SARS-CoV-2 bioaerosol transmission in experimentally infected American mink

SARS-CoV-2 bioaerosol transmission in experimentally infected American mink | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SARS-CoV-2 bioaerosol transmission in experimentally infected American mink Rasmus Malmgren, Vinaya Venkat, Jenni Virtanen, Kristel Kegler, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5980382/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract The SARS-CoV-2 BA.1 (Omicron) variant, which emerged in late 2021, is more transmissible than earlier variants but causes milder symptoms in humans. Mink farms, where animals are housed in close quarters, present a high risk for virus transmission and mutation, necessitating strict control measures due to documented cases of mink-to-human and human-to-mink transmission. Hence, we aimed to detect viable airborne SARS-CoV-2 using BioSampler-air collectors and to investigate aerosol transmission between groups of American mink ( Neovison vison ). Two groups (male and female) were infected with the BA.1 variant, and samples were collected from aerosols, saliva, feces, and surfaces. The results indicated that infectious viruses were predominantly detected in aerosol samples over a three-day period in both groups. Surface, saliva, and fecal samples also showed potential for virus transmission. Notably, infectious viruses were cultivated from aerosol samples, confirming aerosol transmission among American mink. This study highlights the importance of immediate sample culturing to improve infectious virus detection and emphasizes the need for enhanced preventive measures on mink farms to mitigate the spread of viruses. Biological sciences/Microbiology/Virology/Sars cov 2 Biological sciences/Microbiology/Virology/Viral transmission Figures Figure 1 Figure 2 Introduction SARS-CoV-2, a single-stranded RNA virus and the causative agent of the COVID-19 pandemic, is known to transmit among humans through the air via aerosols and droplets, and contaminated surfaces [ 1 ]. Since the emergence of the pandemic, the virus has been able to mutate and develop variants such as the Alpha, Beta and Delta variants. In this study, we focused on the more transmissible variant of SARS-CoV-2, BA.1 Omicron [ 2 ], which has shown milder symptoms in humans [ 3 , 4 ]. Besides humans, SARS-CoV-2 has been shown to infect various animals, including American mink ( Neovison vison ) [ 5 , 6 ]. American mink, though solitary in the wild, are co-housed in large numbers at fur farms, increasing the risk of virus transmission among the animals [ 7 ]. Such an environment serves as a reservoir for viral transmission [ 8 ] and the accumulation of mutations, increasing the possibility of animal-to-human transmission [ 9 , 10 ]. Due to the open design of these farms, stray and wild animals, as well as birds, may encounter mink and their excreta, potentially carrying infections outside the farm [ 11 , 12 ]. Cases of human-to-mink and mink-to-human transmission of SARS-CoV-2 have been documented [ 13 , 14 , 15 ]. Thus, as a biosafety measure, most infected farms resorted to mass culling of millions of animals [ 16 ]. To prevent future crises, a thorough investigation on the sources and transmission of virus infections needs to be conducted to help inform the measures taken during future outbreaks. Some studies have found SARS-CoV-2 on the surfaces of mink farms using PCR and animal bedding, but the presence of infectious viruses have not been detected [ 6 , 12 ]. Additionally, SARS-CoV-2 is known to be transmitted via aerosols among humans [ 17 , 18 , 19 ]. The virus was found to be viable in aerosols for up to 3 hours in laboratory conditions [ 20 ] but detecting viable viruses from air samples in clinical and environmental settings has proven difficult [ 21 , 22 ]. Likely, this difficulty arises from the long delays between sample collection and culturing, as samples are often transported to distant laboratories for analysis. This manuscript is a part of multiple publications on a study of American mink experimentally infected with SARS-CoV-2. In a previous publication, we proved that the Omicron variant, despite being known for mild infections in humans, can also infect mink [ 23 ]. In this manuscript, we focused on consistently capturing infectious viruses in aerosols collected around the experimentally infected mink, to provide evidence of aerosol transmission of SARS-CoV-2 at fur farms. Materials and methods Virus stock, cells lines and cell media Virus stocks, cell lines, and cell media were used as previously published in Virtanen et al. (2022) [ 23 ]. SARS-CoV-2 ba.1 was acquired from the Finnish Institute of Health and Welfare ((original patient sample: hCoV-19/Finland/THL-202126660/2021, EPI_ISL_8768822 (Gisaid)). TMPRSS2-expressing VeroE6 (VE6T) cells were used for virus cultivation and were grown according to Rusanen et al. (2021) [ 