Stability of Feline Coronavirus in aerosols and dried in organic matrices on surfaces at various environmental … – Nature.com

The investigation of virus tenacity in the environment plays a crucial role in enhancing our understanding of potential transmission routes for infectious diseases. Our study focused on assessing the tenacity of airborne FCoV and FCoV in dried organic matrices on surfaces. Overall, airborne FCoV showed a remarkable level of stability over a wide range of RH conditions. However, it is important to note that relative humidity has an impact on FCoV stability. The virus showed higher stability at both low and high RH levels, whereas medium RH conditions (around 5060%) were associated with a higher probability of decay. Remarkably, FCoV remained infectious for over 7h in the airborne state at medium RH levels. Moreover, on surfaces, FCoV showed the ability to remain infectious for extended periods, even up to several months. The stability on surfaces was influenced by factors such as temperature and the presence of organic material.

FCoV was used as a surrogate for SARS-CoV-2 in our study. Working with infectious pathogens of biosafety level 3 (SARS-CoV-2) is only possible in a limited number of laboratories, and especially with virus aerosols, it is very challenging. FCoV, on the other hand, can be studied under biosafety level 2 (BSL2) conditions, making it a safer and more cost-efficient option. FCoV belongs to the genus Alphacoronavirus, while SARS-CoV-2 is a Betacoronavirus. Although they share only 44.044.5% similarity at their nucleotide level3, previous research has shown that non-zoonotic animal coronaviruses like FCoV, canine coronavirus (CCV), transmissible gastroenteritis virus (TGEV) or mouse hepatitis virus (MHV) could be a suitable surrogate for survival of zoonotic SARS-CoVs27,28,29. To the best of our knowledge, this is the first study investigating the tenacity of FCoV in aerosols and on surfaces.

Our aerosol experiments were conducted in an aerosol chamber with a volume of 7m3, allowing individual airflows and climatic conditions. The chamber has been previously utilized in a study investigating the stability of Escherichia coli in aerosols30. Other studies investigating pathogen stability in aerosols, including SARS-CoV-2, have utilized a rotating drum, as described by Goldberg et al.14,31, to generate a dynamic aerosol. Our chamber offers a good opportunity to create a more realistic setting for exploring pathogen behavior within a room. To investigate FCoV stability, we worked with both dynamic (air exchange) and static (no air exchange) aerosol setups.

We observed a slight U-shaped trend in the stability of FCoV in dynamic aerosols at different RH levels, indicating that FCoV was more likely to decay at medium RH levels, ranging from 50 to 60%. This U-shaped pattern has also been observed for other enveloped viruses, including TGEV and influenza virus32,33. However, studies on human coronaviruses have shown varying results. For example, Sars-CoV and MERS have been found to be more stable at medium humidity levels34,35. Oswin et al. demonstrated that at low humidity levels, the initial stability decreases significantly but then remains relatively stable compared to higher humidity levels. If this initial decrease is neglected, a U-shaped pattern could also be observed21. Overall, coronaviruses in the aerosol state appear to be more stable than influenza or filoviruses at medium humidity levels34. When comparing studies on stability of viruses at different humidity levels, it is important to consider the medium used, as significant differences in stability can arise due to this factor. The most important fact to take into consideration when talking about the relationship between stability of viruses and RH is the microenvironment of the droplet and therefore the medium in which it resides36. Simulating human respiratory fluids accurately is still challenging due to the unknown exact components and concentrations. Therefore, many studies used cell culture medium such as DMEM as a model medium. One previous study compared DMEM with porcine respiratory fluid (PRF) and found that they differed greatly in the NA:K ratio. In addition, PRF contained significantly more protein37. It is important to note that studies using simulated respiratory fluids or real respiratory fluids instead of model medium have shown differences in virus stability38. These studies suggest that virus stability might be underestimated in most cases34,36,39,40. To make studies more representative, changes should be made to the virus suspension medium in further aerovirology studies37.

In our study, we modified DMEM by supplementing it with 10% FBS as a protein source, as respiratory droplets contain a variety of salts and proteins41,42. Yang et al. investigated the influence of different model media on the stability of Influenza A viruses in droplets, comparing DMEM and PBS, each with or without the addition of 5% FBS as a protein source. In general, they found better viability in DMEM than in PBS especially at medium and low RH36. Notably, the addition of FBS significantly affected virus stability at medium RH levels, suggesting a protective effect of proteins43,44. When a droplet leaves the respiratory tract, it evaporates by approximately half its original size depending on the ambient RH. This leads to a high concentration of substances within the droplet, such as salts, which are usually harmless but can become toxic to the virus. This effect is only relevant at medium RH levels just before the salts crystallize36. The exact RH at which the salts crystallize (efflorescence RH) depends on the droplet's composition and medium45. These findings support our own observations, as we observed a slight decrease in stability at medium RHs in the aerosol. Overall, we observed a high stability of FCoV in the aerosol, likely due to the presence of 10% FBS. The dynamic aerosol setup aimed to simulate a ventilated room where a virus emitter is present. The results indicate that the ambient RH in a room can significantly impact the stability of the emitted virus in the aerosol and thus its transmission potential. To minimize the risk of infection, it is advisable to keep the relative humidity at medium levels in indoor places.

