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

Emily A. Rex; Dahee Seo; Don B. Gammon

doi:10.3791/58244

Araçlar Viral Immunomodulators ve ana bilgisayar Antiviral faktörler tanımlanması için eleme olar...

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Immunology and Infection

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JoVE Journal Immunology and Infection

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

Araçlar Viral Immunomodulators ve ana bilgisayar Antiviral faktörler tanımlanması için eleme olarak Abdovirüs enfeksiyonlar

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06:02 min

September 13, 2018

DOI: 10.3791/58244-v

Emily A. Rex¹, Dahee Seo¹, Don B. Gammon¹

¹Department of Microbiology,University of Texas Southwestern Medical Center

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Overview

This article presents protocols to identify virus-encoded immunomodulators that promote arbovirus replication and eukaryotic host factors that restrict it. The methods described utilize fluorescence and luminescence to provide quantitative readouts of arbovirus replication in simplified assays.

Key Study Components

Area of Science

Viral immunology
Arbovirus replication
Host-pathogen interactions

Background

Understanding host factors that restrict arbovirus replication is crucial.
Identifying virus-encoded immune evasion proteins can reveal mechanisms of viral persistence.
Lepidopteran insect cells provide a unique model for studying arbovirus replication.
Minimal background replication in these cells aids in detecting conditions for successful viral replication.

Purpose of Study

To elucidate host factors that limit arbovirus replication.
To identify viral proteins that counteract host restriction mechanisms.
To develop a reliable assay for studying arbovirus behavior in a controlled environment.

Methods Used

Maintenance of LD652 cells in tissue culture dishes.
Passaging cells at 80% confluency.
Using luminescence and fluorescence assays to measure viral replication.
Transferring diluted cell cultures to 24-well plates for experimentation.

Main Results

Successful identification of host factors that restrict arbovirus replication.
Characterization of virus-encoded proteins that facilitate replication.
Demonstration of effective assay conditions for studying arbovirus behavior.
Insights into the dynamics of host-pathogen interactions in lepidopteran cells.

Conclusions

The methods developed provide a framework for future studies in viral immunology.
Understanding the interplay between host factors and viral proteins can inform therapeutic strategies.
This research enhances our knowledge of arbovirus replication mechanisms.

Frequently Asked Questions

What are arboviruses?

Arboviruses are viruses transmitted by arthropods, such as mosquitoes and ticks, that can cause diseases in humans and animals.

Why use lepidopteran cells for this study?

Lepidopteran cells have minimal background replication of arboviruses, making them ideal for studying viral replication conditions.

What is the significance of identifying host factors?

Identifying host factors helps understand how viruses evade immune responses and can lead to the development of targeted therapies.

How do fluorescence and luminescence assays work?

These assays measure the light emitted by fluorescent or luminescent markers, providing quantitative data on viral replication.

What are the implications of this research?

The findings can inform future research on viral pathogenesis and the development of antiviral strategies.

Burada, Abdovirüs çoğaltma ve Abdovirüs çoğaltma kısıtlamak 2) ökaryotik ana faktörler teşvik 1) virüs kodlanmış immunomodulators tanımlamak için protokolleri mevcut. Floresan ve ışıldama dayalı yöntemlerin araştırmacılar hızla düşük sinyal noise oranları ile basit deneyleri Abdovirüs çoğaltma nicel veriler elde etmek için izin verir.

Bu yöntem, viral immünoloji alanında, hangi konak faktörlerinin arbovirüs replikasyonunu kısıtladığı ve buna karşılık hangi virüs tarafından kodlanmış immün kaçırma proteinlerinin bu konak kısıtlama mekanizmalarının üstesinden geldiği gibi temel soruların yanıtlanmasına yardımcı olabilir. Bu tekniğin ana avantajı, arbovirüslerin lepidopteran böcek hücrelerinde aksi takdirde abortif bir enfeksiyonda çoğalmasına izin veren hücresel koşulları tanımlamak için basit lüminesans ve floresan boşluk tahlillerinin kullanılmasına izin vermesidir. Lepidopteran hücrelerinde arbovirüslerin minimum arka plan replikasyonu olduğu için, arbovirüs replikasyonuna izin veren koşulları tespit etmek nispeten basit hale gelir.

Başlamak için, LD652 hücrelerini 10 santimetrelik doku kültürü ile muamele edilmiş kaplarda tutun ve hücreleri% 80 birleşmeye ulaştıklarında geçirin. Hücreleri yerinden çıkarmak için ortamı hücre tek tabakasına tekrar tekrar pipetleyin, ardından numuneyi büyüme ortamında mililitre başına yaklaşık 100 bin hücre yoğunluğuna kadar seyreltin. Bundan sonra, seyreltilmiş kültürün bir mililitresini 24 oyuklu bir plakanın her bir oyuğuna aktarın.

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