ABSTRACT

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Complete smell loss (anosmia) or partial smell loss (hyposmia) is a key diagnostic factor in the diagnosis of the COVID-19 disease. In this study, we will be evaluating the factors that potentiate SARS-CoV-2 infectivity and how it correlates with anosmia. Preliminary research done on SARS-CoV-2 infectivity has found that Neuropilin 1 (NRP-1), a protein expressed in the olfactory epithelium, along with the ACE2 enzyme and TMPRSS2 protease potentiate infectivity through furin cleavage of the S1 fragment of the spike protein on SARS-CoV-2. The research on NRP-1 thus far shows that infectivity of SARS-CoV-2 correlates with the expression of NRP-1 in the olfactory epithelium. The relationship between NRP-1 expression and anosmia has not yet been explored and is an important factor for understanding the mechanism of smell loss in COVID-19 positive patients. In this study, we will be using the golden Syrian hamster to evaluate whether NRP-1 expression correlates with the severity of anosmia in SARS-CoV-2 infected hamsters by conducting a food-finding behavioral assay and using immunohistochemistry to analyze the expression of NRP-1 in the hamster olfactory epithelium. It is imperative to study how the SARS-CoV-19 virus infects the human olfactory epithelium and its implications in anosmia to understand the broader implications the virus has on smell loss. 

 

OBJECTIVES

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Due to the recency of protein neuropilin-1 (NRP-1)’s discovered function in facilitating SARS-COV-2 to infect epithelial cells, experiments monitoring its exact role are in production, but limited in their current publication. Preliminary research suggests that in the presence of NRP-1, increased infectivity of cells is seen, which was evaluated through observing decreased infectivity when NRP-1 availability was decreased. This included trials where NRP-1 was deleted, an anti-NRP1 monoclonal antibody was applied, or a selective antagonist, EG00229, was injected (1). NRP-1 was concluded to allow the furin-cleaved S1 fragment of the spike protein on SARS-COV-2 to bind, potentiating infectivity when in the presence of ACE2 and TMPRSS2 (2). Due to NRP-1’s abundant presence within the olfactory epithelium, scientists have theorized that its expression may contribute to the loss of smell, or anosmia, reported by a large fraction of COVID-19 patients (2, 3). Support for its role was confirmed through five autopsies of COVID-19 patients expressing a high expression of NRP-1 within the olfactory epithelium in comparison to control autopsies (2). Although the correlation between high expression of NRP-1 and SARS-COV-2 infection within the olfactory epithelium has been explored, the relationship between the behavioral expression of anosmia and the expression of NRP-1 in COVID-19 cases has not (4). Because of this, we will use an animal model, the golden Syrian hamster, to investigate whether the frequency of NRP-1 expression correlates with the severity of anosmia through behavioral assays to evaluate olfaction loss and immunohistochemistry to analyze the expression of NRP-1 in the olfactory epithelium (OE). These efforts are directly aligned with the priorities of the CDC, specifically to determine NRP-1’s mechanism of action with respect to olfaction (5). 

 

Specific Aim I. Determine behavioral effects of anosmia in golden Syrian hamsters after SARS-COV-2 injection. 

Animal models with similar physiological mechanisms as humans have been used while studying COVID-19 in order to determine SARS-COV-2’s mechanism of action (8). These include the golden Syrian hamster, which was originally used as a model for SARS-COV-1. Recently, this species has been shown to be a good model for SARS-COV-2 due to its similar expression profile of ACE2 within the OE to humans (6,8). Behavioral assays evaluating anosmia in this golden Syrian hamster model exposed to SARS-COV-1 have not been publicly published, but the loss of smell has been assumed via analyzed destruction of cilia and disorganization of the OE structure (6). We hypothesize that the golden Syrian hamsters injected with SARS-COV-2 will show a reduction in smell via a food-finding behavioral assay testing for anosmia. We propose an experiment to characterize the response to infection by performing a food-finding behavioral assay designed to evaluate odor detection. 

 

Specific Aim II. Determine whether frequency of NRP-1 expression in presence of SARS-COV-2 infection correlates with severity of anosmia. 