Furthermore, our study demonstrated that FCoV remained infectious in static aerosols for over 7h with a half-life of 34.8min. The static aerosol setup aimed to simulate an enclosed room without regular air exchange, where a virus was released for a specific duration. During these experiments, we considered the possible natural loss of virus due to sedimentation. We observed a 31.4% loss of infectious virus through sedimentation, which occurred within the initial 10min and was than constant over the subsequent 7h. Moreover, the particle count remained stable throughout the entire experiment, indicating that virus-containing particles relevant for aerosol transmission remained suspended in the aerosol. It is known that aerosol particles <5m, which are relevant for inhalation, remain suspended as droplet nuclei in the air for hours, while larger droplets >10m settle to the ground within minutes due to gravity11,12,46. However, re-aerosolization of these sedimented infectious virus particles may also occur. In these experiments, the RH averaged 33%. Previous studies on related viruses have found that SARS-CoV-1 and SARS-CoV-2 remain stable in aerosols for over 3h, with respective half-lives of 1.1 and 1.2h, at an RH of 65%14. Similarly, MERS-CoV was found to be infectious in aerosols for over 3h35. In our study, we observed that FCoV has a half-life of 34.8min in aerosols and was detectable for over 7h. There was one other research group that investigated the stability of SARS-CoV-2 in aerosols over a longer period and found infectious virus after 16h at an average RH of 53%. However, this was a single observation without replication18. Comparing the half-lives of SARS-CoV-2 and FCoV indicates that both exhibit relatively short durations, suggesting similar behavior in aerosol stability. Observed differences may be more likely attributed to different aerosol generation processes and sampling methods. When considering influenza A viruses, their infectivity in aerosols varies lasting from 1 to 24h, depending on RH levels. Furthermore, influenza A viruses adapted to animals tend to demonstrate longer stability compared to human influenza A viruses47,48. It is important to note that comparisons between these studies are challenging due to variations in RH levels and medium used, as both factors strongly influence the stability of airborne viruses, as mentioned earlier. In general, our findings underscore the potential risk of aerosol transmission of enveloped respiratory viruses, especially in enclosed and unventilated environments over an extended period. This aligns with previous studies that have demonstrated aerosol transmission of SARS-CoV-2 between animals using hamsters as an animal model49,50.

At optimal environmental conditions the recovery rate of airborne FCoV was approximately 13% in our study. Several factors may have an influence on recovery rates of airborne viruses, including inactivation during aerosolization, loss through sedimentation, as well as sampling losses. We assume that our ultrasonic nebulizer and the aerosilization settings used resulted in the production of a suitable viral aerosol. In a study by Kim et al. various nebulizers and settings like pressure and nebulization time were tested to evaluate their impact on the stability of TGEV, and it was concluded that the stability of TGEV was not significantly affected32. Dhla et al. emphasized the importance of selecting an appropriate sampling method, as it can influence the stability of viruses in the sample. Since there is no generally recommended virus air sampling method, the choice of air sampler needs to be individually determined based on the specific experimental setup51. Most commonly used air samplers for collecting SARS-CoV-2 include filters, impactors, cyclone samplers and impingers52. For our experiments we chose the Coriolis cyclone air sampler. Previous studies aiming to detect SARS-CoV-2 in hospitals or healthcare settings have also utilized cyclone samplers due to their high collection volume53,54,55,56. While SARS-CoV-2 RNA has been detected in these studies, the identification of infectious SARS-CoV-2 was reported in only a few cases. It should be noted that cyclone samplers may be less efficient in detecting low levels of viruses compared to other air samplers, as the centrifugal forces affecting the viruses during collection could potentially cause stress57. However, in our study, we worked with high concentrations of viruses in a controlled environment, which made the Coriolis sampler suitable for our purposes, and we were able to detect infectious viruses.

