Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants

Abstract

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Fig. 1. Clinical profile of hamsters infected with SARS-CoV-2 original virus (Wuhan), the recombinant Wuhan/ΔORF7ab or the variants of concern (VoC) Gamma, Delta, and Omicron/BA.1.

A, B Body weight variation over four days post-infection. C Lung weight measured at 4 dpi. D, E Clinical score over four days post-infection. The clinical score is based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity. F Lung weight-to-body weight ratio measured at 4 dpi. A–F Horizontal lines indicate median and the interquartile range (n = 8/group). B, C, E, F Kruskal-Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). G, H Olfactory performance measured at 3 days post-infection (dpi). The olfaction test is based on the hidden (buried) food finding test. Curves represent the olfactory performance of animals during the test (G) and bars represent the final results (H) (n = 8/group). Chi-square test for trend (the adjusted p value is indicated when significant). See Supplementary Figs. 1, 2.

Fig. 2. Virologic assessment and gene expression of selected immune mediators in different tissues of hamsters infected with SARS-CoV-2 original virus (Wuhan), the recombinant Wuhan/ΔORF7ab or the variants of concern (VoC) Gamma, Delta, and Omicron/BA.1.

A Infectious viral titers in the lung, nasal turbinates, and olfactory bulbs at 4 days post-infection (dpi) expressed as TCID₅₀ per 100 mg of tissue. Horizontal lines indicate median and the interquartile range (n = 4/group). B SARS-CoV-2 viral RNA load detected in the lung, nasal turbinates, and olfactory bulbs at 4 dpi. Genomic and sub-genomic viral RNA were assessed based on the E gene sequence. Horizontal lines indicate median and the interquartile range. Gray lines connect symbols from the same animals (n = 4/group). Gene expression values in the lung (C), nasal turbinates (D) and olfactory bulbs (E) of Mx2, Ifn-β, Il-6, Cxcl10, Tnf-α and Il-10 at 4 dpi. A–E Horizontal lines indicate median and the interquartile range (n = 4/group). Kruskal-Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). nd: not detected. See Supplementary Figs. 1, 2.

Fig. 3. Imaging assessment of SARS-CoV-2 neuroinvasion.

A, B iDISCO+ whole head clearing and immunolabeling against the SARS-CoV-2 nucleocapsid in hamsters infected with the SARS-CoV-2 original virus (Wuhan) at 4 days post-infection (dpi). A Representative nasal turbinate (sagittal section from the skull) showing a diffuse distribution of SARS-CoV-2 (scale bar = 500 µm). B Representative olfactory bulb section stained for SARS-CoV-2 nucleocapsid and for podoplanin (CD31/Podo) to identify the vascular compartment. Note presence of SARS-CoV-2 in olfactory bulb neurons, with no macroscopic alterations in the vascular network of the olfactory bulb. Scale bar: 200 µm. See Supplementary Movie 1. C, F Bioluminescent recombinant SARS-CoV-2 viruses induce a clinical disease with infection of the olfactory bulbs. C Clinical profile of hamsters infected with the recombinant viruses SARS-CoV-2 Wuhan_nLuc and Wuhan/ΔORF7ab_nLuc (based on the original SARS-CoV-2 Wuhan) and Delta_nLuc (based on the Delta variant). The body weight loss and the clinical score are comparable with those induced by wild-type viruses (n = 4/group). Horizontal lines indicate median and the interquartile range (See Fig. 1A). The clinical score is based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity (See Fig. 1D). D Ex vivo bioluminescence values from the lungs, the olfactory bulbs (ventral view), the brains (ventral view) at 4 dpi (n = 4/group). Horizontal lines indicate median and the interquartile range. Kruskal-Wallis test followed by the Dunn’s multiple comparisons test (the adjusted p value is indicated when significant). The gray crosshatched area corresponds to background signals obtained from the same tissues of a mock-infected hamster. E, F Representative ex vivo imaging of a lung (C) and a brain (D) of a hamster infected with a recombinant SARS-CoV-2 expressing the nLuc at 4 dpi. Note that in the brain, the major bioluminescent focus is localized in the ventral face of olfactory bulbs. See Supplementary Fig. 3.

Fig. 4. SARS-CoV-2 travels inside axons in in vitro neuron-epithelial networks.

A Schematic view of an axon diode in a microfluidic device containing neurons and epithelial cells in both the left and right chambers. The chambers are connected exclusively by the axons of neurons whose cell bodies are located in the left chamber. In this study, SARS-CoV-2 was added in the right chamber (to assess the retrograde axonal transport) or in the left chamber (to assess the anterograde axonal transport). B Bright field view of a neuron-epithelial network showing neuronal and A549-ACE2-TMPRSS2 co-culture in both chambers, separated by funnel-shaped micro-channels (scale bar = 200 µm). C Detail of β-Tubulin III-positive axons crossing the funnel-shaped micro-channels and entering in the right chamber (scale bar = 50 µm). Neuron-epithelial networks infected at the right chamber with SARS-CoV-2 Wuhan (D), Wuhan/ΔORF7ab (E), Gamma (F), Delta (G) and Omicron/BA.1 (H) showing infected cells in the left chambers (scale bar = 50 µm). Neuron-epithelial networks infected at the left chamber with SARS-CoV-2 Wuhan (I), Wuhan/ΔORF7ab (J), Gamma (K), Delta (L), and Omicron/BA.1 (M) showing infected cells in the right chambers (scale bar = 50 µm). Hoechst: nuclei (blue). TUBB3: β-Tubulin III (red). N: SARS-CoV-2 nucleocapsid (green). The black and white striped zones in the right (D–H) and in the left (I–M) of the photomicrographs represents the microchannels, which are not accessible during immunostainings. B–M These photomicrographs are representative of 3 independent experiments. See Supplementary Figs. 6-9. Panel A was created with BioRender.com.