Immunohistochemistry has been used to evaluate the expression of NPR-1 within animal models including the golden Syrian hamster OE (1,2,7). Although studies have looked at the expression of NPR-1 and SARS-COV-2 within human autopsies and ACE2 within golden Syrian hamsters, identification of NPR-1 within anosmic hamsters due to SARS-COV-2 has not been published (2,6). We plan to address if an increased expression of NPR-1within the OE will be expressed within anosmic hamsters compared to those not infected with SARS-COV-2. In addition, we will note whether the severity of anosmia, determined by increased latency time, correlates with the expression of NPR-1. We hypothesize that golden Syrian hamsters expressing more severe anosmia will present a higher expression of NPR-1 when infected with SARs-CoV-2.  

 

BACKGROUND AND SIGNIFICANCE

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The ability to perceive, differentiate, and identify smell may be categorized as inferior on the totem-pole of senses to some; however, loss of sense of smell not only decreases pleasure but is also a crucial mechanism essential to avoid harmful stimuli with volatile cues (9). Unfortunately, the coronavirus disease (COVID-19), caused by SARS-COV-2, dissipates smell within a large population of those exposed. Though numbers vary, approximately 80% or more of patients infected with the disease report symptoms of smell loss, ranging from full loss, called anosmia, to partial loss, also known as hyposmia (10). Despite the unknown cause of anosmia within COVID-19 patients, recent studies have identified a series of proteins that assist with SARS-COV-2 infectivity in the olfactory epithelium (2). Studies identifying targets of SARS-COV-2 will further both our understanding of the virus as a whole, as well as its direct implications on olfaction.

 

Similar to SARS-COV, a related coronavirus, COVID-19 infection seems to be mediated by angiotensin-converting enzyme, or ACE2 (14). Using a spike protein that acts as the major binding protein, SARS-COV-2  is able to attach to ACE2 and fuse its viral membrane into the cellular barrier at the amino terminal region (14). Sequentially, host proteases, such as the identified transmembrane protease serin 2 (TMPRSS2), cleave at a polybasic furin cleavage site located on the spike protein (15). This cleavage function enables both the availability for further transmission as well as completion of membrane fusion (15). To date, the best understanding we have is that the expression of ACE2 and proteases seem to dictate tropism of the virus (14). 

 

With a high expression of ACE2 found in the olfactory epithelium and increasing reports of anosmia, it is logical to analyze the virus’ impact on olfaction and the factors that lead to this smell loss. Due to anosmia’s common appearance, it was speculated by the public that SARS-COV-2 directly destroyed the olfactory receptor neurons (ORNs) within the olfactory epithelium (16). However, after analyzing the expression of ACE2 within various cell types, many studies have found that ACE2 was not expressed on the ORNs themselves, but on sustentacular cells surrounding these projections (2). As supporting cells to the ORNs, their assistance in regulating the environment for the binding of odorants to the ORNs may explain the loss of smell, as well as its relatively quick return, and be a potential target to study for anosmia in COVID-19 (17). 

 

In order to monitor nasal destruction throughout the progression of the virus, a study used the golden Syrian hamster, an animal model with a similar expression of ACE2 in the OE to humans (6). Destruction of the sustentacular cells was recorded following SARS-COV-2 exposure intranasally (6). Within two days of exposure, the olfactory epithelium (OE) had deteriorated, as quantified through OE thickness and odorant sensory neuron (OSN) cilia quality (6). This loss of cilia, which functions to assist odorants into the ORNs, may contribute to the anosmia experienced due to their deterioration. In this study, the cellular mechanism behind the loss of smell was attributed to the abundance of both ACE2 and TMPRSS2 found in the sustentacular cells (6). However, it is noted that behavioral assays would need to be conducted to confirm that the level of deterioration correlated with anosmia (6). We believe that conducting an experiment suggesting the hamster’s anosmia with the loss of cilia would increase its viability as a good animal model for human COVID-19 anosmia and our understanding of the symptom. 

