In a recent study published on the website of the bioRxiv*, researchers assessed the impact of coronavirus 2019 (COVID-19) on human lung cells.
Widely studied pathogenesis models suggest the involvement of various cell types in the manifestation of symptoms related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the origin and order of molecular signals that target certain groups of cells are still unknown.
About the study
In this study, researchers described an experimental model of COVID-19 infection to explore the early molecular events as well as the pathogenic mechanism of SARS-CoV-2 at the cellular level in human lung tissue.
The team defined the early stages of SARS-CoV-2 infection by obtaining sections of fresh lung tissue from organ donors or surgical resections and then exposing them to SARS-CoV-2. A plaque test was then performed on the culture supernatants. The expression of viral genes and host genes during COVID-19 infection was further characterized by analyzing lung tissue using single-cell RNA sequencing (scRNA-seq). The team also explored the junctional processing and structure of viral RNA molecules by evaluating the scRNA-seq dataset against the SICILIAN framework which identifies RNA sequences that map discontinuously in a viral genome. .
SARS-CoV-2 lung cell tropism was determined by directly comparing the infection of different lung cell types in their natural environment. To do this, the most specific and sensitive markers among the cells present in a healthy human lung were used to detect the types of cells present in the lung tissue cultures and to assess their levels of viral RNA.
Results
Study results showed that the generation of infectious SARS-CoV-2 virions increased between 24 and 72 hours after culturing SARS-CoV-2 infected lung tissue. Productive infection was then reduced by preactivation of viral stocks achieved by the addition of ultraviolet light, heat, or remdesivir, which is a ribonucleic acid (RNA)-dependent RNA polymerase inhibitor.
The team noted that the number of viral RNA molecules found in each infected lung cell varied widely. Indeed, almost 99% of the infected cells contained few or no viral RNA molecules, while the rest of the infected cells contained from 10 to more than 100 viral RNA molecules in each cell after 24 hours of culture. After 72 hours of culture, almost 0.01% of infected cells contained more than 1000 molecules of viral RNA per cell, indicating the large increase in viral production during this period.
Analysis of the scRNA-seq dataset identified subgenomic junctions among RNA sequence reads, suggesting production of canonical SARS-CoV-2 mRNAs in cultured lung tissue. Furthermore, the team recognized several new subgenomic junctions that indicate the production of various non-canonical and canonical subgenomic viral RNAs during SARS-CoV-2 infection.
The team also identified a total of 55 different molecular cell types in the human lung, distributed across major tissue compartments. These cells included 80% of the cell types that define healthy human tissue with five additional varieties of lymphocytes such as cytotoxic T cells (CD4+), γδ T cells, regulatory T cells, memory CD8+ T cells residing in the tissue and granzyme K CD8+ T cells (GZMK+). Healthy tissue also included culture-induced proliferative forms of alveolar cell type 2 (AT2-s), natural killer (NK) cells, dendritic cells (DC), and fibroblasts.
Notably, 10 to 20 viral RNAs were found in almost a third of lung cell types in infected cell cultures. Cells with higher numbers of viral RNA molecules were rare and mostly limited to six cell types, namely AT2 cells, lipofibroblasts, myofibroblasts, NK cells, T cells, and macrophages. The team noticed that macrophages accounted for 75% of infected cells with 50 or more viral unique molecular identifiers (UMI) per cell. However, those cells with higher viral levels only accounted for 0.5% of all macrophages, indicating inefficient viral access.
Furthermore, the clustering of macrophage gene expression features in cultured lung tissue showed the separation of gene profiles into three different groups. Of these, one showed higher expressions of genes including functions related to mature alveolar macrophages (AMs), such as major histocompatibility complex class II (MHCII) genes, and lipid homeostasis. . The second group included interstitial macrophages (IM) that expressed low levels of AD markers, while the third group included genes expressing nuclear factor kappa light chain signaling and activated B cell activator (NF -KB) and MI-like cells.
Overall, the study results showed that activated interstitial macrophages were the most sensitive target of SARS-CoV-2 infection in the lungs and the main focus of inflammation. The researchers believe the current study can be used to develop therapeutic approaches for lung infections like COVID-19.
*bird is important
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health behavior, or be treated as established information.