key: cord-0874398-1l9ht4id
authors: Jeong, Gi Uk; Kwon, Hyung-Jun; Moon, Hyun Woo; Yoon, Gun Young; Shin, Hye Jin; Chae, Ji Soo; Kim, Seong-Jun; Lee, In-Chul; Ahn, Dae-Gyun; Kim, Kyun-Do; Mahalingam, Suresh; Kwon, Young-Chan
title: Ocular tropism of SARS-CoV-2 with retinal inflammation through neuronal invasion in animal models
date: 2022-04-18
journal: bioRxiv
DOI: 10.1101/2022.04.17.488607
sha: 98b03c030e811760d94d6b1ac9348f5240bf4ff3
doc_id: 874398
cord_uid: 1l9ht4id

Although ocular manifestations are commonly reported in patients with coronavirus disease 2019 (COVID-19), there is currently no consensus on ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To investigate this, we infected K18-hACE2 mice with SARS-CoV-2 using various routes. We observed ocular manifestation and retinal inflammation with cytokine production in the eyes of intranasally (IN) infected mice. An intratracheal (IT) injection resulted in virus spread from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasion were confirmed by an intracerebral (IC) infection. Notably, eye-dropped (ED) virus did not infect the lungs and was undetectable with time. Using infectious SARS-CoV-2-mCherry clones, we demonstrated the ocular and neurotropic distribution of the virus in vivo by a fluorescence-imaging system. Evidence for the ocular tropic and neuroinvasive characteristics of SARS-CoV-2 was confirmed in wild-type Syrian hamsters. Our data provides further understanding of the viral transmission; SARS-CoV-2 clinical characteristics; and COVID-19 control procedures. Summary SARS-CoV-2 can spread from the respiratory tract to the brain and eyes via trigeminal and optic nerves in animal models. This ocular tropism of SARS-CoV-2 through neuronal invasion likely causes ocular manifestation and retinal inflammation. Graphical Abstract

* Correspondence to Young-Chan Kwon : yckwon@krict.re.kr 22

Mailing address : 141 Gajeongro, Bldg E5, Rm 101, Yuseong, Daejeon 34114, Republic of Korea 23 ORCID : 0000-0003-1100-5168 to Y.-C.K. 24

Introduction 50

As of 9 January 2022, the ongoing the coronavirus disease 2019 pandemic 51 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has threatened global 52 public health with over 300 million confirmed cases and over 5 million deaths 53 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports). SARS-CoV-2 54 preferentially infects cells in the respiratory tract; however, it has a broad organotropism beyond the 55 respiratory tract, affecting the kidney, small intestine, heart, and brain, and is believed to cause multi-56 organ dysfunction in patients with COVID-19 (Liu et al., 2021a; Puelles et al., 2020; Thakur et al., 57 2021) . 58

Several respiratory viruses, such as species D adenoviruses and subtype H7 influenza viruses, 59 possess an ocular tropism and cause ocular diseases in humans (Belser et al., 2013) . While ocular 60 manifestations and abnormalities have been commonly reported in patients with COVID-19 (Chen et 61 al., 2020; Wan et al., 2021) , the ocular tropism and pathologies of SARS-CoV-2 are still unclear. In 62 some clinical and post-mortem cases, viral RNA was not detected by the PCR analysis of 63 conjunctival swabs of three patients with COVID-19 with bilateral conjunctivitis (Pirraglia et al., 64 2020) and in 16 aqueous humor and vitreous samples (List et al., 2020) . In contrast, a cross-sectional 65 study in Italy reported that viral RNA was found on the conjunctival swabs of 52 of 91 patients with 66

Institute of Chemical Technology (KRICT). All protocols were approved by the Institutional Animal 268

Care and Use Committee (Protocol ID 8A-M6, IACUC ID 2021-8A-02-01 & 2021-8A-03-03) . 269

Eleven-week-old female Golden Syrian hamsters were purchased from Janvier Labs 271 France). The hamsters were kept in standard cages and exposed to a 12:12 hour light/dark cycle at The virus was serially diluted in Eagle's minimum essential medium supplemented with 2% fetal 292 bovine serum for the plaque assay. Culture medium was removed from 24-well plated Vero E6 cells 293 (approximately 1 × 10 5 per/well) a day before the assay, and the inoculum was transferred onto 294 triplicate cell monolayers. After incubation at 37 °C for 1 h, the inoculum was removed, and infected 295 cells were overlaid with 1.8% carboxymethyl cellulose in MEM. Samples were incubated for four 296 days, followed by fixation and staining with 0.05% crystal violet containing 1% formaldehyde. The 297 counts of plaques were measured by an ImmunoSpot analyser (Cellular Technology Ltd, Shaker 298

Heights, OH, USA). 299

Median tissue culture infectious dose (TCID 50 ) assay 300

To determine the titer of SARS-CoV-2, TCID 50 assays were performed with Vero cells, as described 301 elsewhere (Sia et al., 2020). Ten-fold serial dilutions of stock viruses were prepared in DMEM. From 302 these dilutions, 100 µl each was transferred to monolayers of Vero cells grown in 96-well plates in 303 DMEM supplemented with 1 μ g/ml TPCK-treated trypsin and incubated at 37°C in a 5% CO 2 304 incubator. Virus titers were calculated at 4 days post-infection and expressed as TCID 50 /ml values 305 based on the method of Reed and Muench (Reed & Muench, 1938) . The virus dilutions were made 306 based on a prediction of the number of plaque forming units (PFU) using a conversion formula as 307 follows: PFU (ml) / TCID 50 (ml) = 0.7 (Davis et al., 1972) . 308 309

