In a recent study posted to the bioRxiv* preprint server, researchers demonstrated ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through neuronal invasion in transgenic mice with human angiotensin-converting enzyme 2 (hACE2) expression.
Since ocular mucosa serves as an additional mucosal surface for infectious aerosols, the eyes might become a potential transmission route of SARS-CoV-2, causing ocular diseases in humans.
In vitro studies have evidenced that SARS-CoV-2 infects human embryonic stem cell-derived ocular epithelial cells and conjunctival epithelial cells. In animals, e.g., rhesus macaques, studies have demonstrated SARS-CoV-2 entry via the conjunctiva. Likewise, post-mortem studies have detected SARS-CoV-2 ribonucleic acid (RNA) in the conjunctival swabs of coronavirus disease 2019 (COVID-19) patients, with or without ocular manifestations.
Although studies have reported ocular abnormalities in COVID-19 patients, the pathology and ocular tropism of SARS-CoV-2 are still unknown. Elucidation of whether the eyes are a primary or secondary site for viral entry is crucial for developing therapeutic strategies against ocular and neuronal infection-mediated disorders in COVID-19 patients.
About the study
In the present study, researchers demonstrated SARS-CoV-2 ocular tropism via neuronal invasion of trigeminal (TN) and optic nerves (ON) in the K18-hACE2 mice. They challenged K18-hACE2 mice intranasally (IN) with approximately 104 plaque-forming units (PFU) of SARS-CoV-2 and the mock group with the same volume of phosphate buffer saline (PBS).
To validate the hypothesis that SARS-CoV-2 transmits to the brain and eyes via TN and ON, the team infected mice with 104 PFU of SARS-CoV-2 using four injection routes, viz., intratracheal (IT), intracerebral (IC), eye-drop (ED), and intravenous (IV). They assessed weight loss in mice and viral loads in the lungs, brain, eyes, TN, and ON of mice between three and six days post-infection (dpi).
Furthermore, they showed the ocular tropic and neuroinvasive characteristics of SARS-CoV-2 in wild-type Syrian hamsters.
The researchers examined the neurotropic and ocular distribution of SARS-CoV-2 in vivo using a fluorescence-imaging system. At six dpi, the team assayed the virus quantity in the lungs and eyes using a plaque assay. Similarly, they used nucleocapsid (N) gene-targeting quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to assess SARS-CoV-2 RNA copies in the lacrimal gland.
Furthermore, the researchers examined inflammatory and histopathological changes due to SARS-CoV-2 infection using hematoxylin and eosin (H&E)-stained eye sections from mock- and SARS-CoV-2-infected mice. They also measured retinal thickness, from the ganglion to the nuclear cell layers, post-infection to estimate retinal inflammation.
Lastly, the researchers evaluated the functional consequences of retinal inflammation on visual behavior using the visual cliff test. They tested each mouse only once to avoid a memory effect and randomized the test order.
The authors observed around 20% weight loss at seven dpi and mortality at eight dpi. On six dpi, they also observed tearing and eye discharge in one-fourth of the infected mice. The infectious viral titer of the eyes was as high as that of the lung (~106 PFU/g).
Surprisingly, the viral load of the lacrimal gland was much diminished than that of the eyes but similar to that of the spleen (~104 viral RNA copies/mg of total RNA), barely susceptible to SARS-CoV-2 infection. These results demonstrated the presence of infectious viruses in the eyes, suggesting that the eyes are a target of SARS-CoV-2 infection.
SARS-CoV-2 infection increased the retinal thickness by 1.62-fold due to the accumulation of infiltrating inflammatory cells in the ganglion and nuclear cell layers. Compared to the mock group, there were apparent lesions in retinal tissues in the SARS-CoV-2-infected mice. Accordingly, the mean retinal thickness of the infected mice was 75.14 µm, much higher than that of mock mice (46.27 µm).
The multiplex immuno-analysis of the eyes showed augmentation in pro-inflammatory chemokines and cytokines in response to SARS-CoV-2 infection on six dpi in mice. The authors observed substantially elevated levels of the following cytokines: granulocyte colony-stimulating factor (G-CSF), interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and interleukin 12 (IL-12).
These chemokines and cytokines were augmented, particularly in the brain, where the viral load was higher than in the eyes, indicating that IN-administered SARS-CoV-2 promoted retinal inflammation and cytokine production in the eyes of K18-hACE2 mice.
The mice of mock and SARS-CoV-2-infected groups dismounted the visual cliff test platform on an average within 4.28 and 3.56 seconds, respectively. Specifically, SARS-CoV-2-infected mice with ocular symptoms showed a prolonged latency for the dismounting up to 42.92 seconds. Notably, the retinal inflammation due to SARS-CoV-2 infection did not worsen retinal degeneration or visual loss.
The IN injection resulted in ocular tropism with increased viral RNA levels in the eyes between three and six dpi. However, the authors observed mortality only in mice injected via the IC route after two dpi. The viral RNA titers in the TN and ON were almost equal in the brain and eyes, indicating viral transmission to the eyes and brain via the neuronal invasion of these nerves.
In the present study, an infectious SARS-CoV-2-mCherry clone (with a fluorescent reporter gene) generated by a reverse genetics system helped the researchers trace viral infection and tropism in vivo.
Robust fluorescence signals in the TN and ON of infected mice validated the neuronal invasion of SARS-CoV-2. It demonstrated that SARS-CoV-2 used TNs, along with ONs to reach the brain and the eyes.
Future studies should investigate the clinical relevance of SARS-CoV-2-induced ocular inflammation and manifestations because not only respiratory organs but other organs, including eyes and TNs, are susceptible to SARS-CoV-2 infection.
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.