliquoted into flasks, and...

Source: Allaire, A., et al. Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus. J. Vis. Exp. (2015).The video demonstrates the procedure for preparing cells to assess the intracellular uptake and activity of an antiviral agent against the hepatitis C virus.

Source: Olety, B., et. al., Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes. J. Vis. Exp. (2016)This study demonstrates a fluorescence-based assay to observe the membrane-binding behavior of human immunodeficiency virus type-1 structural protein. Using synthetic giant unilamellar vesicles as model membranes, the assay visualizes protein recruitment and assembly, mimicking early steps of viral particle formation.

Source: Lê-Bury, G., et al. Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1. J. Vis. Exp. (2016)This video demonstrates live confocal imaging of HIV-1-infected human macrophages to visualize and quantify phagosome migration using GFP fluorescence and bright field tracking, providing insights into how HIV alters intracellular trafficking during Fc receptor–mediated phagocytosis.

Source: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015)This video demonstrates the step-by-step procedure for preparing mouse retinal cryosections to visualize adeno-associated virus permeability across disrupted retinal barriers.

Source: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015).This video demonstrates the method of AAV-mediated gene delivery in a transgenic mouse retina, followed by tissue dissection and staining to assess inner limiting membrane integrity.

Source: Moore, M. D., et al. Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity. J. Vis. Exp.(2017).This video demonstrates how purified viral capsids are heat-treated, prepared, and imaged using transmission electron microscopy (TEM) to reveal structural changes and heat-induced damage.

Source: Shah, R., et al. Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection. J. Vis. Exp. (2020)This video demonstrates simultaneous visualization of HIV-1 DNA, RNA, and capsid protein in infected human lymphocytes using branched DNA hybridization and immunofluorescence, enabling detailed analysis of viral replication stages within individual cells.

Source: Pohl, M. O. & Stertz, S. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry. J. Vis. Exp. (2015)This video demonstrates the use of a streptavidin blocking assay to distinguish surface-bound from internalized influenza virus in human lung epithelial cells. Comparing fluorescence signals shows that a higher intensity after incubation indicates successful viral internalization.

Source: Niu, Y. D., et al. Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings. J. Vis. Exp. (2021)This video demonstrates a microplate-based assay to evaluate the lytic activity of phage cocktails against E.coli bacterial cultures. It highlights how changes in optical density reveal phage potency and the concentration needed for complete bacterial...

Source: Dembowski, J. A., Deluca, N. A. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017)This video demonstrates the infection of stationary-phase mammalian cells with HSV-1 (Herpes Simplex Virus-1), followed by replication of the viral genome using host and viral replication machinery. A synthetic nucleoside analog is incorporated into newly synthesized viral DNA to label replicating genomes, which are subsequently isolated for...

Source: Hiller, T. et. al. Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2. J. Vis. Exp. (2016)This video demonstrates immunohistochemical staining to evaluate viral gene transfer in a recellularized rat liver model. Reporter expression, confirmed by antibody detection and co-localization with nuclear staining, indicates successful viral transduction in the cells.

Source: Bou Khalil, J. Y., et al. A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis. J. Vis. Exp. (2016).This video demonstrates imaging of faustovirus particles from an infected amoeba culture using transmission electron microscopy. It covers grid preparation, negative staining with ammonium molybdate to enhance contrast, and visualization of the virus under the microscope. The protocol concludes with measuring the size of the...

Source: Vallery, T. K. & Steitz, J. A. Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells. J. Vis. Exp. (2019)This video demonstrates fluorescence in situ hybridization to visualize viral RNA in virus-infected cells. It outlines the steps involved in probe hybridization, signal amplification, and confocal imaging.

Source: Rghei, A. D., et al. Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models. J. Vis. Exp. (2021)The video demonstrates purification of recombinant adeno-associated virus (AAV) vectors from crude host lysate using a heparin-based affinity matrix. Viral particles bind to immobilized heparin, while impurities are washed away. A high-salt buffer releases intact viruses, which is collected in enriched fractions. These fractions are...

Source: Rozelle, D. K., et al. Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression. J. Vis. Exp. (2014)This video demonstrates a method to monitor vaccinia virus replication using fluorescent reporters regulated by stage-specific promoters. Sequential expression of green, red, and blue fluorescent proteins marks the early, intermediate, and late phases of infection, enabling high-throughput analysis of stage-specific viral gene expression.