 

Despite the abundance of ACE-2 presented within the OE, not all who are exposed to SARS-COV-2 present symptoms of smell loss, and those who do seem to range in intensity and duration (18). In addition, studies have shown that varying populations express different levels of ACE-2, though no clear correlation between expression and anosmia (19). Because of this, further research and experimentation is needed to better understand the proteins that may modulate the infectivity of the virus. As more studies have looked into SARS-COV-2’s mechanism of action, more proteins have been identified as modulating the effectiveness of the virus. One of these, neuropilin-1 (NRP-1), was studied after looking for proteins that the polybasic cleavage site in SARS-COV-2 could bind to (2). Through using Human embryonic kidney (HEK) cells transfected with NRP-1, ACE2, and TMPRSS2, NRP-1 was concluded to allow the furin-cleaved S1 fragment of the spike protein on SARS-COV-2 to bind and potentiate infectivity (2). However, this infectivity was only seen when NRP-1 was transfected alongside ACE2 and TMPRSS2, showing its modulatory effect (2). Due to its high expression in the olfactory epithelium, NRP-1 is one of the newer proteins being analyzed for its function within causing anosmia (20). 

 

When analyzing six OE samples from COVID-19 exposed autopsies, it was seen that the infected olfactory epithelium cells showed high expression of NRP-1 (2). This increased expression indicated that NRP-1 may play a role in anosmia (2). It should be noted that this study did not give information on the symptoms of these patients, which may provide support towards NRP-1’s potential function within smell loss.

 

Given our current understanding of SARS-CoV-2 and its common symptom of anosmia, it is imperative to expand knowledge of where the virus is able to bind and its implication. Since data may be drawn ethically and relatively quickly through animal models, further research may benefit us by increasing our confidence in the golden Syrian hamster’s potential to allow insight into anosmia. Though ACE2 expression and deterioration of the OE were studied in the presence of SARS-CoV-2, analysis of behavioral assays may confirm anosmic effects. In addition, given NRP-1’s recently discovered role in binding the furin-cleaved S1 site, quantifying its expression within infected cells of hamsters with anosmic behaviors will provide insight. This may include whether the frequency of NRP-1’s expression correlates with a more drastic loss of smell. 

 

Through using the golden Syrian hamster in behavioral assays to identify anosmia and conducting immunohistochemistry to identify NRP-1 expression in affected cells, the results of the work described in this proposal will shed light on how expression of NRP-1 correlates to anosmia caused by COVID-19. Despite loss of smell seeming inferior to the symptoms resulting in respiratory failure and termination of life, studying the increased expression of NRP-1 within the olfactory epithelium may inform the population on NRP-1’s role in acting as a host factor for SARS-CoV-2. In addition, if NRP-1 expression correlates with anosmia severity, these results could provide support towards a future study into NRP-1 acting as a potential therapeutic target for anosmia. 

 

METHODS

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We will obtain 12-18 Golden Syrian Hamsters from Charles River Laboratory. We will perform a  food-finding behavioral assay on the hamsters to act as a baseline before infecting half of the hamsters with SARS-CoV-2. 

In order to infect the hamsters with SARS-CoV-,2 we will anesthetize the hamsters with a 0.5ml intramuscular injection of an anesthetic (18ml ketaject, 0.6ml xylazine, 1.1ml glycopyrate, and 0.5ml acepromazine). Half of the hamsters will remain uninfected. The other half will be infected with tissue culture infective doses of SARS-CoV-2 (Invitrogen) administered intranasally in a dose of 100µl. We will then run the food-finding behavioral assay on day 3 to test for the behavioral effects of anosmia. The hamsters will be euthanized at 7 days post-infection in order to observe the expression of NRP-1 in the olfactory epithelium and test for the presence of SARS-CoV-2. The lungs will be removed, homogenized, and purified with centrifugation for the RNA extraction. RNA extraction will be done with a QIAamp Viral RNA mini-kit using the manufacturer's instructions and followed by PCR to determine SARS-CoV-2 infectivity. 

 

Specific Aim I. Determine presence of anosmia after SARS-COV-2 injection via behavioral assays. 