24 ]. Experimental setup, animal infection and euthanasia The mink were purchased from a commercial mink farm. Animals were acclimatized to the BSL3-laboratory room and custom-made enclosures for three days before infection. The enclosures were 84x76x58.4 cm metal bar cages, 10–20 cm apart, with a 26.2x31.5x40 cm closed metal nest with an open bar cage roof, containing hay as nesting material. The laboratory room was 59 m 2 with ventilation flowing from left to right (Fig. 1 ) at a rate of approximately 242 l/s. Airflow in the room was turbulent or partially turbulent and could flow through animal enclosures. Animal waste was collected under the enclosures on a removable metal tray. Refer to animal enclosure setup in Fig. 1 . Before infection, animals were anesthetized using 30 µl of Ketaminol (100 mg/ml, Intervet, Netherlands) and Domitor (1 mg/ml, Orion Pharma, Finland). Three male and two female mink were nasally infected with 200 µl of SARS-CoV-2 ba.1 stock (pfu = 10 6 ), as indicated in Fig. 1 . Naïve recipient mink received PBS instead of the virus stock. Sedation was reversed using Revertor (5 mg/ml, Scanvet, Poland). One female mink had to be excluded from the study due to stress, resulting in only four animals in the female group. The nasally infected animals were euthanized in a CO 2 chamher on 7 dpi (days post infection), while the recipient males and females were euthanized at 10 and 11 dpi respectively. The experimental infection, sedation and euthanasia of the American mink are described in more detail in Virtanen et al. (2022) [ 23 ]. Experiment 1 consisted of five males while experiment 2 consisted of four females. The experiments were conducted successively. Reversing of the sedation of the animals indicated the starting point of the experiment. Sample collection Three air samples were collected simultaneously twice a day, before and after feeding, for 30 min using three 5 ml BioSamplers (SKC Inc., USA) positioned 30 cm from the enclosures, at the height of the animals. The samplers’ inlets faced the table the enclosures were on (Fig. 1 ). All samplers were operated with a single air pump, with individual airflows monitored using Mass Flowmeter 3063-devices (TSI Inc., USA). BioSamplers were thoroughly disinfected and washed with ethanol and MilliQ-water between collections. Aerosols were collected into 5 ml of minimal essential eagle’s medium (MEM, Sigma-Aldrich, USA) using a flow rate of 12.5 LPM. Amphotericin B (Gibco, USA) was added to MEM for the female group after some samples from the male group had fungal contaminations. Due to dehydration, aerosol samples were refilled with fresh media to 3 ml before analysis. All animal and surface sample collection was done using Sigma Virocult®-swabs (MWE, UK). Saliva was collected as animals chewed on the swab, fecal samples collected from swabbing the feces, and surface samples collected using MEM dipped swabs swabbing a comprehensive surface area. Samples were collected in duplicates and post sampling the swabs were stored in 1ml of MEM awaiting further processing. One of the saliva and surface duplicates were transferred to cells for virus cultivation while the others were stored at -80°C until PCR analysis. Virus cultivation 1 ml of samples were mixed with 2 ml of culture media and added to VE6T-cells on 6-well plates. Cells were incubated in 37°C for 9 days or until cytopathic effect (CPE) could be detected. A 140 µl sample was taken from wells with CPE for RNA extraction to confirm SARS-CoV-2 as the causing agent. Extracted RNA was analyzed using PCR. PCR Saliva and surface samples were extracted using the QIAamp Viral RNA Mini Kit (QIAGEN), while fecal and cell culture samples were extracted using QIAamp 96 Virus QIAcube HT kit (QIAGEN, off-board lysis). All samples were PCR tested for SARS-CoV-2 using Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit (NEB), targeting 2019-nCoV_N1 and 2019-nCoV_N2. Samples were positive (+) if they gave a signal with both probes, weak positive ((+)) if they only gave a signal with one probe, and negative (-) if they gave no signal. Cell culture samples were considered positive (+) if their Ct values were more than 5 cycles lower than the original non cell-cultured sample, possibly positive ((+)) if their Ct values were 1–5 cycles lower, and negative (-) if the Ct values were similar or higher. Ethics statement Experimental procedures were approved by the Animal Experimental Board of Finland (ESAVI/33259) and carried out accordingly. This study is performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. Results SARS-CoV-2 Omicron transmission among mink Infectious viruses were detected from aerosol samples in both experiments. For the males, infectious viruses were detected mostly on the first 3 days post infection (dpi) while in the female group they were detected later, mostly on 5–7 dpi (Fig. 2 ). PCR positive aerosol samples were