Source: Zeigler Allen, L., et al. Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'. J. Vis. Exp. (2013).This video demonstrates how flow cytometry enables the isolation of individual, genome-containing viruses from mixed populations through fluorescence-based detection and precise particle sorting, followed by agarose embedding for subsequent genomic characterization.

Source: Kalveram, B. et al. Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis. J. Vis. Exp. (2013)This video demonstrates the use of click chemistry and fluorescence microscopy to visualize nascent RNA transcription in mammalian cells. It highlights how viral infection suppresses host transcription by comparing fluorescent signals between infected and control cells.

Source: Marzook, N. B., et al., Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins. J. Vis. Exp. (2014)This video demonstrates the generation of recombinant vaccinia viruses using plasmid-mediated homologous recombination in infected epithelial cells. Fluorescent and metabolic markers enable the selection and identification of viruses expressing tagged proteins.

Source: Blancett, C. D., et al. Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment. J. Vis. Exp. (2017).This video demonstrates the safe viral specimen preparation for transmission electron microscopy. The viral particles are fixed with glutaraldehyde, stained with an electron-dense agent, and subjected to vapor fixation with osmium tetroxide to stabilize structures and ensure complete inactivation, ensuring safe handling of viral samples while improving...

Source: Chen, C. C., et al. Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods. J. Vis. Exp. (2018)This video demonstrates the preparation and negative staining of hepatitis E virus-like nanoparticles for visualization by transmission electron microscopy. The method highlights the structural integrity of self-assembled capsid-only particles, confirming their uniformity and purity through high-resolution imaging.

Source: Contreras, D., et al. Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons. J. Vis. Exp. (2016)The video demonstrates the detection of Zika viral dsRNA intermediates in infected epithelial cells using immunocytochemistry. Infected cells are fixed and then blocked with a blocking solution. The primary and secondary antibodies are added. The nuclei are stained with a fluorescent dye. The labeled viral dsRNA is then seen under a fluorescence...

Source: Rustad, M., et al. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp. (2017).This video demonstrates the synthesis of bacteriophages using a cell-free transcription–translation (TXTL) system. A master mix is prepared using a bacterial crude extract, an energy mix, an amino acid mix, an inhibitor for a DNA-degrading enzyme, and bacteriophage DNA, then incubated. The in vitro molecular machinery enables viral protein expression and DNA...

Source: Jamieson, T. R., et al. Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)This video demonstrates the procedure for infecting mammalian cells with green fluorescent protein (GFP)-encoding pseudovirus, monitoring viral replication and spread by fluorescence microscopy, and collecting virus-containing supernatant for downstream applications.

Source: Yuan, M., et al. A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors. J. Vis. Exp. (2016)This video demonstrates the expansion of plaque-derived recombinant virus in adherent mammalian cells. The virus carries a fluorescent reporter in place of a virulent gene, enabling infection tracking. Infected cells are harvested for PCR verification of the genetic modification.

Source: Liu, P., et al. Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae. J. Vis. Exp. (2017).This video demonstrates the process of isolating recombinant Aedes aegypti densovirus (rAaeDV) from infected mosquito-derived epithelial cells. The infected cells are collected and exposed to repeated freeze-thaw cycles for lysis. Lysate is centrifuged, and the supernatant containing viral particles is filtered, aliquoted, and...

Source: Philip, A. A., et al. Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)This video demonstrates the recovery of recombinant rotavirus by transfecting plasmids encoding viral genome segments into hamster kidney cells, followed by amplification in monkey epithelial cells, enabling studies of rotavirus biology, replication, and vaccine development.

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System. J. Vis. Exp. (2020)This video demonstrates the in vitro transformation of murine epithelial cells through viral delivery of Cre recombinase and mutation-inducing components. It outlines the steps for Cas9 activation, cancer-gene modification, and the establishment of a tumorigenic murine cell line.

Source: Ehrke-Schulz, E., et al. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5. J. Vis. Exp. (2016)This video demonstrates the large-scale amplification of high-capacity adenoviral vectors in suspension-adapted human embryonic kidney cells expressing Cre recombinase for use in gene delivery applications requiring high-titer, helper-free vector preparations.

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021)This video demonstrates the production of influenza pseudoviruses in human cells using calcium phosphate-mediated plasmid transfection, enabling safe generation of single-cycle viral particles for downstream applications such as antibody neutralization assays under biosafety level 2 conditions.

