key: cord-283193-8qj41kpp
authors: Chak-Yiu Lee, Andrew; Zhang, Anna Jinxia; Fuk-Woo Chan, Jasper; Li, Can; Fan, Zhimeng; Liu, Feifei; Chen, Yanxia; Liang, Ronghui; Sridhar, Siddharth; Cai, Jian-Piao; Kwok-Man Poon, Vincent; Chung-Sing Chan, Chris; Kai-Wang To, Kelvin; Yuan, Shuofeng; Zhou, Jie; Chu, Hin; Yuen, Kwok-Yung
title: Oral SARS-CoV-2 inoculation establishes subclinical respiratory infection with virus shedding in golden Syrian hamsters
date: 2020-09-22
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2020.100121
sha: 
doc_id: 283193
cord_uid: 8qj41kpp

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted largely by respiratory droplets or airborne aerosols. Despite being frequently found in the immediate environment and faeces of patients, evidence supporting oral acquisition of SARS-CoV-2 is unavailable. Utilizing Syrian hamster model, we demonstrate that the severity of pneumonia induced by intranasal inhalation of SARS-CoV-2 increases with virus inoculum. SARS-CoV-2 retains its infectivity in vitro in simulated human fed-gastric and fasted-intestinal fluid after two hours. Oral inoculation with the highest intranasal inoculum(105PFU) causes mild pneumonia in 67% (4/6) of the animals with no weight loss. The lung histopathology score and viral load are significantly lower than those infected by the lowest intranasal inoculum(100PFU). However, 83% oral infection (10/12 hamsters) have similar level of detectable viral shedding from oral swabs and faeces as that of intranasally infected hamsters. Our findings indicate oral acquisition of SARS-CoV-2 can establish subclinical respiratory infection with less efficiency.

The Coronavirus Disease 2019 pandemic caused by severe acute respiratory 36 syndrome coronavirus 2 (SARS-CoV-2) has affected over 14 million patients with more than 0.6 37 million deaths within a period of 6 months. 1 Because of the high degree of genome homology 38 between SARS-CoV-2 and bat SARS-related coronaviruses 2-4 and its growth in intestinal 39 organoids derived from Chinese horseshoe bats, 5 SARS-CoV-2 is likely to originate from bats. 6,7 40 Although how SARS-CoV-2 jumped the species barrier from animals to humans is still uncertain, CoV-2 (10 2 , 10 3 , 10 4 and 10 5 PFU). The clinical signs of lethargy, ruffled fur, hunched back 71 posture, and rapid breathing were observed which were accompanied by a maximum body 72 weight loss of 4% to 10% at 4 day-post-infection (dpi) in the four groups ( Figure 1A) . The viral 73 loads in the trachea and lung tissues at 2dpi increased with the virus inoculum, while similar lung 74 viral loads were detected among the different groups at 4dpi ( Figure 1B ). These results indicated 75 that the viral load peaked earlier with higher virus inoculum and were consistent with our 76 previous report. 10 Immunohistochemical staining for viral nucleocapsid (N) protein in the lung 77 tissues at 2dpi showed more focal distribution in bronchiolar epithelium with lower virus 78 inoculum and more diffuse and intense N protein expression in the alveoli with higher inoculum 79 J o u r n a l P r e -p r o o f ( Figure 1C ). But by 4dpi, similar intensity and distribution of N protein expression was observed 80 in all groups ( Figure 1C ), which also correlated with their lung viral loads. 81 Histological examination of H&E stained lung tissue sections at 2dpi showed mainly 82 bronchiolar epithelial cell death ( Figure 1D , open arrows) and patchy alveolar septal congestion 83 and infiltration without severe lung parenchymal damage in the hamsters infected with 10 2 or 10 3 84 PFU of SARS-CoV-2 ( Figure 1D , solid arrows); while the lungs of the hamsters infected with 85 10 4 or 10 5 PFU of virus showed large patches of inflammatory consolidation in addition to 86 bronchiolar epithelial cell death, with alveolar septal congestion, oedema, alveolar space 87 infiltration, protein rich fluid exudation, and patchy alveolar haemorrhage ( Figure 1D ). These 88 findings indicate that a higher intranasal virus inoculum is associated with earlier onset of lung 89 parenchymal damage at 2dpi. By 4 dpi, the hamsters infected with 10 2 or 10 3 PFU of SARS-90 CoV-2 also developed lung parenchymal damage which were milder than those observed in the 91 hamsters infected with 10 4 or 10 5 PFU of virus ( Figure 1D including both arteries and veins. But no viral N protein antigen was detected in the blood vessel.

The inflammatory cytokines and chemokines in the lung tissues inoculated with the 99 lowest inoculum of 10 2 PFU and highest inoculum of 10 5 PFU were determined at 2 and 4dpi.