collected throughout the experiment from both groups (Fig. 2 ). Most sampled surfaces tested PCR positive, however, infectious viruses were recovered only from one sample (Supplement Tables 1–2). A few infectious samples were collected from animal saliva in both groups, however, most samples were culture negative (Fig. 2 ). In the male group, infectious viruses were detected in the saliva of infected animals only during 1–3 dpi, and in the recipient mink’s saliva already on 1 dpi. In the female group infectious viruses were observed on 1 and 7 dpi (Fig. 2 ). All saliva samples tested PCR positive for the infected mink in both groups. The male recipient mink saliva was consistently PCR positive from 3 dpi onwards, while only one of the female recipients was (Fig. 2 ). PCR tests from infected mink feces were variably positive for both groups, however, more so in the male group. In the recipient mink feces, PCR positive results were observed only in the male group (Fig. 2 ). Discussion Studies conducted in ferrets [ 24 ] and hamsters [ 26 ] show the production of infectious SARS-CoV-2 containing aerosols, but this has not been demonstrated in American mink ( Neovison vison ). Our study bridges that gap, as we show that American mink infected with SARS-CoV-2 BA.1 (Omicron) variant can transmit viral aerosols from infected to healthy animals in a laboratory setting. We were able to come up with better aerosol sampling methods to capture these live viruses, which were not possible in previous studies. In our work, infectious viruses were detected primarily in aerosol samples, indicating that virus transmission among animals is likely to occur when animal enclosures are in close proximity. Most culture positive samples were obtained with aerosol collectors, suggesting that aerosol transmission is the main mode of transmission for the Omicron variant when animals are in separate enclosures. However, culture positive surface- and saliva samples were also detected, making surface and contact transmission also possible routes for infection. Previous studies on viral bioaerosol transmission have had difficulties in detecting infectious viruses from bioaerosols [ 27 , 28 ] or have only focused on RNA detection [ 29 , 30 , 31 ]. Additionally, while filter collectors have been the most used device for SARS-CoV-2 air sampling [ 32 ], liquid-based collectors have been found to retain virus infectivity much better [ 33 , 34 ]. Here, we successfully detected infectious viruses in multiple bioaerosol samples by using 5 ml BioSamplers and culturing the samples immediately after collection. A pattern was observed in the aerosol sample results, where both groups of mink had a three-day period when most of the culture positive samples were collected. Interestingly, this period happened in the first 1–3 dpi in the male group and later at 5–8 dpi in the female group. It is likely that the recipient male mink were infected already on 1 dpi, resulting in an earlier peak in culture positive aerosol samples. Additionally, SARS-CoV-2 has been reported to have more severe symptoms in males [ 35 , 36 , 37 ], which could be a reason for earlier infection and detection of virus-containing aerosols. However, as this experiment only consisted of two groups of animals, more studies are needed to confirm these differences. Still, the three-day period observed here is much shorter than reported in humans [ 38 , 39 ]. Moreover, as not all culture positive samples were PCR positive from the original sample, the virus concentrations in aerosols were likely low. A previous study has also shown that viral loads on surfaces and air can be quite low [ 1 ]. While the SARS-CoV-2 Omicron variant has been shown to survive on surfaces for multiple days [ 40 ], we observed infectious viruses in only one surface sample. By contrast, most surface samples were PCR positive, indicating high environmental contamination, however the longevity of virus viability appears to be brief. Additionally, it is important to acknowledge that the initial titer of the virus affects its survival [ 41 ]. Therefore, viruses originating from aerosols or droplets might not survive as long on surfaces compared to in vitro experiments in which higher titers of infectious particles are used. SARS-CoV-2 was also variably found in the feces of both infected and recipient mink with PCR. Previous studies have suggested that infected mink feces could spread SARS-CoV-2 to other wildlife and farm animals, such as cats [ 42 ], foxes, and other animals [ 43 ], if accessible. Although birds have not been observed to be susceptible to SARS-CoV-2 [ 44 , 45 ], they could still transport the virus from the feces to other housing units or even other farms. As the animals often defecate through their enclosures onto the ground below, it is accessible by wild animals. This area should be restricted to prevent the spreading of viruses. Additionally, dust