In a recent study published in Nature Communications, a group of researchers investigated the neurotropic potential of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants by studying their ability to invade the brain through the olfactory pathway in hamsters.
Study: Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants. Image Credit: Lightspring/Shutterstock.com
Background
The coronavirus disease 2019 (COVID-19) pandemic remains a global health crisis, with over 760 million confirmed cases of all SARS-CoV-2 variants. Different variants, including Alpha, Beta, Gamma, Delta, and Omicron, have emerged, impacting the clinical presentation of the disease.
COVID-19 affects the respiratory system and can cause various symptoms, including neurological manifestations like headaches and loss of smell. The neurological effects are believed to be a result of inflammation and reduced oxygen supply rather than direct viral invasion into the central nervous system.
About the study
In the present study, hamsters were housed in groups of four and handled in a safe environment. The SARS-CoV-2 virus and its variants were obtained from reputable sources and stored at -80°C. Recombinant viruses expressing nanoluciferase (nLuc) were generated using reverse genetics.
The complete genome of the Delta_nLuc virus was generated by recombining viral fragments in yeast. The virus was rescued by transfecting Vero-E6 cells with viral messenger ribonucleic acid (mRNA) and the nucleoprotein gene. Viral growth curves were determined by infecting Vero-E6 cells and measuring viral titers at different time points. The animals were infected intranasally and monitored for clinical signs and olfactory function.
Frozen lung fragments, nasal turbinates, and olfactory bulbs were weighed and homogenized using the FastPrep-24 system. Viral titers were determined using the tissue culture infective dose (TCID50) method, while viral RNA loads were quantified using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).
For transcriptomics analysis, RNA preparations from different tissues were subjected to reverse transcription-quantitative polymerase chain reaction RT-qPCR. The n-fold change in gene expression was calculated using the 2-ΔΔCt method.
In the immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) clearing process, hamsters were perfused and their heads were collected for further analysis. The iDISCO+ protocol was used to detect SARS-CoV-2 nucleocapsid in the snout and brain tissues. Sample staining and immunostaining were performed using specific antibodies. Light sheet imaging was conducted to capture 3D images of the samples.
In vivo and ex vivo bioluminescence imaging was carried out using the spectrum in vivo Imaging System. Histopathology and immunohistochemistry were performed on lung fragments using hematoxylin and eosin staining.
Human neural stem cells (hNSCs) and human alveolar A549-angiotensin-converting enzyme 2-transmembrane serine protease 2 (A549-ACE2-TMPRSS2) cells were cultured in specific media and maintained in microfluidic chips.
Neuron-epithelial networks were generated by seeding hNSCs in the chambers and incubating them. Finally, cells of A549-ACE2-TMPRSS2 were added over the neurons, and SARS-CoV-2 infection was further assessed.
Study results
The authors investigated the clinical presentation of various variants of SARS-CoV-2 compared to the ancestral virus, the original Wuhan SARS-CoV-2 strain (hereby referred to as the original strain), in hamsters.
In vitro growth curves of the viruses in Vero-E6 cells showed no significant differences. Male golden hamsters were then intranasally inoculated with different variants of SARS-CoV-2 and followed for 4 days post-infection.
The authors found that all infected animals experienced weight loss, but the severity varied among the variants. SARS-CoV-2 original strain-infected animals had the most significant weight loss, followed by Gamma, Delta, and Omicron/BA.1-infected animals. Non-specific clinical signs such as ruffled fur, slow movement, and apathy followed a similar pattern.
Original strain-infected animals had the highest incidence of olfaction loss, while Gamma-infected animals had a partial loss, and Delta and Omicron/BA.1-infected animals showed no signs of olfactory impairment.
Mutations in the spike protein distinguished the different variants, and some variants had deletions in the open reading frame 7 (ORF7) sequence. A recombinant SARS-CoV-2 virus with the ORF7ab sequence replaced by green fluorescent protein (GFP) showed a similar clinical profile to the original strain, indicating that ORF7ab was not necessary for viral infection and replication.
However, the incidence of olfaction loss decreased in the recombinant virus-infected animals. Lung pathology, including lung enlargement and lung weight-to-body weight ratio, was more severe in original strain and recombinant virus-infected animals compared to the other variants.
All infected hamsters had detectable viral titers and RNA loads in the upper and lower airways, with the highest values observed in Gamma-infected animals and the lowest in Omicron/BA.1-infected animals.
Both the upper and lower airways showed a tissue-specific inflammatory response to the infection, with different gene expression patterns observed in the lungs and nasal turbinates among the variants.
Viral titers and genomic viral RNA were detected in the olfactory bulbs of all infected animals, with original strain and Gamma-infected animals having the highest levels. The olfactory bulbs showed an upregulation of antiviral and inflammatory genes in all infected animals, regardless of their olfactory performance.
In a whole-brain analysis, viral distribution was observed in the nasal cavity, olfactory epithelium, and olfactory bulbs of original strain-infected animals. Recombinant viruses expressing nLuc confirmed the infection of the olfactory bulbs.
The incidence of olfactory dysfunction did not correlate with viral load or infection of the olfactory bulbs. Animals infected with a lower infectious dose of the original strain showed a lower incidence of olfactory dysfunction despite the presence of an infectious virus in the olfactory bulbs.
Moreover, while some studies showed infection and senescence in certain neurons, others suggested additional permissive cells are needed. In the present study, authors developed a co-culture system using axonal diodes in microfluidic devices to assess SARS-CoV-2's ability to infect neurons and move through axons.
The virus was observed to travel bidirectionally inside axons and blocking axonal transport halted infection. Such viral movements were confirmed by luminescence assays.
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