Source: Nishitsuji, H., et al. Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle. J. Vis. Exp. (2017)This video demonstrates the production and purification of recombinant Hepatitis B virus particles, enabling safe, replication-deficient viral preparations for studying early infection events and for high-throughput antiviral drug screening applications.

Source: Liu, P., et al. Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae. J. Vis. Exp. (2017)This video demonstrates the use of recombinant mosquito densoviruses encoding microRNA to silence specific genes in mosquito larvae. Infected early instar larvae produce functional microRNAs that suppress target gene expression for downstream studies.

Source: Eggers, C. H. Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi. J. Vis. Exp. (2022).This video demonstrates the transduction of genetically modified Borrelia spirochetes using polyethylene glycol–precipitated, non-infectious bacteriophages carrying a kanamycin-resistance plasmid. After incubation with the bacteria, the culture is plated on antibiotic-supplemented medium. Only cells that receive the plasmid survive and form colonies. These colonies...

Source: Van Lidth de Jeude, J. F., et al. Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015)This video describes the lentiviral transduction of mouse intestinal organoids from a crypt-like structure containing suspension. Using a basement membrane matrix as a scaffold and growth factor–supplemented media, lentiviral vectors are introduced to enable stable gene...

Source: Chen, H., et al. Live Cell Imaging of the TGF-β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System. J. Vis. Exp. (2018).This video demonstrates a method to study SMAD pathway activation in breast cancer cells using dual adenoviral infection. One virus delivers a constitutively expressed GFP gene, while the other carries an RFP reporter under a SMAD-sensitive promoter. Upon TGF-beta stimulation, SMAD proteins are activated, forming a complex that induces RFP...

Source: Meister, T. L., et al. A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks. J. Vis. Exp. (2020).This video demonstrates the process of isolating hepatitis E virus (HEV) particles from transfected human hepatocellular carcinoma cells. Extracellular viral particles are collected from filtered supernatants, while intracellular particles are released by subjecting the cells to repeated freeze-thaw cycles. The lysate is centrifuged, and the supernatant containing HEV...

Source: Rim, Y. A., et al. Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation. J. Vis. Exp. (2016).This video demonstrates the generation of induced pluripotent stem cells (iPSCs) from human peripheral blood mononuclear cells using Sendai virus–mediated reprogramming. The viral transcription factors activate pluripotency networks and suppress lineage genes, enabling a subset of cells to acquire adhesion properties and establish iPSC colonies. The...

Source: Ehrke-Schulz, E., et al. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5. J. Vis. Exp. (2016)This video demonstrates the purification of high-capacity adenoviral vectors using cesium chloride gradient ultracentrifugation, enabling the separation of genome-containing virions for applications in gene delivery, vector characterization, and preclinical therapeutic...

Source: Lu, A., et al. Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk. J. Vis. Exp. (2022)The video demonstrates a method for ultrasound-guided intramyocardial injection of a recombinant viral vector in a mouse model. This technique enables targeted gene delivery to the heart for studying the effects of transgene expression on cardiac function.

Source: Hiller, T., et al. Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2. J. Vis. Exp., (2016)This video demonstrates adeno-associated virus-mediated gene delivery in a humanized rat liver scaffold. The vector delivers a transgene encoding a fluorescent reporter and gene-silencing RNA into liver cells via receptor-mediated endocytosis. This model supports efficient transduction in a perfused 3D liver tissue system.

Source: Kubala, M. H. & DeClerck, Y. A. Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment. J. Vis. Exp. (2017)This video demonstrates the generation of conditional knockdown cancer cell lines using a Tet-inducible lentiviral shRNA system. It outlines the steps for viral transduction, antibiotic selection, and doxycycline-induced gene silencing.

Source: Ding, J., et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)This video demonstrates the production of recombinant adeno-associated virus particles through plasmid transfection in mammalian cells. It highlights the coordinated expression of Rep, Cap, and helper proteins that drive viral genome replication and capsid assembly within the nucleus.

Source: McAllister, S. C., Hanson, R. S., et al. An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus. J. Vis. Exp.(2017)This video demonstrates retroviral transduction of primary endothelial cells with HPV16 E6/E7 oncogenes. The integrated oncogenes inactivate p53 and Rb tumor suppressors, extending cell lifespan. G418 selection enriches for transduced cells, yielding immortalized endothelial cultures suitable for transformation...

