Herpes Simplex Virus Growth, Preparation, and Assay

Sutter, Sereina O.; Marconi, Peggy; Meier, Anita F.

doi:10.1007/978-1-4939-0428-0_2

Cited by 19 publications

(26 citation statements)

References 6 publications

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“…Traditionally, plaque assays or PCRs are used to evaluate single-step and multistep growth kinetics of herpesviruses, specifically focusing on factors related to viral entry and cell-to-cell spread, respectively (28). Here, we propose ECIS as a novel tool with which to study herpesvirus growth kinetics, with the major advantages that ECIS provides objective quantification in real time, thus avoiding the need for static intermittent sample collection and extensive postexperimental processing.…”

Section: Discussionmentioning

confidence: 99%

“…Next, cells were transfected with an additional 500 ng of donor plasmid and simultaneously infected with approximately 6,500 PFU of FHV-1. A pure FHV-1-gD-DsRed stock was then obtained by three rounds of limiting-dilution assays (28). …”

Section: Methodsmentioning

confidence: 99%

“…For the latter, a standard curve with primers targeting a region in the US7 gene homology arm was created with the linearized donor plasmid used to create FHV-1-gD-DsRed as the template (Table 1). Efficiency of amplification was confirmed to be >98% with R 2 = 0.998, and qPCR was performed as previously described (28). For samples containing large amounts of virus, template DNA was first diluted 1:100 to keep the copy number within the limits of the standard curve.…”

Section: Methodsmentioning

confidence: 99%

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Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

Pennington

Walle

2017

mSphere

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Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

Pennington

Walle

2017

mSphere

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“…Supernatants from the HSV-2 infection experiments were harvested at 6 h and 24 h, and the viral yields were quantified using a modified plaque assay method ( 33 ) on Vero cells. Briefly, DC supernatants were incubated in 2- or 10-fold dilutions in 24-well plates with a confluent monolayer of Vero cells for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Complement Opsonization Promotes Herpes Simplex Virus 2 Infection of Human Dendritic Cells

Crisci

Ellegård

Nyström

et al. 2016

J Virol

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Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections globally, with a very high prevalence in many countries. During HSV-2 infection, viral particles become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of immune responses. In genital mucosa, the primary target cells for HSV-2 infection are epithelial cells, but resident immune cells, such as dendritic cells (DCs), are also infected. DCs are the activators of the ensuing immune responses directed against HSV-2, and the aim of this study was to examine the effects opsonization of HSV-2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV-2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV-2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV-1- or HSV-2-specific antibodies more or less abolished HSV-2 infection of DCs. Our results clearly demonstrate the importance of studying HSV-2 infection under conditions that ensue in vivo, i.e., conditions under which the virions are covered in complement fragments and complement fragments and antibodies, as these shape the infection and the subsequent immune response and need to be further elucidated.IMPORTANCE During HSV-2 infection, viral particles should become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of the immune responses. The dendritic cells are activators of the immune responses directed against HSV-2, and the aim of this study was to examine the effects of complement alone or complement and antibodies on HSV-2 infection of dendritic cells and their ability to mount inflammatory and antiviral responses. Our results demonstrate that the presence of antibodies and complement in the genital environment can influence HSV-2 infection under in vitro conditions that reflect the in vivo situation. We believe that our findings are highly relevant for the understanding of HSV-2 pathogenesis.

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“…The plaque assay was used to determine viral titers[22]. In brief, pre-cultured Vero cells were seeded onto 6-well plates and infected with serial dilutions (up to 10 logs) of virus samples.…”

Section: Methodsmentioning

confidence: 99%

Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic Herpes Simplex Virus Type 1 (oHSV-1) for Generation and Isolation of HSV-Based Vectors

Abdoli

Roohvand

Teimoori‐Toolabi

et al. 2017

IBJ

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Background:Oncolytic herpes simplex virus (oHSV)-based vectors lacking γ34.5 gene, are considered as ideal templates to construct efficient vectors for (targeted) cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and γ34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors.Methods:Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein (GFP) and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines.Results:We generated three recombinant viruses, HSV-GFP, HSV-GR (Green-Red), and HSV-Red. The HSV-GFP showed two log higher titer (1010 PFU) than wild type (108 PFU). In contrast, HSV-GR and HSV-Red showed one log lower titer (107 PFU) than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 (P<0.001). Moreover, HSV-GFP showed higher infection potency (98%) in comparison with HSV-GR (82%).Conclusion:Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2nd and 3rd generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy.

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Herpes Simplex Virus Growth, Preparation, and Assay

Cited by 19 publications

References 6 publications

Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

Complement Opsonization Promotes Herpes Simplex Virus 2 Infection of Human Dendritic Cells

Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic Herpes Simplex Virus Type 1 (oHSV-1) for Generation and Isolation of HSV-Based Vectors

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