in mink sheds has previously been shown to contain SARS-CoV-2 RNA [ 12 ], providing another possible route for the viruses to spread at the farms and outside them. Viral RNA has also been detected in outside air of recently infected mink farms, however only near the entrance [ 11 ]. Moreover, antibodies against SARS-CoV-2 have also been found in escaped mink around mink farms [ 46 ], though the infections could have occurred before escaping. Overall, more PCR positive samples were collected during the male group (63% vs. 45%, Supplement tables 1–2), suggesting that male mink have higher virus shedding than female mink. However, this difference was not observed in viable virus detections (13% vs. 15%, Supplement tables 1–2). Similar findings regarding viral RNA shedding have been made in humans [ 47 , 48 , 49 ], however, more studies are needed with better infectious virus detection methods to understand whether there is also a difference in infectious virus shedding between males and females. Our study demonstrated that American mink infected with the SARS-CoV-2 BA.1 variant produce aerosols containing infectious virus particles, capable of spreading the virus to nearby animals. We also found that culturing aerosol samples immediately after collection significantly improves the detection of viable viruses, making diagnostics for both infected animals and humans more accurate. To prevent animal suffering and avoid mink farms becoming reservoirs for respiratory viruses, enhanced preventive measures and surveillance are essential. However, these measures can only be effective if the various modes of transmission, including aerosol spread, are thoroughly researched and understood. Declarations Acknowledgements We thank Jari Elemo, Mari Elemo, and other animal caretakers for handling the animals and assessing their health, and Esa Pohjalainen, Sanna Mäki, Tiina Sihvonen, Johanna Rintamäki, Hanna Valtonen, Marika Skön, Larissa Laine,and Elina Väisänen for technical assistance. We thank Kati Kuipers, Anne Kujanpää, Laura Vähälä, and the Finnish Centre for Laboratory Animal Pathology (FCLAP) for expert technical help, as well as Johanna Korpela and Jussi Peura from Finnish Fur Breeders Association and Jan Segervall and Maarit Mohaibes from the Kannus Research Farm Luova Ltd. for providing the animals. We also thank E3 Excellence in Pandemic Response and Enterprise Solutions co-innovation project and all its parties. Ethics statement Experimental procedures were approved by the Animal Experimental Board of Finland (ESAVI/33259). Funding This study was funded by the Academy of Finland (grant no. 336490, 339510), VEO–European Union’s Horizon 2020 (grant no. 874735), Business Finland E3 (4917/31/2021), Finnish Institute for Health and Welfare, and the Jane and Aatos Erkko Foundation. Conflicts of interest We report no conflicts of interest. Data availability statement The datasets used and/or analysed during the current study are available from the corresponding author on a reasonable request. References Santarpia, J. L. et al. Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care. Sci. Rep. 10 (1), 12732. https://doi.org/10.1038/s41598-020-69286-3 (2020). Vauhkonen, H. et al. 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Zheng, S., Fan, J., Yu, F., Feng, B., Lou, B., Zou, Q., Xie, G., Lin, S., Wang, R.,Yang, X., Chen, W., Wang, Q., Zhang, D., Liu, Y., Gong, R., Ma, Z., Lu, S., Xiao,Y., Gu, Y., … Liang, T. (2020). Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: Retrospective cohort study. BMJ , 369 , m1443. https://doi.org/10.1136/bmj.m1443. Shastri, A. et al. Delayed clearance of SARS-CoV2 in male compared to female patients: High ACE2 expression in testes suggests possible existence of gender-specific viral reservoirs (s. 2020.04.16.20060566). medRxiv. (2020). https://doi.org/10.1101/2020.04.16.20060566 Additional Declarations No competing interests reported. Supplementary Files Supplementtables.xlsx Supplement.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 May, 2025 Reviews received at journal 10 May, 2025 Reviews received at journal 21 Apr, 2025 Reviewers agreed at journal 14 Apr, 2025 Reviewers agreed at journal 14 Apr, 2025 Reviewers invited by journal 14 Apr, 2025 Submission checks completed at journal 12 Apr, 2025 First submitted to journal 01 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5980382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":442754095,"identity":"8c1f48c0-a7ad-4118-962c-750d8ce5e658","order_by":0,"name":"Rasmus Malmgren","email":"data:image/png;base64,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","orcid":"","institution":"University of Helsinki","correspondingAuthor":true,"prefix":"","firstName":"Rasmus","middleName":"","lastName":"Malmgren","suffix":""},{"id":442754096,"identity":"c65bf0c3-8f40-4818-8f21-de8b3e5e86ba","order_by":1,"name":"Vinaya