SARS-CoV-2 disease severity and transmission efficiency is increased for airborne compared to fomite exposure in Syrian hamsters

Since the emergence of SARS-CoV-2 in late 2019, the virus has caused a global pandemic with a wide spectrum of clinical outcomes, from asymptomatic infection to severe lower respiratory disease. Peak respiratory shedding in humans typically occurs around symptom onset and declines thereafter, and gastrointestinal shedding is less frequent and generally not associated with infectious virus. However, a clear relationship between COVID-19 disease severity and the duration or magnitude of SARS-CoV-2 shedding has not been established. It remains unclear how different exposure routes—direct contact, fomites, and airborne (large droplets and aerosol droplet nuclei)—contribute to human-to-human transmission, and how exposure route affects disease manifestation. Environmental sampling in healthcare settings frequently detects SARS-CoV-2 RNA on surfaces and in air, but recovery of infectious virus is limited, complicating inference about transmission routes. Using the Syrian hamster model, this study aims to delineate the relative roles of airborne and fomite transmission and to determine how exposure route influences disease severity, shedding dynamics, immune responses, and pathology. The authors hypothesize that exposure route determines initial viral deposition and replication kinetics, which in turn modulate disease severity and transmission efficiency.

Environmental studies in hospitals have repeatedly detected SARS-CoV-2 RNA on surfaces and in air, though isolation of viable virus has been limited. Experimental work shows RNA can persist on surfaces up to seven days while infectious virus declines rapidly within two days, highlighting a gap between RNA detection and viable virus presence. Prior animal studies in Syrian hamsters established susceptibility to SARS-CoV-2 and showed potential for transmission, typically using intranasal inoculation. Limited prior work suggested lower fomite transmission efficiency versus airborne routes in hamsters. Observational human studies indicate airborne exposure in confined spaces with directed airflow can lead to clusters, and both asymptomatic and symptomatic individuals can shed virus. There is no definitive human evidence linking route of transmission to disease severity. These findings provide the rationale for experimentally comparing aerosol and fomite exposure under controlled conditions to quantify their relative contributions and effects on disease.

Ethics: All animal procedures were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories and conducted under BSL-3 with IBC approval. Virus: SARS-CoV-2 strain nCoV-WA1-2020 (MN985325.1) propagated in VeroE6 cells. Stocks confirmed free of contaminants and sequence-identical to reference. Animals and exposure: Female Syrian hamsters (4–6 weeks). Three exposure routes were tested (n=12 per route): intranasal (I.N.), aerosol, and fomite. Unexposed controls (n=12) included. For each exposed group, 4 animals euthanized at 1 DPI, 4 at 4 DPI, and 4 monitored to 14 DPI. - Intranasal: 40 µL DMEM containing 8×10^4 TCID50 applied to nares. - Aerosol: Non-anesthetized hamsters exposed 10 min in a wire mesh cage using AeroMP platform with 3-jet collision nebulizer generating 1–5 µm droplet nuclei. Target dose 1.5×10^3 TCID50; inhaled dose estimated using D = R × Caero × T. - Fomite: A polypropylene dish with 40 µL of 8×10^4 TCID50 per hamster (total 3.2×10^5 TCID50 per 4-animal cage) placed for 24 h; interaction confirmed. Clinical monitoring: Daily weights and observation. Oropharyngeal and rectal swabs daily until day 7, then three times per week to 14 DPI. Transmission experiments: - Airborne: Custom cages with a 3D-printed perforated divider preventing contact and bedding transfer; alpha-dri bedding used. Donors I.N.-infected; sentinels placed downflow (with airflow) or against airflow. Particle size distribution and reduction across divider measured with an aerodynamic particle sizer; glycerol aerosol used for characterization. - Fomite: Donors (2/cage) I.N.-infected and housed 4 days; then donors euthanized and sentinels (2/cage) placed into contaminated cages for 21 days without disturbing bedding. Sampling and assays: - Virology: Infectious virus titers (TCID50/mL or per g adjusted for tissue weight) in trachea, lung, brain, and GI tissues. SARS-CoV-2 genomic (gRNA) and subgenomic RNA (sgRNA) quantified by qRT-PCR from swabs and environmental cage/bedding swabs. - Histopathology and IHC: Formalin-fixed tissues (nasal turbinates, trachea, lungs) processed and stained. IHC for SARS-CoV-2 N protein to assess cellular tropism at 1 DPI. - Serology: Spike ELISA endpoint IgG titers at 14 DPI (or 21 DPE for transmission studies) and reciprocal live virus neutralization (VN) titers. - Cytokines: Serum TNF-α, IFN-γ, IL-6, IL-4, IL-10 at 4 DPI via hamster ELISAs. - Lung transcriptomics: RNA-Seq at 1 and 4 DPI; differential expression (DESeq2); pathway analysis by Ingenuity Pathway Analysis (IPA); clustering. Statistics: Mann-Whitney, Kruskal–Wallis with Dunn’s multiple comparisons, two-way ANOVA with Tukey’s, Spearman correlations. Significance: ns p>0.05; * p≤0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001.