All viral inoculations (SARS-CoV-2 diluted in PBS, approximately 10 4 PFU) were administered by 311 three and more groups, one-way analysis of variance (ANOVA) was used. P < 0.05 was considered 384 statistically significant. Specific analysis methods are described in the figure legends. Statistical 385 significance is indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001. 386

Online supplemental material 387 Fig. S1 shows the up-regulated cytokines in the brains of SARS-CoV-2-infected mice using the 388 multiplex analysis. Fig. S2 shows the results of IT and IC infections with Evans blue. Fig S3  389 indicates the body weight loss and mortality by diversely administered SARS-CoV-2 infections in 390 the mouse model. analysed by plaque assay. (E) Viral RNA levels in the lungs, brain, eyes, lacrimal gland, parotid 574 gland, spleen, and colon were assessed by RT-qPCR. Viral RNA copies were cut-off by the limit of 575 detection (10 4 copies/μg). A dashed line indicates the viral RNA levels of spleen, barely susceptible 576 to SARS-CoV-2 infection. Symbols represent means ± SEM. Statistically significant differences 577 between the groups were determined by multiple t-test (A), Student's t-test (D), or one-way ANOVA 578 (E). **P < 0.01; ***P < 0.001; ****P < 0.0001. SARS-CoV-2: severe acute respiratory syndrome 579 coronavirus 2; PFU: plaque-forming unit; dpi: days post-infection Symbols represent means ± SEM. Statistically significant differences between the groups were 592 determined by Student's t-test (B) or one-way ANOVA (C, E). *P < 0.05; **P < 0.01; ****P < 593 0.0001. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; GCL: ganglion cell layer; 594 IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear 595 layer; IS: inner segments; OS: outer segments; dpi: days post-infection 596 

K18-ACE2 mice were inoculated with 600 approximately 10 4 PFU SARS-CoV-2 via five different injection routes (n = 8 per injection route; n = 601 4 for 3 dpi and 6 dpi, respectively). (C) Viral RNA levels in the lungs, brain, eyes, trigeminal nerve, 602 and optic nerve were analyzed on 3 and 6 dpi by RT-qPCR (3 dpi, Blue; 6 dpi, Red). Viral RNA 603 copies were cut-off by the limit of detection

-2: severe acute respiratory syndrome coronavirus 2; PFU: plaque-forming unit; dpi: days 605 post-infection

In vivo viral distributions of infectious SARS-CoV-2-mCherry in IN-infected mice

K18-hACE2 mice were IN-inoculated with approximately 10 4 PFU of SARS-CoV-2-mCherry (n = 6 608 for the mock-infected and infected mice, respectively). (A) Body weight and (B) survival were 609 monitored on the indicated dpi. Symbols represent means ± SEM

A brain-dissected dorsal view of the murine cranial cavity showing optic nerves, optic chiasm, 612 trigeminal ganglia

Representative in vivo fluorescence images of organs, including the lungs, brain, eyes, and spleen in 614 mock-or SARS-CoV-2-mCherry-infected mice (n = 5 for the mock-infected and infected mice, 615 respectively; Upper, Mock; Lower, SARS-CoV-2-mCherry) were acquired by sequential imaging

Color bars indicate radiant efficiency

-2: severe acute respiratory 617 syndrome coronavirus 2; PFU: plaque-forming unit

or eye-drop (ED)-infected wild-type Syrian hamsters. Eleven-week-old female Golden Syrian 620 hamsters were IN-mock-infected or infected with approximately 10 4 PFU of SARS-CoV-2 (n = 6 for 621 mock-infected and n = 5 for infected mice; mock, gray; SARS-CoV-2, red). (A) Body weight 622 changes are shown as a percentage of the starting weight at the

ED-mock-infected, and ED-infected). A graph showing the 625 percent body weight change is shown. (C) Representative digital images of lungs harvested from 626 mock-infected or infected hamsters on 6 dpi. (D-E) Viral loads in the lungs and eyes of

ED-infected (G) animals were assessed by RT-qPCR. Viral RNA copies were 630 subjected to a cut-off based on the limit of detection (10 4 copies/μg). A dashed line indicates the viral 631 RNA levels in the spleen, which is barely susceptible to SARS-CoV-2 infection. Symbols represent 632 means ± SEMs. Statistically significant differences between the groups were determined by a 633 multiple t-test

SARS-CoV-2: severe acute respiratory syndrome coronavirus 2

PFU: plaque-forming unit; dpi: days post-infection

Multiplex cytokine analysis of the brain tissues of SARS-CoV-2-infected mice. The 639 chemokine and cytokine levels of the brain were measured by multiplex immuno-analysis (n = 4 per 640 indicated dpi). G-CSF, Granulocyte-macrophage colony-stimulating factor

MKC, mouse keratinocyte-derived chemokine; MCP-1, Monocyte

Macrophage-inflammatory protein; RANTES, Regulated 643 upon Activation

Statistically significant differences between the groups were determined by one-way

Body weight (Left) and survival (Right) were monitored 653 at the indicated dpi. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; PFU: plaque-654 forming unit