SARS-CoV-2 develops differently in the brain and provides important insights into viral tropism

Study: The evolution of SARS-CoV-2 in the central nervous system of mice drives virus diversification. Photo credit: Stock_Good / Shutterstock.com

Numerous sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infections have been associated with neurological complications, possibly resulting from direct infection of the central nervous system (CNS). A recent Natural Microbiology Study compares the development of SARS-CoV-2 in the lungs and the CNS.

The pathology of COVID-19

SARS-CoV-2 is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. This virus, which replicates in lung epithelial cells, can also affect the CNS and cause acute kidney injury, myocarditis and thromboembolism. To date, the underlying host and viral properties leading to these pathologies are not well understood.

SARS-CoV-2 entry into a host cell is mediated by the viral spike glycoprotein (S), which consists of S1 and S2 subunits at the furin cleavage site (FCS). The continued evolution of SARS-CoV-2 has led to the emergence of more infectious variants of concern (VOCs). SARS-CoV-2 VOCs typically contain mutations that affect FCS cleavage efficiency and the stability of the S1/S2 interaction.

Most studies investigating FCS have monitored viral load in the lungs. The dynamics of altered FCS virus variants and how these mutations alter viral tropism and disease pathogenesis are still unclear.

About the study

In the current study, two different mouse models were used to investigate how SARS-CoV-2 evolves in different host tissues and whether pre-existing immunity influences viral evolution. To this end, mice were randomly divided into experimental groups and vaccinated either intranasally or intracranially with two types of Ad5 vector vaccines.

The vaccines investigated in the present study encoded either the SARS-CoV-2 open reading frame S (Ad5-S) or the nucleocapsid (N) open reading frame (Ad5-N). Phosphate-buffered saline (PBS) was used as a control. After three weeks, mice were challenged with a high mutation rate in the spike FCS.

Focus formation assays and real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed. In addition, whole genome sequencing of viral RNA was performed to elucidate the determinants of viral evolution. Shannon entropy was calculated to compare and evaluate intrahost diversification in different animals and tissues.

Study results

SARS-CoV-2 strains lacking FCS are attenuated in the lung, which may be due to increased dependence of ΔFCS pseudoviruses on the transmembrane serine protease 2 (TMPRSS2)-independent endosomal entry pathway. This finding is consistent with the results of a previous study that found that the absence of FCS in other coronaviruses increases CNS tropism.

The ΔFCS pseudovirus cannot penetrate lung cells as efficiently as visceral adipose tissue (VAT) cells. The present study hypothesized that the attenuated growth of ΔFCS viruses after intranasal administration was due to reduced virus entry into airway cells, lower virus titers in the lung, and reduced pathology.

No influence of vaccination status and formulation on virus divergence in the lungs was observed. However, brain isolates varied regardless of vaccination type.

In PBS and Ad5-N mice, viral diversity was higher in the lungs than in the brain. No difference in overall diversity was observed in Ad5-S and Ad5-N + Ad5-S mice. There were no statistical differences in viral diversity in the lungs across all groups.

In the brain, Ad5-S and Ad5-N + Ad5-S mice showed higher diversity than the control group. Thus, Ad5-S reduces viral diversity in the lungs, while maintaining higher diversity in the brain.

A significant enrichment in S diversity was observed, with the highest diversity in and around the FCS. These observations were further explored in an alternative model in which neuroinvasion triggered a selective pressure for mutations or deletions of the FCS, regardless of the prior immunity status.

Previous studies have hypothesized that deletion of the FCS is selected because it maintains pressure on the virus to favor endosomal-mediated entry. However, further research is needed to clarify whether this selection pressure is driven by the tropism of a particular transported cell type or other factors associated with population bottlenecks.

Based on immunohistochemical results, both wild-type and FCS mutant viruses were found to successfully infect neuronal cells. However, the FCS mutant strain replicated faster, suggesting that selective pressure may be present within the target cells of the CNS.

Consistent with the results of the present study, previous studies have shown that viral replication in the CNS is associated with mutational variants, particularly deletions within the S protein.

Conclusions

The current study highlights the occurrence of selective pressure on the SARS-CoV-2 S protein during neuroinvasion and compartmental transport. In the future, further investigations are needed to determine the role of compartmentalization in the emergence of new virus variants. Furthermore, it is important to assess whether direct viral infection in the CNS is responsible for the neurological complications observed in acute COVID-19 and Long COVID.