Virale Groei en Technieken

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.

Bron: Mackler, R. M., et al. Assembly and Purification of Prototype Foamy Virus Intasomes. J. Vis. Exp. (2018)Deze video demonstreert de zuivering van prototype schuimvirus (PFV) intasomen uit een gemengde eiwit-DNA-suspensie met behulp van zoutextractie en grootte-uitsluitingschromatografie, wat resulteert in actieve complexen voor verdere analyse.

Bron: 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).Deze video demonstreert de procedure voor het voorbereiden van virale monsters voor kwantitatieve PCR-analyse (Polymerase chain reaction). Virale monsters en RNA-standaarden worden serieel verdund, behandeld met een proteinase K-buffer en geïncubeerd onder gecontroleerde temperaturen om RNA vrij te maken. Het bewerkte RNA wordt...

Bron: Dembowski, J. A., et al. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017).Deze video demonstreert de isolatie van eiwit-viraal DNA-complexen uit celkernen met behulp van click-chemie en purificatie met magnetische beads. Het protocol omvat het labelen van virale genomen met alkyn-gemodificeerde nucleotiden, biotinylering via een click-reactie en binding aan streptavidine-gecoate magnetische beads. Na het wassen en hitte-elutie...

Bron: Ding, J. et. al., Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)Deze video laat de zuivering van recombinante viruspartikels uit getransfecteerde menselijke niercellen zien. Het virus wordt vrijgegeven door vries-dooi-lyse en gezuiverd met behulp van iodixanolgradiënten met blokcopolymeer-ondersteunde filtratie.

Bron: 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)Deze video demonstreert het terugwinnen van ruw adeno-geassocieerd virus uit getransfecteerde zoogdiercellen middels chemische lyse en centrifugatie, wat een hoge opbrengst van AAV mogelijk maakt voor verdere zuivering en preklinische gentherapie-toepassingen.

Bron: Lopez Jr., M. A., et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)Deze video demonstreert de zuivering van polyhistidine-getagde virale integrase met behulp van nikkelaffiniteitschromatografie. Het protocol maakt gebruik van imidazolgradiënt-elutie om de integrase selectief te isoleren op basis van de affiniteit voor nikkelgeladen hars.

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.

Bron: Zhao, J., et al. Potato Virus X-Based microRNA Silencing (VbMS) In Potato. J. Vis. Exp. (2020).Deze video demonstreert het gebruik van Agrobacterium tumefaciens voor het afleveren van een construct op basis van Potato Virus X (PVX) in in vitro gekweekte aardappelplanten voor virus-gebaseerde microRNA-silencing (VbMS). Deze methode maakt systemische onderdrukking van een doelwit-microRNA mogelijk, waardoor onderzoekers de rol ervan in de plantontwikkeling kunnen bestuderen via...

Bron: 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)Deze video demonstreert een fluorescentie-assay voor co-cultuur om virale transmissie van cel naar cel te monitoren. De assay maakt gebruik van een rood-naar-groen reportercassette om virale membraanfusie te visualiseren en de effectiviteit van testverbindingen bij het blokkeren van virale entry te evalueren.

Bron: Yates, J. G. E., et al. Productie van recombinant Newcastle disease-virus met een hoge titer uit allantoïsvloeistof. J. Vis. Exp. (202)De video demonstreert de zuivering en concentratie van viruspartikels uit geklaarde allantoïsvloeistof, verzameld uit virus-geïnfecteerde geëmbyoneerde kippeneieren. Er wordt een reeks filtratie- en scheidingsstappen getoond, waaronder dieptefiltratie, tangential flow-filtratie en dichtheidsgradiënt-ultracentrifugatie om intacte virussen te isoleren. De...

Bron: 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)Deze video demonstreert de groei van het coronavirus in menselijke nasale epitheelcellen gekweekt op een lucht-vloeistofinterface, wat de infectie en virale replicatie in de menselijke neusholte nabootst.

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.

Bron: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015)Deze video demonstreert de stapsgewijze procedure voor het bereiden van cryosneden van de muizenretina om de permeabiliteit van adeno-geassocieerde virussen door verstoorde retinale barrières te visualiseren.

Bron: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015).Deze video demonstreert de methode voor AAV-gemedieerde genoverdracht in een retina van een transgene muis, gevolgd door dissectie en kleuring van het weefsel om de integriteit van het intern begrenzend membraan te beoordelen.

Bron: Moore, M. D., et al. Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity. J. Vis. Exp.(2017).Deze video demonstreert hoe gezuiverde virale capsiden worden hittebehandeld, geprepareerd en afgebeeld met behulp van transmissie-elektronenmicroscopie (TEM) om structurele veranderingen en door hitte veroorzaakte schade bloot te leggen.