Source: Koza, P., et al. Generation of Transgenic Rats using a Lentiviral Vector Approach. J. Vis. Exp. (2020)This video demonstrates microinjection-assisted delivery of lentiviral vectors into fertilized rat embryos to achieve stable transgene integration for the generation of transgenic rats.

Source: Zhang, Y., et al., Direct Reprogramming of Mouse Fibroblasts into Melanocytes. J. Vis. Exp. (2021)This video demonstrates the production and concentration of recombinant lentivirus in mammalian cells, enabling efficient gene delivery of transcription factors or other genetic elements for applications such as cellular reprogramming, functional studies, and potential therapeutic research.

Source: Hoenen, T., et al. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes. J. Vis. Exp. (2014)This video demonstrates how a tetracistronic minigenome plasmid mimicking the Ebola virus genome is transfected into human cells along with helper plasmids. Inside the cells, viral proteins and luciferase are expressed, leading to the assembly and budding of non-pathogenic, replication-competent virus-like...

Source: Heidbuechel, J. P., Engeland, C. E. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J. Vis. Exp. (2019)This video demonstrates the generation of recombinant measles viruses using liposomes to deliver plasmids encoding the viral antigenome and essential helper proteins into producer cells. The transcribed and replicated RNA leads to the formation of viral particles expressing immunostimulatory and fluorescent reporter proteins. Fluorescence microscopy...

Source: Zjablovskaja, P., et al., Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells. J. Vis. Exp. (2018)This video demonstrates the production of recombinant retroviral particles by transfecting human kidney-derived packaging cells, enabling stable gene delivery to target cells for downstream applications such as genetic modification, functional assays, or lineage differentiation studies.

Source: Bouillier, C. et. al., Amplification, and Titration of Recombinant Respiratory Syncytial Viruses. J. Vis. Exp. (2019)This video demonstrates the generation of a green fluorescent protein (GFP)-expressing recombinant respiratory syncytial virus using a T7-based reverse genetics system. The co-transfection of plasmids encoding viral proteins and a GFP-tagged genome leads to protein expression and initiates virus rescue.

Source: Natilla, A.,and Hammond, R. W. Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs. J. Vis. Exp. (2013)This video demonstrates site-specific fluorescent labeling of mutant virus-like particles (VLPs) produced in Nicotiana benthamiana using a thiol-reactive dye, followed by purification and gel electrophoresis to confirm surface modification.

Source: Chen, Z., et al. Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry. J. Vis. Exp. (2018)This video demonstrates the fluorescent functionalization of virus-like particles by re-bridging reduced disulfides with a polyethylene glycol (PEG) linker, producing stable, trackable conjugates suitable for cellular imaging and targeted delivery in biomedical applications.

Source: Yun, S. et al. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses. J. Vis. Exp. (2015)This video demonstrates the recovery of infectious Japanese encephalitis virus from synthetic RNA transcribed from a bacterial artificial chromosome clone. It shows how cells are electroporated with the synthetic RNA, allowing the viral genome to direct the production of new virions. The resulting plaques formed in infected cell monolayers confirm...

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021)This video demonstrates a luciferase-based pseudovirus titration assay to determine the optimal target cell density for maximum infection. By comparing luminescence across cell densities, the method ensures high assay sensitivity and reproducibility for downstream applications.

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021).This video demonstrates a luciferase-based assay to quantify pseudovirus infection in epithelial cells. The pseudovirus encodes the luciferase gene, which is expressed in infected cells. After washing, and cells are incubated with lysis buffer at an ultra-low temperature, and then thawed to induce lysis.

Source: Klaus, J. P. et al. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment. J. Vis. Exp. (2016)This video demonstrates the extraction of viral RNA from surface-bound virions on epithelial cells using silica spin columns. The method includes chemical lysis, RNA stabilization, and membrane-based purification for downstream quantification of virus-cell attachment.

Source: Ren, S., et al. A Protocol for Analyzing Hepatitis C Virus Replication. J. Vis. Exp. (2014).This video demonstrates the procedure for quantifying Hepatitis C viral RNA using quantitative reverse transcription PCR to measure viral load in infected cell cultures.