- Susceptibility by route: Syrian hamsters were infected via intranasal, aerosol, and fomite exposure. - Clinical severity varies by route: I.N. and aerosol led to significant weight loss (I.N. from 3 DPI; aerosol from 2 DPI). Fomite exposure caused limited, transient weight loss or reduced weight gain. By 14 DPI, weights were not significantly different across groups. - Early viral deposition and load: At 1 DPI, infectious virus detected in trachea of all I.N. and aerosol animals; in lungs of all aerosol and a subset of I.N. animals. Aerosol-exposed hamsters had higher tracheal and lung titers than I.N. (two-way ANOVA/Tukey: trachea p=0.0115; lung p<0.0001) despite a 10-fold lower inoculum, indicating efficient lower respiratory deposition by aerosols. No infectious virus detected at 1 DPI in fomite group in respiratory tract. No infectious virus in GI tract at 1 DPI for any route. - Cellular tropism at 1 DPI: Aerosol exposure showed widespread antigen in nasal ciliated epithelium, trachea, bronchioles, type I/II pneumocytes, pulmonary macrophages, and olfactory epithelium. I.N. showed antigen in nasal ciliated/olfactory epithelium, with antigen in pulmonary macrophages in 2/4; minimal epithelial antigen in lung/trachea. Fomite: rare nasal ciliated epithelial antigen in 1/4; none in trachea/lung. - Pathology: At 1 DPI, nasal turbinate pathology most consistent/severe in I.N., followed by aerosol; minimal in fomite. Tracheitis present in all aerosol and half of I.N.; none in fomite. Lung pathology minimal across groups at 1 DPI. By 4 DPI, infectious virus present in lungs of all groups with no significant titer differences (I.N. vs aerosol p=0.4114; I.N. vs fomite p=0.9201). Moderate broncho-interstitial pneumonia in aerosol and I.N.; milder and less consistent in fomite. Clustering linked 1 DPI tracheal pathology with trachea/lung viral load; fomite animals resembled controls at 1 DPI and formed a distinct cluster at 4 DPI with upper airway-focused pathology. - Systemic cytokines (4 DPI): Fomite had reduced TNF-α (I.N. vs fomite p=0.0360); IL-4 elevated most in fomite (vs unexposed p=0.0109); IL-10 increased in fomite and I.N., decreased in aerosol (fomite vs aerosol p=0.0286). No significant IL-6 differences; trend toward decreased IFN-γ. - Humoral response: All exposed animals seroconverted by 14 DPI. I.N. elicited higher spike ELISA titers than fomite (p<0.0209). Aerosol had highest neutralizing titers and fomite lowest, differences not significant (p=0.2026). - Lung transcriptomics (4 DPI): I.N. and aerosol showed extensive pathway modulation (>50 canonical pathways), including macrophage activation, dendritic cell maturation, interferon signaling, and T/B/NK-cell pathways; fomite showed only ~10 significantly modulated pathways, including interferon signaling, Th17, and pattern recognition. Coronavirus pathway genes differentially regulated in aerosol vs I.N. (e.g., downregulation of MAVS, ELK1, BCL2, SERPINE1, IFNAR1; upregulation of IL1B in aerosol). Th1/Th2 pathway markers upregulated in I.N. and aerosol (IL-18, IFN-γ, IL-6, IL-2; CD4/CD8; co-stimulatory molecules; chemokine receptors), but minimal in fomite. - Shedding dynamics by route: Oropharyngeal sgRNA shedding began earlier in aerosol and I.N.; fomite showed delayed increase with peaks around 5 DPI. No significant differences in peak respiratory shedding between groups (p=0.8400). Intestinal shedding was lower overall, intermittent (1–3 consecutive days), with no group differences (p=0.1512). Cumulative respiratory shedding (AUC) was lower in aerosol versus I.N. and fomite (p=0.0263); cumulative rectal shedding highest in aerosol. - Early shedding predicts severity: Lung titers at 4 DPI positively correlated with oropharyngeal RNA at 2 DPI (Spearman p=0.047), upper/lower respiratory pathology, and weight loss. TNF-α negatively correlated with IL-4 and IL-10 (p=0.048 and p=0.008). Early rectal shedding correlated with TNF-α and olfactory pathology (p=0.0002; p=0.001). Olfactory pathology correlated with IgG ELISA and VN titers (p=0.001; p=0.021). - Transmission efficiency: Environmental contamination of donor cages by RNA/sgRNA was high up to 7 DPI. Fomite transmission to sentinels occurred in 4/8 without weight loss; seroconversion at 21 DPE confirmed infection. Airborne transmission with directional airflow led to 100% sentinel seroconversion and no weight loss; reversing airflow reduced seroconversion to 1/4, demonstrating airflow direction dependence. Sentinel shedding after airborne exposure started by 1–2 DPE and remained high until ~6 DPE; cumulative shedding did not differ between airborne and fomite-exposed sentinels, suggesting asymptomatic but potentially transmissive infection in both indirect routes.

