Since the emergence of SARS-CoV-2 in Wuhan, China, in December 2019, it has spread globally, causing millions of cases and deaths. Infection can range from asymptomatic to severe lower respiratory tract infections. Peak respiratory shedding in humans usually occurs around symptom onset, declining afterward. While shedding from the intestinal tract has been observed, it's not typically associated with infectiousness or transmission. The relationship between COVID-19 severity and the duration/magnitude of SARS-CoV-2 shedding remains unclear. Understanding the relative contributions of different exposure routes (direct contact, fomites, airborne transmission) is crucial for effective control measures. Most cases occur in households or social gatherings, suggesting a significant role for these routes. Airborne transmission involves large droplets and smaller droplet nuclei, while fomites are surfaces contaminated by respiratory secretions. SARS-CoV-2 RNA has been detected on surfaces and in air samples in various settings, but detecting infectious virus has been more limited. This discrepancy between RNA detection and viable virus presence makes it difficult to assess the relative contributions of fomites and airborne transmission. This study utilizes the Syrian hamster model to experimentally determine the relative contributions of fomite and airborne transmission and assess the impact of transmission route on disease severity. The goal is to establish a well-characterized model to study SARS-CoV-2 transmission and potential changes due to viral evolution.

Existing research indicates SARS-CoV-2 can be transmitted through various routes, including direct contact, fomites, and airborne transmission. Studies have shown the detection of SARS-CoV-2 viral RNA on surfaces and in air samples in healthcare settings. However, the detection of infectious virus in these samples has been less consistent, hindering the understanding of the relative contribution of each transmission route. Animal models, such as the Syrian hamster, have been used to study SARS-CoV-2 infection and transmission, offering a controlled environment to study various aspects of the disease. Previous studies using hamsters have primarily focused on intranasal inoculation, which doesn't fully replicate natural infection routes. There is a need for more research utilizing aerosol and fomite exposure models to better understand the transmission dynamics and disease severity associated with different exposure routes. The inconsistencies in findings highlight the need for a more comprehensive understanding of the various factors affecting SARS-CoV-2 transmission.

The study used four- to six-week-old female Syrian hamsters, divided into groups for intranasal (I.N.), aerosol, and fomite exposure to SARS-CoV-2. The I.N. group received 8 × 10⁴ TCID₅₀ intranasally. The aerosol group was exposed to 1.5 × 10³ TCID₅₀ for 10 minutes using an aerosol management platform generating 1–5 µm particles. The fomite group was exposed to 8 × 10⁴ TCID₅₀ on a polypropylene dish placed in their cage for 24 hours. A control group remained unexposed. Four hamsters per group were euthanized at 1 and 4 days post-inoculation (DPI), and the remaining four at 14 DPI. Daily weight measurements, oropharyngeal and rectal swabs were collected to monitor weight loss and viral shedding. Viral titers in respiratory and intestinal tissues were determined using TCID₅₀ assays at 1 and 4 DPI. Immunohistochemistry (IHC) was used to assess SARS-CoV-2 antigen distribution at 1 DPI. Histopathological evaluations were performed on lung, trachea, and nasal turbinate sections at 1 and 4 DPI. Serum cytokine levels (TNF-α, IFN-γ, IL-6, IL-4, IL-10) were measured at 4 DPI using ELISAs. ELISA and neutralization assays were performed at 14 DPI to assess antibody responses. RNA sequencing was performed on lung samples at 1 and 4 DPI to analyze gene expression changes. Airborne and fomite transmission experiments were conducted using specially designed cages with a divider to prevent direct contact but allow airflow. Particle sizing was performed to assess the effectiveness of the divider in blocking particle flow. Statistical analysis included Mann-Whitney, Kruskal-Wallis, two-way ANOVA, and Spearman correlation tests.

The study found that aerosol exposure caused severe respiratory pathology, high viral loads, and significant weight loss in hamsters. Intranasal inoculation also resulted in weight loss and respiratory pathology but less severe than aerosol. In contrast, fomite exposure led to milder disease with delayed viral replication, an anti-inflammatory immune state, and a delayed shedding pattern. Aerosol exposure led to efficient deposition of virus in the upper and lower respiratory tracts, while fomite exposure caused slower replication in the lung. Histopathological analysis showed more severe lesions in the nasal turbinates and lungs of the aerosol and intranasal groups compared to the fomite group. At 4 DPI, lung viral titers were comparable across all inoculation routes. Fomite-exposed animals showed reduced levels of TNF-α and elevated levels of IL-4 and IL-10 compared to the other groups, indicating an anti-inflammatory immune response. All exposed animals seroconverted at 14 DPI, with the strongest antibody response observed in the I.N. group. Gene expression analysis revealed significant upregulation of immune and infection-related pathways in aerosol and I.N. groups, while fomite exposure showed minimal pathway activation. Viral shedding patterns differed across exposure routes, with aerosol exposure resulting in less cumulative viral RNA shedding in oropharyngeal swabs. Early oropharyngeal shedding correlated positively with lung viral titers and respiratory pathology. Airborne transmission experiments demonstrated high transmission efficiency (100% seroconversion), and the direction of airflow significantly influenced transmission. Fomite transmission also occurred, albeit at a lower rate (50% seroconversion).

The findings demonstrate that SARS-CoV-2 transmission efficiency and disease severity are significantly influenced by the exposure route. Aerosol exposure leads to more severe disease compared to fomite exposure, likely due to direct deposition of virus in the lower respiratory tract. The anti-inflammatory immune response observed in the fomite group may contribute to milder disease. Early viral shedding is a strong predictor of disease severity, emphasizing the importance of early detection and intervention. The high efficiency of airborne transmission highlights the relevance of measures to improve ventilation and reduce aerosol spread. The occurrence of fomite transmission underscores the need to maintain surface hygiene practices. The differential immune responses observed across the exposure routes may have implications for understanding re-infection with novel variants and the development of effective countermeasures. The study provides crucial data for improving our understanding of SARS-CoV-2 transmission and informing public health strategies.

This study demonstrated that SARS-CoV-2 transmission and disease severity in Syrian hamsters vary significantly depending on the exposure route (airborne vs. fomite). Airborne transmission was more efficient and resulted in more severe disease than fomite transmission. Early viral shedding was a strong predictor of disease severity. These findings highlight the importance of both airborne precautions and surface disinfection in controlling SARS-CoV-2 spread. Future research should focus on investigating the impact of novel SARS-CoV-2 variants on transmission dynamics and disease severity, as well as exploring the potential of the Syrian hamster model for testing interventions aimed at blocking human-to-human transmission.

The study used a Syrian hamster model, which may not fully recapitulate human responses to SARS-CoV-2. The exact dose of virus delivered via the fomite route could not be precisely determined. The systemic cytokine analysis was limited by the availability of hamster-specific reagents. The study focused on a single SARS-CoV-2 strain; therefore, the results might not be generalizable to other strains or variants. The airborne transmission experiments did not definitively distinguish between true aerosol transmission and droplet transmission. The sample size of the study might have limited the statistical power of some analyses.