# A novel ZIKV-targeted scRNA-seq method for precise quantification of ZIKV RNA

## TL;DR

This study introduces a new single-cell RNA-sequencing method to accurately measure Zika virus RNA in individual cells, identifying key cell types infected and improving understanding for vaccine and treatment development.

## Contribution

The first scRNA-seq method tailored for ZIKV, enabling precise quantification of viral RNA in individual cells and identifying susceptible cell types.

## Key findings

- The ZIKV-targeted scRNA-seq method showed higher efficiency in capturing exogenous cells compared to 10× Genomics.
- Neurons, microglia, and peripheral blood-derived monocytes/macrophages were identified as key cell types infected by ZIKV.
- IFA validation confirmed significant reductions in infected neuron and microglia numbers post-infection.

## Abstract

Zika virus (ZIKV), transmitted by mosquitoes, poses a serious public health threat. Currently, precise quantitative diagnostic methods are lacking. Existing single-cell RNA-sequencing (scRNA-seq) techniques are challenged to detect ZIKV RNA due to its absence of a poly(A) tail, hindering the identification of infected cells. In this study, we developed a novel ZIKV-targeted scRNA-seq method that enables precise quantification of ZIKV RNA in individual cells. Immunocompetent suckling mice were intracerebrally infected with ZIKV to establish persistent infection in the brain. Samples were collected at 10-day post infection for ZIKV-targeted and 10× Genomics scRNA-seq analysis. Comparative analysis identified 17 distinct cell types in both ZIKV-infected and control suckling mouse brains, with significant changes in cell type distribution and proportion post-infection. The ZIKV-targeted scRNA-seq method suggested higher efficiency in capturing exogenous cells compared to the 10× Genomics scRNA-seq method. Both methods identified multiple endogenous and exogenous cell types susceptible to ZIKV, with peripheral blood-derived monocytes/macrophages (PBDMMs), neurons, and T cells as primary cell types expressing ZIKV RNA. IFA validated scRNA-seq findings, revealing that neurons and microglia could be infected by ZIKV, with significant reductions in their numbers post-infection. This study presents a novel ZIKV-targeted scRNA-seq that enables accurate quantification of ZIKV RNA within individual cells, identifies key susceptible cell types, and offers advantages in detecting exogenous cells, making it a scalable solution for providing valuable insights into therapeutic and vaccine development.

This study marks the first use of a scRNA-seq method tailored for ZIKV, allowing accurate measurement of ZIKV RNA in individual cells and identification of critical susceptible cell types. A comparative analysis with the 10× Genomics scRNA-seq method highlighted the advantages of ZIKV-targeted scRNA-seq in terms of accuracy and practicality, particularly its superior ability to capture exogenous cells. Beyond ZIKV, this method also helps establish precise quantification of viral RNA at the single-cell level for other viruses by designing target-specific beads based on conserved regions of the viral genome. This advancement is set to greatly enhance studying pathogenesis of ZIKV infection and then significantly contribute to improve prevention and research in therapeutics and vaccines.