Bron: 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)Deze video demonstreert de gelijktijdige visualisatie van HIV-1 DNA, RNA en capsid-eiwit in geïnfecteerde menselijke lymfocyten met behulp van branched DNA-hybridisatie en immunofluorescentie, waardoor een gedetailleerde analyse van de virale replicatiestadia binnen individuele cellen mogelijk...

Bron: Pohl, M. O. & Stertz, S. Het meten van aanhechting en internalisatie van influenza A-virus in A549-cellen via flowcytometrie. J. Vis. Exp. (2015) Deze video demonstreert het gebruik van een streptavidine-blockeringsassay om oppervlaktegebonden influenza-virus te onderscheiden van geïnternaliseerd virus in humane longepitheelcellen. Vergelijking van fluorescentiesignalen laat zien dat een hogere intensiteit na incubatie wijst op een succesvolle virale internalisatie. Pohl, M. O. &...

Bron: Niu, Y. D., et al. Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings. J. Vis. Exp. (2021)Deze video demonstreert een op microplaten gebaseerde assay om de lytische activiteit van faagcocktails tegen E.coli bacterieculturen te evalueren. Er wordt toegelicht hoe veranderingen in de optische dichtheid de potentie van de fagen en de concentratie onthullen die nodig is voor volledige bacteriële...

Bron: Dembowski, J. A., Deluca, N. A. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017)Deze video demonstreert de infectie van zoogdiercellen in de stationaire fase met HSV-1 (Herpes Simplex Virus-1), gevolgd door replicatie van het virale genoom met behulp van de replicatiemachinerie van de gastheer en van het virus. Een synthetisch nucleoside-analoog wordt ingebouwd in nieuw gesynthetiseerd viraal DNA om replicerende genomen te...

Grootschalige productie van virale vectoren afgeleid van adeno-geassocieerde virussen...

Bron: 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).Deze video demonstreert de beeldvorming van faustovirusdeeltjes uit een geïnfecteerde amöbenkweek met behulp van transmissie-elektronenmicroscopie. Er wordt ingegaan op de preparatie van het raster, negatieve kleuring met ammoniummolybdaat om het contrast te verhogen en de visualisatie van het virus onder de microscoop. Het protocol...

Bron: 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)Deze video demonstreert fluorescentie in situ hybridisatie om viraal RNA te visualiseren in virusgeïnfecteerde cellen. Er wordt een overzicht gegeven van de stappen die betrokken zijn bij probe-hybridisatie, signaalamplificatie en confocale beeldvorming.

Bron: 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)De video demonstreert de zuivering van recombinante adeno-geassocieerde virus (AAV) vectoren uit ruw gastheerlysaat met behulp van een op heparine gebaseerde affiniteitsmatrix. Virale deeltjes binden aan geïmmobiliseerde heparine, terwijl onzuiverheden worden weggewassen. Een buffer met een hoog zoutgehalte laat intacte virussen vrij, die...

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.

Bron: 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)Deze video demonstreert de productie van influenzapsudovirussen in menselijke cellen met behulp van plasmidtransfectie via calciumfosfaat, wat de veilige generatie van single-cycle virusdeeltjes mogelijk maakt voor downstream toepassingen, zoals antilichaamneutralisatie-assays onder biosafety level...

Bron: 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)Deze video demonstreert de productie en zuivering van recombinante hepatitis B-virusdeeltjes, wat veilige, replicatie-deficiënte virale preparaten mogelijk maakt voor het bestuderen van vroege infectiegebeurtenissen en voor high-throughput screeningtoepassingen van antivirale geneesmiddelen.

Bron: 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)Deze video demonstreert het gebruik van recombinante muggendensovirussen die microRNA coderen om specifieke genen in muggenlarven stil te leggen. Geïnfecteerde larven in een vroeg stadium produceren functionele microRNA's die de expressie van doelgenen onderdrukken voor vervolgstudies.

Bron: Eggers, C. H. Fage-gemedieerde genetische manipulatie van de ziekte van Lyme spirochaet Borrelia burgdorferi. J. Vis. Exp. (202). Deze video demonstreert de transductie van genetisch gemodificeerde Borrelia-spirocheten met behulp van niet-infectieuze bacteriofagen, geprecipiteerd met polyethyleenglycol, die een kanamycin-resistentieplasmid dragen. Na incubatie met de bacteriën wordt de cultuur geplaatst op medium aangevuld met antibiotica. Alleen cellen die het plasmid ontvangen,...

