Study: Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification. Image Credit: Stock_Good / Shutterstock.com

Multiple sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infections have been associated with neurological complications that are potentially attributed to direct infection of the central nervous system (CNS). A recent Nature Microbiology study compares the evolution of SARS-CoV-2 in the lungs and 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, as well as induce acute kidney injury, myocarditis, and thromboembolism. To date, the underlying host and viral characteristics that lead to these pathologies are not well understood.

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

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

About the study

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

The vaccines investigated in the current study encoded either the SARS-CoV-2 S open reading frame (Ad5-S) or the nucleocapsid (N) open reading frame (Ad5-N). A phosphate-buffered saline (PBS) solution was used as a control. After three weeks, mice were challenged with a high frequency of mutations in the spike FCS.

Focus-forming assays and real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed. Whole-genome sequencing from viral RNA was also conducted to elucidate the determinants of viral evolution. Shannon entropy was calculated to compare and assess intra-host diversification in different animals and tissues.

Study findings

SARS-CoV-2 strains lacking FCS get attenuated in the lungs, which could be due to enhanced dependence of ΔFCS pseudoviruses on the transmembrane serine protease 2 (TMPRSS2)-independent endosomal entry pathway. This finding is consistent with those of a previous study, which revealed that the absence of FCS in other coronaviruses increased CNS tropism.

The ΔFCS pseudovirus cannot enter lung cells as efficiently as visceral adipose tissue (VAT) cells. The present study assumed that the attenuated growth of ΔFCS viruses after intranasal inoculation was due to reduced viral entry in respiratory cells, lower viral titers in the lungs, and reduced pathology.

Vaccination status and formulation were not found to impact viral divergence in the lungs. However, brain isolates varied, irrespective of the type of vaccination.

In PBS and Ad5-N mice, viral diversity was higher in the lungs as compared to the brain. No difference in overall diversity was observed in the Ad5-S and Ad5-N + Ad5-S mice. Across all groups, variations in viral diversity in the lungs was not statistically different.

In the brain, Ad5-S and Ad5-N + Ad5-S mice exhibited higher diversity than the control treatment. Thus, Ad5-S reduces viral diversity in the lungs, whereas higher diversity is maintained in the brain.

Significant enrichment in S diversity was observed, with the highest diversity in and around the FCS. These observations were further examined in an alternative model, in which neuroinvasion elicited selective pressure for mutations or deletions of the FCS, irrespective of 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, additional research is needed to elucidate whether this selective pressure is derived through the tropism of a specific trafficked cell type or other factors associated with population bottlenecking.

Based on immunohistochemistry results, both wild-type and FCS mutant viruses were found to successfully infect neuronal cells. However, the FCS mutant strain proliferated more rapidly, thus indicating the possibility of selective pressure occurring within the CNS’s target cells.

Consistent with the present study’s findings, previous studies have indicated that viral replication within the CNS is associated with variant mutations, specifically 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 trafficking. In the future, more research is needed to determine the role of compartmentalization in the emergence of novel viral variants. Furthermore, it is important to assess whether direct viral infection in the CNS is responsible for the neurological complications observed during acute COVID-19 and long COVID.