The study demonstrates that exposure route strongly shapes SARS-CoV-2 disease dynamics in Syrian hamsters. Aerosol exposure, even at a lower inoculum, efficiently deposits virus in the lower respiratory tract, resulting in earlier and higher early lung and tracheal viral loads, accelerated weight loss, and more severe pulmonary pathology than intranasal inoculation. Fomite exposure leads to delayed replication in the respiratory tract, milder pathology, and a systemic immune profile indicative of reduced pro-inflammatory signaling (lower TNF-α) alongside elevated IL-4/IL-10. Transcriptomic profiling indicates robust activation of antiviral and adaptive immune pathways in aerosol and intranasal exposure, whereas fomite exposure minimally perturbs these pathways at 4 DPI, consistent with reduced disease severity. Shedding patterns varied by route: aerosol infection produced overall less cumulative respiratory RNA but earlier onset, while fomite infection showed delayed peaks. Importantly, early oropharyngeal shedding predicted subsequent lung viral burden and disease severity, highlighting potential early markers of severe outcomes. Transmission experiments establish that airborne transmission is more efficient than fomite transmission and is highly dependent on airflow direction, aligning with human outbreak observations in confined, poorly ventilated settings. Notably, both airborne and fomite indirect exposures yielded asymptomatic infections with measurable shedding and robust humoral responses, underscoring the potential for transmission from minimally symptomatic hosts. These findings suggest that interventions which reduce aerosol exposure and limit lower respiratory deposition (e.g., masking, ventilation) may not only reduce transmission but could also mitigate disease severity by altering initial deposition sites. The hamster model with naturalistic exposure routes provides a platform to evaluate transmissibility and pathogenicity of emerging variants and to test targeted countermeasures across distinct transmission modalities.

This work establishes that SARS-CoV-2 disease severity and transmission efficiency in Syrian hamsters depend on the exposure route. Aerosol exposure deposits virus efficiently in the lower respiratory tract, producing earlier, more severe respiratory disease and distinct immune activation, while fomite exposure results in delayed replication, milder disease, and anti-inflammatory systemic signatures. Airborne transmission between hamsters is highly efficient and airflow-dependent, exceeding fomite transmission efficiency, and both indirect routes can yield asymptomatic but shedding infections. Early respiratory shedding predicts later lung viral burden and severity, offering a potential early indicator of disease trajectory. The study advances an experimentally validated hamster model encompassing aerosol and fomite routes to assess transmissibility and pathogenicity, which will be vital for evaluating emerging variants and informing public health interventions. Future research should: (1) quantify dose-response relationships across routes with refined dosimetry, especially for fomites; (2) resolve aerosol versus large-droplet contributions with improved particle separation; (3) expand immune profiling with hamster-specific reagents and single-cell resolution; (4) assess variant-dependent changes in environmental stability, transmission route efficiency, and disease severity; and (5) test targeted interventions (masking, filtration/ventilation strategies, surface decontamination) under controlled transmission scenarios.

- Use of cultured virus: Particle-to-infectivity ratios may differ from those in naturally shed aerosols or fomites, potentially affecting comparability to natural transmission. - Fomite dose uncertainty: Exact infectious dose delivered via fomite exposure could not be confirmed, limiting precise dose-response interpretation. - Cytokine profiling: Systemic cytokine analyses were constrained by availability of hamster-specific reagents, providing a limited snapshot of immune status. - Aerosol vs droplet distinction: The divider reduced but did not eliminate larger particles (>10 µm); thus, the relative contributions of true aerosols (<5 µm) versus larger droplets could not be fully disentangled. - Species/behavioral differences: Hamster interactions with fomites differ from humans, potentially inflating fomite transmission in this model relative to typical human exposure patterns.