Bron: Van Lidth de Jeude, J. F., et al. Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids. J. Vis. Exp. (2015)Deze video beschrijft de lentivirale transductie van muis-intestinale organoïden vanuit een suspensie die crypt-achtige structuren bevat. Met behulp van een basaalmembraanmatrix als steunstructuur en medium aangevuld met groeifactoren worden lentivirale vectoren geïntroduceerd om stabiele genintegratie mogelijk te...

Bron: 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).Deze video demonstreert een methode om de activatie van de SMAD-route in borstkankercellen te bestuderen met behulp van dubbele adenovirale infectie. Het ene virus levert een constitutief tot expressie gebracht gen voor GFP, terwijl het andere een RFP-reporter draagt onder een SMAD-gevoelige promoter. Na stimulatie met TGF-beta worden...

Bron: Meister, T. L., et al. A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks. J. Vis. Exp. (2020).Deze video demonstreert het proces van het isoleren van hepatitis E-virus (HEV) deeltjes uit getransfecteerde humane hepatocellulaire carcinoomcellen. Extracellulaire virusdeeltjes worden verzameld uit gefiltreerde supernatanten, terwijl intracellulaire deeltjes worden vrijgemaakt door de cellen bloot te stellen aan herhaalde vries-dooi-cycli. Het lysaat wordt...

Bron: Rim, Y. A., et al. Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation. J. Vis. Exp. (2016).Deze video demonstreert de generatie van geïnduceerde pluripotente stamcellen (iPSCs) uit mononucleaire cellen van menselijk perifeer bloed met behulp van Sendai-virus-gemedieerde herprogrammering. De virale transcriptiefactoren activeren pluripotentienetwerken en onderdrukken lineage-genen, waardoor een subset van de cellen adhesie-eigenschappen...

Bron: 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)Deze video demonstreert de zuivering van high-capacity adenovirale vectoren met behulp van cesiumchloride-gradiënt ultracentrifugatie, waardoor de scheiding van genoombevattende virionen mogelijk wordt voor toepassingen in genoverdracht, vectorcharacterisering en preklinische therapeutische...

Bron: 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)Deze video demonstreert genoverdracht via een adeno-geassocieerd virus in een gehumaniseerd ratleversteunweefsel. De vector brengt een transgen dat een fluorescerende reporter en gene-silencing RNA codeert over in levercellen via receptor-gemedieerde endocytose. Dit model ondersteunt efficiënte transductie in een geperfuseerd...

Bron: 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)Deze video demonstreert het genereren van conditionele knockdown-kankercellijnen met behulp van een Tet-induceerbaar lentiviraal shRNA-systeem. Er wordt een overzicht gegeven van de stappen voor virale transductie, antibioticaselectie en doxycycline-geïnduceerde gen-silencing.

Bron: Ding, J., et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)Deze video demonstreert de productie van recombinante adeno-geassocieerde viruspartikels via plasmidtransfectie in zoogdiercellen. Er wordt ingezoomd op de gecoördineerde expressie van Rep-, Cap- en helperproteïnen die de replicatie van het virale genoom en de assemblage van de capside in de nucleus aansturen.

Bron: McAllister, S. C., Hanson, R. S., et al. An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus. J. Vis. Exp.(2017)Deze video demonstreert de retrovirale transductie van primaire endotheelcellen met HPV16 E6/E7-oncogenen. De geïntegreerde oncogenen inactiveren de tumorsuppressoren p53 en Rb, waardoor de levensduur van de cellen wordt verlengd. Selectie met G418 verrijkt de populatie met getransduceerde cellen, wat resulteert in geïmmortaliseerde...

Bron: Koza, P., et al. Generation of Transgenic Rats using a Lentiviral Vector Approach. J. Vis. Exp. (2020)Deze video demonstreert de via micro-injectie ondersteunde aflevering van lentivirale vectoren in bevruchte rattenembryo's om stabiele integratie van het transgen te bereiken voor de generatie van transgene ratten.

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.

Bron: 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).Deze video demonstreert het gebruik van een p24 ELISA om virale titers van lentivirale vectoren te schatten. De assay omvat het lyseren van virale deeltjes om p24 vrij te maken, het vastleggen hiervan met specifieke antilichamen en het detecteren hiervan met een peroxidase-gekoppeld systeem dat een meetbare...

Bron: Krishnamurthi, R., et al. Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics. J. Vis. Exp. (2024).Deze video demonstreert de temporele enumeratie van spontane fageninductie in lysogene Pseudomonas aeruginosa door het kwantificeren van levensvatbare bacteriële cellen (CFU's) en infectieuze fagenpartikels (PFU's) over tijd. Door monstername op vaste intervallen tijdens de groei en het analyseren van zowel kolonie- als plaquevorming, onthult deze...

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.