The gene expression levels of inflammatory cytokines and chemokines including IL-6, TNF-α, 101 MIP-1α, RANTES, IP-10, IFN-α and IFN-γ were significantly upregulated in hamsters infected 102 J o u r n a l P r e -p r o o f with 10 5 PFU at 2 and 4dpi ( Figure 1F ). Though the cytokine and chemokine response peaked at 103 2dpi in both groups, the response induced by lower virus inoculum (10 2 PFU) mostly decreased 104 at 4dpi ( Figure 1F ). We then investigated the effect of SARS-CoV-2 on hamsters by oral inoculation. 10 5 PFU of 125 SARS-CoV-2 in 200µl of DMEM was delivered by plastic pipettes to their mouth which were 126 swallowed spontaneously by the alert animals without anaesthesia. 16, 17 For comparison, a parallel 127 group of animals were inoculated with 10 5 PFU of SARS-CoV-2 by intranasal inhalation under 128 anaesthesia. Intranasally infected hamsters developed ruffled fur, laboured breathing, and lost 129 about 8% body weight. All the orally infected hamsters did not show sign of disease while 130 gaining similar weight as the mock controls ( Figure 2B ). At 12hpi, a low viral load was 131 detectable in homogenised oral mucosa, but not detectable in the oesophagus, stomach, small 132 intestinal tissues and blood samples of orally infected hamsters (n=3, Figure 2C ). The 133 intranasally infected hamsters had significantly higher viral load in oesophagus and stomach, but 134 no detectable virus in small intestinal tissues (n=3, Figure 2C ). Interestingly, viral N protein was 135 detected in one of the three lymph nodes taken from the oropharyngeal tissues at 12hpi in the intranasally infected controls ( Figure 5A ). As expected, the viral titers in the lung tissues were 191 significantly lower than that of intranasally infected control hamsters ( Figure 5B ). Histological 192 J o u r n a l P r e -p r o o f examination showed that the orally infected hamster lung tissues had milder alveolar septal 193 infiltration and capillary congestion than the diffuse alveolar infiltration, exudation, epithelium 194 desquamation and pulmonary vasculitis observed in intranasally infected hamsters ( Figure 5C ).

In order to quantitatively compare the severity of lung damage after oral and intranasal 196 inoculation, we performed semi-quantitative histopathological evaluation of the bronchioles, 197 alveoli and blood vessels using a method modified from our previous influenza infection mouse 198 model and a reported hamster infection model (Table S1 ). 18, 19 The results showed that same (Table S2) ; the expressions of lung cytokines and 202 chemokines were also significantly lower in orally infected hamsters ( Figure 5D and Table 1 ).

Notably, when comparing the lung pathological damage caused by oral infection through the 204 intranasal route, oral inoculation with 10 5 PFU caused significantly milder infection than the 205 intranasal route even with the lowest inoculum (10 2 PFU), while the hamsters in the latter group 206 showed significantly higher lung viral load, lung histology scores and lung cytokines and 207 chemokines, among which RANTES and IP-10 were significantly higher (Table 2) . These results 208 suggested that oral inoculation of SARS-CoV-2 can establish respiratory infection but with much 209 less efficiency, delayed course and mild lung damage. Follow up of the histopathology showed 210 resolution of the inflammation in the lung at 7dpi which was similar to our previous report. 10 211 Serum neutralizing antibody was detected at a similar level from both orally (titer=10-40) and 212 intranasally (titer=20-40) inoculated hamsters at 7dpi, the neutralizing antibody titer in 213 intranasally inoculated hamsters increased to a higher level at 14 dpi (titer=40-160) but not 214 significantly different when compared with orally inoculated hamsters (titer=10-80) ( Figure 5E ).

After oral inoculation, viral load was detected from oral swabs for 10 days with higher titer 218 before 8dpi, then decreasing until 12dpi ( Figure 5F ). Oral swabs from intranasally infected 219 hamsters had similar viral load as orally infected hamsters, except at 2dpi when significantly 220 higher viral load was detected in intranasally infected hamsters ( Figure 5F ). The faecal samples 221 had lower viral load compared to oral swab, which became undetectable after 8dpi ( Figure 5G ).

Overall, despite the lack of disease signs and weight loss, orally infected hamsters had similar 223 viral load and similar duration of virus shedding in oral swabs (11.33 days) and faecal samples 224 (6.67 days) compared to intranasally infected hamsters (9.20 days and 6.8 days, respectively). CoV-2 related acute respiratory distress syndrome in a recent observational study. 28 We therefore 282 caution against indiscriminate PPI use during this pandemic. 

Hamsters were orally inoculated with 10 5 PFU of SARS-CoV-2, intranasal inoculation group 446 were included as control. Nasal turbinate, trachea and lung were examined at 12hpi. Tissues fixed in 10% formalin immediately after sampling were processed into paraffin- Table S1 was applied to assess the severity of lung damage. Briefly, each sample was firstly 594 examined under lower power lens (4x) to determine the scope of tissue damage and assign a 595 score from 0 to 3 as normal morphology to most diffuse tissue lesions. Then, inflammatory 596 lesions in bronchiolar, alveolar and vascular structures was assessed, 0-3 scores were given to 597 each category of pathological changes. A total of 27 scores would be accumulated for the most 598 severely damaged lung tissue. with PrimeScript TM RT reagent kit (Takara). Real-time quantitative PCR was performed with 615 gene specific primers (Table S4 ) using SYBR Premix Ex Taq II kit (Takara). The expression of 616 house-keeping gene β-actin was analyzed in parallel to normalize the amount of RNA. The ∆∆Ct 617 method was applied for the comparison of the differential gene expressions between samples.

Microneutralization assay 620 Serum taken from orally or intranasally inoculated hamsters at 7dpi and 14dpi was tested for 621 viral neutralizing antibody titer by microneutralizing assay in Vero E6 cells as we described 622 previously. 10 Briefly, the 2-fold serial diluted (1:10 to 1:1280) serum samples were mixed with 623 100 TCID 50 of SARS-CoV-2 virus and incubated at 37°C for 1 hour. The mixture was then 624 added to Vero E6 cells and further incubated at 37°C for 72 hours. The neutralizing antibody 625 titer was defined as the highest dilution that inhibits 50% of cytopathic effect. All data were analysed with Prism 8.0 (GraphPad Software Inc). One-way ANOVA, Two-way 629 ANOVA and multiple Student's t-test were used to determine significant differences between 630 groups. Categorical variables were analysed by Chi-square test. P value of <0.05 was considered 631 statistically significant. 

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