In a recent study posted to the bioRxiv* preprint server, researchers evaluated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the peripheral nervous system (PNS).
Previous studies have reported that neurons lack angiotensin-converting enzyme 2 (ACE2) receptors, critical for SARS-CoV-2 entry, and therefore, do not acquire SARS-CoV-2 infections. Thus, peripheral neuropathies could arise as a result of unspecific inflammation of sensory neurons. However, neuropathies in the peripheral nervous system have been observed late in the course of coronavirus disease 2019 (COVID-19), during the period of reduced inflammation.
About the study
In the present study, researchers explored the mechanisms used by SARS-CoV-2 to infect the PNS.
Sensory neurons of human origin were generated from iPS (induced pluripotent stem cells) exposed to the SARS-CoV-2 WA1/2020 strain and variants of concern (VOCs), such as the Delta variant and the Omicron variant. Single-cell ribonucleic acid sequencing (scRNAseq) was performed on HI (heat-inactivated) SARS-CoV-2 or active WA1/2020. The team investigated whether the neurons could express ACE2 and acquire SARS-CoV-2 infection. Human embryonic kidney (HEK)293T cells were included in the cell cultures for simultaneous comparison of iPS-sensory neurons to cells with robust ACE2 expression and active SARS-CoV-2 replication.
Genomic expression profiles of the SARS-CoV-2 RNA-positive and SARS-CoV-2-RNA-negative sensory neurons were compared. For investigating SARS-CoV-2 RNA replication in HEK293T cells and sensory neurons, scRNAseq information was analyzed for negative strands of SARS-CoV-2 RNA. To investigate whether sensory neurons could shed infectious SARS-CoV-2, cells were infected with the virus, following which, the amounts of infectious SARS-CoV-2 in the culture media were quantitated using plaque assays. Calu3 cells (human lung carcinoma cell line) were used as positive controls.
For evaluating differences in the sensory neuronal infection by WA1/2020, Delta and Omicron, co-cultures of sensory neurons derived from induced pluripotent stem cells and HEK293T cells were infected for two days, and subsequently, the cells were analyzed by scRNAseq analysis. Further, differential gene expression analysis was performed to identify genetic signatures exhibited by SARS-CoV-2 RNA- negative and SARS-CoV-2 RNA-positive sensory neurons and 293T cells infected with WA1/2020, Delta, or Omicron.
SARS-CoV-2 could infect 20% of the sensory neurons, with Omicron having the least infection rate (three-fold lower than WA1/2020 and Delta). Even though the neurons were infected by SARS-CoV-2, the virus was unable to actively replicate and release viral progeny. After 1.50 months of differentiating human induced pluripotent stem cells into sensory-type of neurons, >90.0% of the cells showed sensory neuronal marker expression, including the receptor tyrosine kinase (RET), tropomyosin receptor kinase B (Trk.B), peripherin 2 (PRPH), Trk.C, and calcitonin-gene related peptide (CGRP). The induced pluripotent stem cell-derived neurons demonstrated elevated ACE2 expression at levels comparable to those expressed by Calu3 lung cells, indicative of the infectivity.
SARS-CoV-2 ribonucleic acid was detected in 30.0% of SARS-CoV-2-infected cells. The team detected SARS-CoV-2 N (nucleocapsid) gene RNA in 18% and 21% of the neurons and HEK293T cells, respectively, indicating that neurons supported infection by SARS-CoV-2 and ribonucleic acid replication. Neural crest cellular and sensory neuronal marker expressions were comparable among the cell populations, indicating that the sensory neuronal maturity did not determine the development of COVID-19. Rather, SARS-CoV-2 ribonucleic acid-positive cells demonstrated a decreased expression of genes encoding for ribonucleic acid metabolism-associated proteins, including nucleolin (NCL), FUS RNA binding protein (FUS), and nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS1). Infected neurons showed lowered RNA metabolism, which might inhibit SARS-CoV-2 ribonucleic acid translation.
Genes associated with olfactory receptors did not show elevated expression in the cells, indicating that the olfactory system’s sensory neuronal cells are not infected by SARS-CoV-2. In sensory neurons, negative-strand RNA was detected in WA1/2020-infected and Delta-infected cells, indicative of active synthesis of SARS-CoV-2 ribonucleic acid in the neurons post-WA1/2020 infection. Two days post-infection the amount of infective SARS-CoV-2 in the culture media was comparable to cells that did not express angiotensin-converting enzyme 2 like human iPS-derived cortical neurons, indicative of lack of viral propagation in sensory neurons of human origin in vitro.
Of note, while the N gene and open reading frame 1ab (Orf1ab) gene RNA levels were the most abundant transcripts, other SARS-CoV-2 genes, such as the M (membrane) and S (spike) genes, were detectable in sensory neurons. The findings were indicative of differential tropism and infectivity of WA1/2020 strain, Delta VOC and Omicron VOC for sensory neurons; however, they could infect sensory neurons. Omicron upregulated ATR (ataxia telangiectasia and Rad3 related), BACE1 (beta-secretase 1) and CALU (calumenin) expression. WA1/2020 suppressed neuronal genes such as KCNMB2 (potassium calcium-activated channel subfamily M regulatory beta subunit 2) and CADM1(cell adhesion molecule 1) in the neurons. Delta suppressed neuronal transcription factor gene expression, including MN1 (meningioma) and KLF7 (Kruppel-like factor 7) in the neurons.
Overall, the study findings showed that the SARS-CoV-2 WA1/2020 strain, Delta VOC, and Omicron VOC could infect the human sensory type of neurons. However, SARS-CoV-2 infection in the neurons is non-productive. The findings could probably explain the peripheral neuropathies observed in COVID-19 patients.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.