Comprehensive quantification of herpes simplex virus latency at the single-cell level

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Quantifying viral genomes within individual cells in complex tissues has been a significant challenge.
• Understanding viral latency requires precise cellular quantification of viral DNA.
• Previous methods lacked the resolution to analyze nucleic acids within intact tissue at the single-cell level.

Purpose of the Study:

• To develop and validate a novel method, Contextual Analysis (CXA), for quantitative analysis of nucleic acids in individual cells within complex solid tissues.
• To characterize herpes simplex virus (HSV) latency in trigeminal ganglia at the single-cell level.
• To determine the cellular reservoirs of HSV DNA and the viral genome copy number per cell.

Main Methods:

• Development of Contextual Analysis (CXA) for quantitative nucleic acid analysis in intact solid tissues.
• Application of CXA to analyze herpes simplex virus (HSV) DNA in latently infected trigeminal ganglia.
• Determination of cell type harboring viral genomes and viral genome copy number per cell.

Main Results:

• HSV-1 DNA is primarily located in neurons within the trigeminal ganglion.
• Viral genome copy number per latently infected neuron varies significantly (from <10 to >1,000).
• Higher viral input titers correlate with increased numbers of infected neurons and higher viral genome copy numbers per neuron.
• Acyclovir (ACV) treatment during acute infection reduced latently infected neurons by 20-fold and resulted in uniformly low (<10) viral genome copy numbers.

Conclusions:

• CXA provides a powerful new tool for quantifying nucleic acids within individual cells in complex tissues.
• This study comprehensively quantifies HSV latency at the single-cell level for the first time.
• CXA has broad applications beyond viral latency, including microbial pathogenesis, tumorigenesis, and gene transfer studies.