We observed that 31.4% of the infectious virus sedimented onto the ground or surfaces within the first 10min in the static aerosol. This finding highlights the potential risk of contact transmission and the importance of studying virus infectivity on commonly encountered surfaces. We focused on stainless steel surfaces, which are frequently found in public buildings and clinical settings and are frequently touched. Previous studies have shown that CoVs exhibit greater stability on non-porous surfaces like metal, glass or plastic compared to porous surfaces, such as paper or fabrics58,59. Furthermore, viruses tend to be more stable at lower humidity levels and temperatures59. In our study, we demonstrated that FCoV remained infectious for 1958days at 20C and low RH, with the organic load significantly influencing the virus's stability. Comparatively, SARS-CoV-2 remained infectious on stainless steel surfaces for 47days at room temperature, while MERS and Sars-CoV-1 remained infectious for 2days14,15,35,60,61. TGEV and MHV, other non-zoonotic CoVs, remained infectious at room temperature for 3days at 50% RH and up to 28days at 20% RH27. It is important to note that differences in the results of various studies may occur due to varying medium used. While most of these studies were conducted using cell culture medium, we enriched our medium with 10g/L yeast extract/BSA or 3g/L sheep blood/BSA, representing a high organic load according to the guidelines for virus inactivation studies on nonporous surfaces62. Exhaled droplets that would sediment on surfaces consist of respiratory tract residues, saliva and organic material from the environment, resulting in a high organic load. Other studies added a tripartite soil load (mucin, BSA and tryptone) following international standard ASTM to the medium and found increased stability of SARS-CoV-2 on stain-less steel surfaces at 20C for 1428days, indicating a protective effect of the organic load16,63. Therefore, we would suggest using a high organic load, such as ASTM Internationals standardized tripartite soil load64, for further studies to avoid underestimating the stability of these viruses in the environment. However, it should be taken into consideration that stability may differ in dried human respiratory fluids. Regarding the influence of temperature, we found that infectious FCoV was detectable at 4C and 50% RH for 54167days, depending on the organic load. Only few studies have investigated CoVs stability at temperatures below 20. Notably, Onianwa et al. observed a reduction in infectiousness of the Delta variant of SARS-CoV-2 at 24C and 65% RH in the first 2.5h, while no reduction was observed at 4C and 85% RH within 2.5h65. TGEV and MHV also remained infectious at 4C for over 28days at all tested RHs, with the lowest losses observed at 20% RH27. Interestingly, we observed prolonged infectivity with yeast extract at 4C, although the reason for this difference remains unclear.

Like in aerosols, evaporation, and thus RH, plays an important role in terms of virus stability in droplets that sediment. French et al. studied the interplay of droplet volume and RH on surfaces and found that loss of infectivity was slower and more affected by RH in larger droplets (50L) than in small droplets (1L)66. Studies investigating stability of CoVs on surfaces, including our study, all used larger droplet volumes, which are not in line with realistically expelled droplet volumes (<0.5L) and may lead to different conclusions about virus stability. Another limitation of our study design is that we could not regulate the RH at the storage place and therefore could not distinguish between the influence of temperature and RH after drying. However, French et al. found that that viral decay during the wet phase was higher than during the dry phase regardless of RH66. In our experiment, all germ carriers were dried under controlled conditions for 45min, allowing us to neglect the influence of RH during the wet phase. Thus, the observed differences in stability may be primarily attributed to temperature and variations in organic load.

In summary, our study demonstrated that FCoV could remain infectious in the airborne state for hours and on surfaces up to months, with the duration depending on environmental conditions. Factors such as RH, temperature, and the presence of organic material significantly impact the pathogen's infectivity outside the host. Comparing studies on virus stability is challenging due to the lack of standardized experimental setups and medium used in these investigations. Additionally, reproducing respiratory fluids in the laboratory is difficult as their exact composition is still unknown. However, existing evidence suggests that viruses may exhibit even greater stability in respiratory fluids. It can be stated that aerosol transmission as well as droplet and contact transmission are possible transmission routes for coronaviruses under various environmental conditions over an extended period. Whether an infection occurs depends on many other factors, such as the viral load in the environment, the minimum infection dose,and the immune state of individuals. Especially enclosed, poorly ventilated rooms and low RH environments may pose a higher risk of infection due to the accumulation and better stability of these enveloped viruses. Given that, different pathogens respond uniquely to environmental conditions based on their biological and physical properties, it is essential to study a wide range of viruses to identify and understand potential correlations. The exact mechanisms that lead to the inactivation or protection of enveloped viruses by environmental components remain unknown and require further research. Our study suggests that FCoV could be a valuable surrogate for studying the behavior of zoonotic coronaviruses like SARS-CoV-2 in the environment. Although surrogates could offer valuable insights into the stability and persistence of these viruses outside the host, enhancing our understanding of zoonotic transmission dynamics, it remains crucial to directly investigate the actual virus.

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Stability of Feline Coronavirus in aerosols and dried in organic matrices on surfaces at various environmental ... - Nature.com