Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposi’s Sarcoma-Associated Herpesvirus

Overview

This protocol outlines a reliable in vitro system for modeling de novo lytic infection of Kaposi's sarcoma-associated herpesvirus (KSHV) using BAC16-derived virions. The method allows for the investigation of early viral replication and dissemination in a tractable manner.

Key Study Components

Area of Science

• Virology
• Infectious Diseases
• Cell Biology

Background

• KSHV exhibits a strong latency bias, complicating the modeling of its lytic infection.
• Primary cells that support KSHV lytic infection are delicate and have limited lifespans.
• Existing models lack reproducibility and scalability.
• The study utilizes HCT116 cells to capture efficient KSHV lytic replication.

Purpose of Study

• To establish a robust in vitro platform for KSHV de novo lytic infection.
• To facilitate the study of early events in herpesvirus replication.
• To improve reproducibility and scalability of KSHV infection models.

Methods Used

• Seed five T75 flasks with dox-inducible iSLK cells containing wild-type KSHV BAC16.
• Maintain cells at approximately 80% confluency.
• Quantify infectious virus production using GFP-based assays.
• Employ real-time PCR for encapsidated viral genomes analysis.

Main Results

• Establishment of a tractable model for KSHV lytic replication.
• Demonstration of efficient viral replication during primary infection.
• Provision of a unique platform for studying early viral events.
• Enhanced understanding of KSHV lytic infection dynamics.

Conclusions

• The developed model offers a reliable system for KSHV research.
• It addresses challenges related to latency and cell viability.
• This platform can significantly advance the study of herpesvirus biology.

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