SARS-CoV-2 evolves differently in the brain, providing important insights about viral tropism

Study: Growth of SARS-CoV-2 in the central nervous system of mice promotes diversification of the virus. Image Source: Stock_Good / Shutterstock.com

Several consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection have been found to be associated with neurological complications, possibly due to direct infection of the central nervous system (CNS). Nature Microbiology The study compares the growth 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, 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.

Entry of SARS-CoV-2 into the 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 (VOC). 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 viral variants and how these mutations alter viral tropism and disease pathogenesis are still unclear.

About the study

The current study used two different mouse models to assess how SARS-CoV-2 evolves in different host tissues and whether pre-existing immunity affects virus evolution. For this purpose, mice were randomly divided into experimental groups and vaccinated intranasally or intracranially with two types of Ad5-vector vaccines.

The vaccines tested 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). 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 assay and real-time quantitative polymerase chain reaction (RT-qPCR) assay were performed. Whole genome sequencing from viral RNA was also performed 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 that lack the FCS are attenuated in the lungs, which may be due to the increased reliance of the ΔFCS pseudovirus on the transmembrane serine protease 2 (TMPRSS2)-independent endosomal entry pathway. This finding is consistent with the findings of a previous study, which showed that the absence of FCS in other coronaviruses increased CNS tropism.

The ΔFCS pseudovirus could not enter lung cells as efficiently as visceral adipose tissue (VAT) cells. In the present study, it is believed that the attenuated growth of the ΔFCS virus after intranasal inoculation was due to reduced viral entry into respiratory cells, lower viral titer in the lungs, and less pathology.

Vaccination status and formulation were found to have no effect on viral divergence in the lungs. However, brain divergences were different, irrespective of the type of vaccination.

In PBS and Ad5-N mice, viral diversity was higher in the lungs than in the brain. No differences in overall diversity were observed in Ad5-S and Ad5-N + Ad5-S mice. In all groups, the variation in viral diversity in the lungs was not statistically different.

In the brain, Ad5-S and Ad5-N + Ad5-S mice displayed greater diversity than the control treatment. Thus, Ad5-S reduces viral diversity in the lungs, while high diversity is maintained in the brain.

A significant increase in S diversity was observed, with the highest diversity in and around the FCS. These observations were further investigated in an alternative model, in which neuroinvasion generated selective pressure for mutation or deletion of the FCS regardless of previous immune status.

Previous studies have hypothesized that deletion of the FCS is selected because it places pressure on the virus to favor endosomal-mediated entry. However, additional research is needed to clarify whether this selective pressure is achieved through tropism for a specific trafficking cell type or other factors associated with a population bottleneck.

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

Consistent with the findings of the present study, previous studies have indicated that viral replication within the CNS is associated with variant mutations, particularly deletions within the S protein.

conclusion

The present 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 new 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.