Prior to infection, all hamsters within the trial will undergo a fasted food-finding assay that associates increased latency time with olfactory impairment in order to assess for behavioral signs of anosmia. This first test will provide a baseline, with a second trial repeated three days after infection of SARS-COV-2. This test will be conducted two days prior to infection, and modeled after previous methods identifying olfactory dysfunction in hamster models via the buried food task (23). Before the randomization of infected and noninfected hamsters, each subject will undergo the same assay. On day 1, the hamsters will be habituated to the smell of the cereal by allowing them to interact with a piece placed in their cage. The following day, or 24 hours prior to the food finding assay, the hamsters will be fasted, though they will have access to water and a clean cage. After the 24 hour fasting period, they will be placed into independent test cages (46 cm L × 23.5 cm W × 20 cm H) for about 10 minutes (fig. 1). Hamsters will then be removed and placed in a similar cage while 5 pieces of cereal that are shown to be highly palatable to hamsters are hidden about 1.5 cm underneath bedding in the corner of the test cage. Afterward, each hamster will be reintroduced to the test cage and placed on the opposite side of the food. The latency to find the food, which will be defined as the time between entering the cage and locating the cereal through digging, will be recorded using a chronometer. If the hamster fails to find the food after 15 minutes, the test will be stopped, and the recorded time will be 900 seconds as its latency score.

 

The same food-finding assay will then be conducted again 3 days post-infection (DPI) by both the infected and non-infected hamster groups. Latency scores will be recorded again, and statistical analysis will be conducted using a paired T-test to determine if there is a significance between the infected and non-infected group. In addition, the percentage of subjects in each group who failed to find the cereal pellet within the 15 minute time period may be computed to see overall trends. We expect to find that those in the infected group will show higher latency times in comparison to the control, non-infected group. It must be noted that other symptoms of COVID-19 aside from anosmia may also affect the performance in the behavioral task; because of this, causation must be avoided, and rather inferences of correlation between infection and latency may be created. 

 

Specific Aim II. Determine whether expression of NRP-1 in presence of SARS-COV-2 correlates with severity of anosmia. 

Previous studies have used the golden Syrian hamster for immunohistochemical staining to detect presence of NRP-1 in the olfactory epithelium (2,12). We hypothesize that there will be higher expression of NRP-1 in SARS-CoV-2 infected hamsters that express more severe anosmia. After the hamsters have been euthanized, we will fix the whole animal head for three days at room temperature in 4% paraformaldehyde PBS and then decalcify for one week. The nasal septum and nasal turbinates will be removed as a block and fixed again in 4% paraformaldehyde PBS and then decalcified for three days. We will cut sections (14µm) of the nasal cavity in order to highlight the vomeronasal organ, olfactory mucosa, Steno’s gland, and the olfactory bulb. The sections will be incubated overnight with primary antibodies against NRP-1. 

 

For immunohistochemistry, the sections will be incubated in a solution of with the primary antibody; Goat Anti-rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Invitrogen, Thermo Fisher), diluted in 10% blocking solution and incubated overnight. The sections will be washed with PBS and then incubated in the secondary antibody; anti-goat counterstained with Hematoxylin (blue) (Thermo Fisher) for two hours at room temperature. Again the sections will be washed in PBS and mounted for imaging. Sections from the hamster olfactory epithelium will be imaged and evaluated for the presence of NPR-1. Imaging for immunohistochemistry will be visualized with a fluorescence microscope (Olympus BX-63 with a color camera (Olympus DP80)). The number of NRP-1 positive cells in the olfactory epithelium will be manually counted from the images produced with the blue stain indicating NRP-1 presence. Comparisons will be made between the uninfected hamsters to the infected hamsters and between hamsters of different expressions of anosmia. 

 

The average number of NRP-1 positive cells within the infected and non-infected group will be compared to each other in an independent sample T-test. In addition, a correlation will be computed to determine the relationship between the number of NRP-1 positive infected cells and increased latency time. We predict that there will be a positive correlation between increased latency time and the number of infected NRP-1 infected cells. Yet, this might be hard to determine from this study due to the challenges with the behavioral assay for anosmia. In addition, NRP-1 expression differences might not be statistically significant due to the limited sample size and uncertainty of positive infectivity. Through this study, we will be able to determine if NRP-1 expression is correlated with behavioral signs of anosmia. By using the food-finding behavioral assay and linking NPR-1 to anosmia in COVID-19 positive hamsters, we will be introducing novel research into the field that will aid the understanding of the effect of COVID-19 on anosmia and its facilitation through NRP-1. 

 

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