In a recent study posted to the bioRxiv* pre-print server, researchers evaluated the neurological complications due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) D614G strain, and variants of concern (VOCs), Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) in a hamster model.
Although the underlying mechanisms of the neurological symptoms are barely understood, scientific evidence suggests that SARS-CoV-2 enters the central nervous system (CNS) via the olfactory nerve. This nerve attaches the olfactory mucosa directly with the olfactory bulb in the brain and thus represents the shortest path between the nasal cavity and the brain.
As demonstrated by several in vivo and in vitro studies, inside the CNS, the neurotropism of SARS-CoV-2 remains restricted to only a few permissive CNS cells, and its replication is often inefficient and abortive in most cells except the choroid plexus epithelial cells. Despite this, SARS-CoV-2 is neurovirulent and triggers neuroinflammatory responses in different anatomical parts of the brain.
About the study
In the present study, researchers used the Syrian golden hamster model to investigate the differences in the neuroinvasive and neurovirulent potential of SARS-CoV-2 variants, D614G, Delta, and Omicron BA.1.
The team inoculated the Syrian golden hamsters intranasally with SARS-CoV-2 variants D614G, Delta or Omicron BA.1. After five days, the team sacrificed the test animals and harvested their nasal turbinates (containing olfactory mucosa), olfactory bulb, cerebral cortex, and cerebellum.
They quantified the infectious SARS-CoV-2 titers and ribonucleic acid (RNA) levels using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Additionally, they computed statistically significant variations (p<0.05) in different samples using a two-way analysis of variance (ANOVA) with a Dunnett’s post hoc test. They compared the mean values of four animals (per infection group) with four mock-treated animals.
They performed in-situ hybridization (ISH) to detect SARS-CoV-2 RNA and immunohistochemistry (IHC) to detect SARS-CoV-2 nucleoprotein (N). In addition, they examined the expression of type-I and type-III-interferon (IFN) response in the olfactory bulb, cerebral cortex, and cerebellum by RT-qPCR to analyze the overall antiviral response. To this end, they used the hematoxylin and eosin (H&E) staining technique.
Further, they analyzed the increased ionized calcium-binding adapter molecule 1 (IBA-1) expression in the different layers of the olfactory bulbs of the D614G, Delta, or Omicron BA.1 inoculated hamsters.
The authors did not observe a substantial difference between the SARS-CoV-2 titers in the nasal turbinates of any test animal. However, Delta-infected hamsters tended to have higher titers and more RNA in nasal turbinates than those infected with D614G and Omicron BA.1.
The olfactory epithelium of the nasal turbinates of D614G infected hamsters showed multifocal mild to moderate attenuation with positive cytoplasmic IHC staining for SARS-CoV-2 antigen.
The researchers detected infectious SARS-CoV-2 in the olfactory epithelium, cerebellum, and cerebral cortex of only one out of the four D614G-infected test animals and in none of the Delta or Omicron-infected animals; however, there were no differences in RNA levels among the different groups.
The SARS-CoV-2 antigen was present in one to several small clusters of periglomerular cells of the glomerular layer. Notably, viral RNA was present at the same sites of the olfactory bulb where the ISH detected SARS-CoV-2 antigens. The authors observed no histological lesions in the olfactory bulbs, cerebral cortex, and cerebellum of the hamsters.
RT-qPCR detected an increase in the messenger RNA (mRNA) for IFN-β and IFN-λ in the olfactory bulb of hamsters infected with D614G. However, the test results did not show induction of IFNs and interferon-stimulated genes (ISGs) in the cerebral cortex or cerebellum of the hamsters.
Interestingly, the authors also observed a significant increase in the allograft inflammatory factor 1 (Aif1) mRNA, encoding the IBA-1 in the olfactory bulbs of hamsters inoculated with D614G or Delta. They did not observe any such increased levels of Aif1 in the hamsters inoculated with the Omicron BA.1 variant.
In the olfactory bulb of the D614G infected hamsters, a significant increase of the chemokines C-X-C motif chemokine 10 (Cxcl10) and C-C motif chemokine ligand 5 (Ccl5) mRNA was detected. However, the gene cluster of differentiation 3 (Cd3) was upregulated only in the cerebral cortex of D614G-infected hamsters.
The study data showed that the most prominent antiviral and inflammatory responses were induced in the olfactory bulb of hamsters inoculated with D614G. Thus, suggesting the neuroinvasive nature of D614G that most likely enters the CNS via the olfactory nerve. These findings also support the observed higher frequency of anosmia in individuals infected with the D614G strain early in the COVID-19 pandemic.
On the contrary, Omicron-infected hamsters showed no antiviral or inflammatory response in the olfactory bulb, and Delta-infected hamsters had upregulated levels of ISG Mx2 and the inflammatory marker IBA-1 in their olfactory bulb. Both Delta and Omicron BA.1 showed a reduced neuroinvasive potential compared to the ancestral D614G variant in the acute phase of SARS-CoV-2 infection (five days post-inoculation).
The abundance of viral antigen and virus-induced lesions in the olfactory mucosa mimicked a pattern previously observed for influenza A viruses. This observation further strengthens the notion that SARS-CoV-2 replicates efficiently in the olfactory mucosa and spreads to the CNS via the olfactory nerve.
For further insights, future studies should evaluate these findings for the post-acute COVID-19 stages for their better application to human situations.
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.
- Lisa Bauer, Melanie Rissmann, Feline Benavides, Lonneke Leijten, Lineke Begeman, Edwin Veldhuis Kroeze, Peter van Run, Marion P.G Koopmans, Barry Rockx, Debby van Riel. (2022). Differences in neuroinflammation in the olfactory bulb between D614G, Delta and Omicron BA.1 SARS-CoV-2 variants in the hamster model. bioRxiv. doi: https://doi.org/10.1101/2022.03.24.485596 https://www.biorxiv.org/content/10.1101/2022.03.24.485596v1