Poster Presentation 12th Australasian Virology Society Meeting 2024

Transcriptomics in virology: we are doing it wrong! Novel sequencing approach uncovers unexpected mechanisms of flavivirus neuropatogenesis. (#248)

Lachlan De Hayr 1 , Mohammed Shaker 1 , Washington Romero 1 , Ziyao Zhao 1 , Ernst Wolvetang 1 , Alexander Khromykh 1 , Andrii Slonchak 1
  1. The University of Queensland, St Lucia, QLD, Australia

Traditional bulk RNA-Seq of tissue homogenates can produce misleading results when used for the analysis of viral infection in complex tissues consisting of multiple cell types and containing infected and uninfected cells. Herein, we employed single-cell RNA-Seq (scRNA-Seq) to overcome this issue and obtain novel insights into neuropathogenesis of West Nile virus (WNV).

We developed flavivirus-inclusive scRNA-Seq method and perform single-cell transcriptomic profiling of human brain organoids (hBOs) infected with WNV. We previously used hBOs as a model for ZIKV and SARS-CoV-2 infection1,2.  Here we demonstrated that 60-day old organoids also support replication of WNV. We then performed scRNA-Seq of WNV-infected and uninfected organoids and found that they contain 11 cell types including neurons and glial cells. We also found that WNV predominantly infect neurons and only a small number of astrocytes. Surprisingly, infected neurons did not express interferons (IFNs). In addition, only ~1% of infected astroglia produced IFN and pro-inflammatory cytokines. Furthermore, infected and uninfected neurons developed little to know response to IFN as indicated by expression of interferon-stimulated genes (ISGs). Instead, they exhibited strong expression of the genes associated with autophagy and cell death. In contrast, profound IFN response was observed in astrocytes, radial glia and progenitor cells, which likely protected them from infection. Further gene expression analysis revealed that neurons lack expression of pattern recognition receptors (PRRs), transcription factors or kinases required for PRR and IFN signal transduction. These findings were also confirmed by scRNA-Seq of ZIKV-infected hBOs, validated by immunostaining and analysis of publicly available human brain scRNA-Seq datasets.

In conclusion, we demonstrated that neurons are vulnerable to flavivirus infection due to the lack of competency in pathogen sensing and IFN response. Our data also suggests that apoptosis of the infected neurons and not the cytokine storm is it the likely cause of flavivirus encephalitis.    

  1. 1. Slonchak A, Wang X, Aguado J, Sng JDJ, Chaggar H, Freney ME, et al. Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis. Science Advances. 2022;8:eadd8095.
  2. 2. Shaker MR, Slonchak A, Al-Mhanawi B, Morrison SD, Sng JDJ, Cooper-White J, et al. Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2. Science Advances. 2024;10(23):eadj4735.