Source: Kadoya, S., Sano, D. Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus. J. Vis. Exp. (2019).This video demonstrates the use of a plaque assay to assess the rotavirus growth curve. The method involves infecting host cells with diluted virus, overlaying with agarose to restrict spread, and staining to visualize plaques formed by viral replication. By counting plaque-forming units at defined time points, the resulting growth curve reveals the lag, exponential, and...

Source: Fout, G. S., Cashdollar, J. L. EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay. J. Vis. Exp. (2016)This video demonstrates the use of a total culturable virus assay to quantify infectious viruses in water samples. It outlines host cell inoculation, observation of cytopathic effect, and statistical quantification of infectious virus concentration.

Source: Scarborough, R. J., et al. Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy. J. Vis. Exp. (2016).This video demonstrates a reverse transcriptase (RT) assay to assess the effect of a test RNA on virus-like particle (VLP) production. Cells are co-transfected with plasmids, and RT activity is measured as a quantitative indicator of VLP output. A decrease in RT activity reflects suppression of viral gene expression by the test RNA.

Source: Manocheewa, S., et al. Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses. J. Vis. Exp. (2015)The video demonstrates the procedure for a growth competition assay to measure the relative replication fitness of two viral strains by comparing their replication in human immune cells.

Source: Vijayraghavan, S., et al. Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J. Vis. Exp. (2017).This video demonstrates the use of a p24 ELISA to estimate viral titers of lentiviral vectors. The assay involves lysing viral particles to release p24, capturing it with specific antibodies, and detecting it with a peroxidase-linked system that produces a measurable color change. By comparing sample absorbance...

Source: Krishnamurthi, R., et al. Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. J. Vis. Exp. (2024).This video demonstrates the temporal enumeration of spontaneous phage induction in lysogenic Pseudomonas aeruginosa by quantifying viable bacterial cells (CFUs) and infective phage particles (PFUs) over time. By sampling at defined intervals during growth and analyzing both colony and plaque formation, the method reveals when spontaneous induction...

Source: Frias-De-Diego and A., Crisci, E. Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays. J. Vis. Exp., (2022)This video demonstrates a crystal violet staining method to visualize cytopathic effects in virus-infected cell cultures. The pattern of stained and unstained wells across serial dilutions enables rapid, cost-effective estimation of the viral infectious dose.

Source: Jamieson, T. R., et al., Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection. J. Vis. Exp. (2021)This video describes the virus plaque assay using coronavirus pseudovirus particles. The diluted pseudovirus is added to a monolayer of mammalian cells, followed by incubation with a methylcellulose overlay. Plaques formed by infected cells are counted visually to determine the infectious virus titer.

Source: Ren, S., et al. A Protocol for Analyzing Hepatitis C Virus Replication. J. Vis. Exp. (2014)This video demonstrates the fluorescence focus assay used to quantify infectious Hepatitis C virus in cell culture. It demonstrates the serial dilution of viral supernatant, the infection of hepatoma cells, and the detection of viral replication sites using antibody-based fluorescence microscopy.

Source: Bouillier, C., et al. Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses. J. Vis. Exp. (2019)The video demonstrates a plaque titration assay used to quantify infectious recombinant respiratory syncytial virus (RSV) in epithelial cell monolayers. It presents the infection of host cells with serially diluted viral suspensions, the restriction of viral spread by a cellulose-based semi-solid overlay, and the formation of localized plaques from cytopathic...

Source: Claiborne, D. T. et al. A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses. J. Vis. Exp. (2014)This video demonstrates the assessment of viral replication capacity using a radiolabeled reverse transcriptase assay. Culture supernatants containing progeny virions are added to a reverse transcription mixture containing a radiolabeled nucleotide. The incorporated signal reflects viral output and replication...

Source: Garcia, V., et al. High-throughput Titration of Luciferase-expressing Recombinant Viruses. J. Vis. Exp. (91), e51890, doi:10.3791/51890 (2014).This video demonstrates the preparation of cell culture samples to assess viral load using a luciferase-based assay. It outlines the process of infecting epithelial cells with a genetically modified virus, transferring viral supernatants and standards into assay wells, and promoting viral entry through centrifugation and incubation. The...

Source: Baer, A. and Kehn-Hall, K. Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. J. Vis. Exp. (2014).This video demonstrates the use of a liquid microcrystalline cellulose-based overlay in a plaque-based assay by comparing its effectiveness in viral plaque formation with that of a traditional semi-solid agarose overlay.