Venkat","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Vinaya","middleName":"","lastName":"Venkat","suffix":""},{"id":442754097,"identity":"c2d6935a-ef14-4cab-8144-90ff1f44599a","order_by":2,"name":"Jenni Virtanen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Jenni","middleName":"","lastName":"Virtanen","suffix":""},{"id":442754098,"identity":"186d023b-1f88-4565-a81c-7eac98af1e42","order_by":3,"name":"Kristel Kegler","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Kristel","middleName":"","lastName":"Kegler","suffix":""},{"id":442754099,"identity":"b9c5f6d9-850b-4a88-bd23-f45503ecde94","order_by":4,"name":"Thanakorn Niamsap","email":"","orcid":"","institution":"University of 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Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Atanasova","suffix":""},{"id":442754103,"identity":"e384f1e5-e0a8-4f2a-9981-ff11ca2b9285","order_by":8,"name":"Pamela Österlund","email":"","orcid":"","institution":"Finnish Institute for Health and Welfare","correspondingAuthor":false,"prefix":"","firstName":"Pamela","middleName":"","lastName":"Österlund","suffix":""},{"id":442754104,"identity":"1ce780a0-d995-4a54-89eb-791a633e42d4","order_by":9,"name":"Teemu Smura","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Teemu","middleName":"","lastName":"Smura","suffix":""},{"id":442754105,"identity":"27dc3de3-b66c-421d-877c-aa5b54857400","order_by":10,"name":"Antti Sukura","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Antti","middleName":"","lastName":"Sukura","suffix":""},{"id":442754106,"identity":"dfa4e39f-384f-4425-a6bb-f5cac0ee8382","order_by":11,"name":"Lara Dutra","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"","lastName":"Dutra","suffix":""},{"id":442754107,"identity":"50541e57-ce95-47b3-a3e0-3dc57eaa16f4","order_by":12,"name":"Olli Vapalahti","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Olli","middleName":"","lastName":"Vapalahti","suffix":""},{"id":442754108,"identity":"d6b18356-7347-400f-a683-fd5e980f6744","order_by":13,"name":"Heli Nordgren","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Heli","middleName":"","lastName":"Nordgren","suffix":""},{"id":442754109,"identity":"4c3ec7dd-bb3e-4343-8256-8f0722221e17","order_by":14,"name":"Ravi Kant","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Ravi","middleName":"","lastName":"Kant","suffix":""},{"id":442754111,"identity":"7007aae0-2eb1-469f-93bf-29dd6bd5ff72","order_by":15,"name":"Tarja Sironen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Tarja","middleName":"","lastName":"Sironen","suffix":""},{"id":442754112,"identity":"37ae9af5-860e-4aa6-9244-5cc30c66a943","order_by":16,"name":"Kirsi Aaltonen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Kirsi","middleName":"","lastName":"Aaltonen","suffix":""}],"badges":[],"createdAt":"2025-02-07 10:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5980382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5980382/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08111-1","type":"published","date":"2025-07-01T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80795387,"identity":"19d2c632-695c-4b1e-87f2-2e6157c2c3af","added_by":"auto","created_at":"2025-04-17 07:34:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":486999,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup for male (A) and female (B) groups in the BSL3 facility. Metal bar enclosures were placed 10-20 cm apart on a table and the BioSamplers were placed 30 cm from the enclosures, at the height of the animals, to collect air samples from the area surrounding the mink. The samplers’ inlets were faced towards the table the enclosures were on. Inoculated animals are marked with virus illustrations and naïve recipients are as indicated in the figure. Figure created in Biorender (https://BioRender.com).\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5980382/v1/8b8dd98eeef8dde5cb4ec2f9.jpeg"},{"id":80796748,"identity":"cd4b1a7e-30c3-4623-bd9b-1a8727125cdf","added_by":"auto","created_at":"2025-04-17 07:42:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443191,"visible":true,"origin":"","legend":"\u003cp\u003ePositive\u003cstrong\u003e \u003c/strong\u003ecell culture and PCR findings. Infectious isolates represent cultivated virus samples, while viral RNA represents all viruses, infectious and inactive. The thickness of the graph describes the relative number of SARS-CoV-2 positive samples on each day post infection (dpi). Individual positive samples are represented with dots on the graphs. Positive sample medians are illustrated with full lines and quartiles with dashed lines. Negative or weakly positive samplesare not shown in the graph, but they are \u0026nbsp;presentedwith all other sample data in Supplement Tables 1-2. On day four, 10 surfaces were sampled instead of four (Supplement Tables 1-2). Graphs created with GraphPad Prism 10.2.1.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5980382/v1/4c0455e5d192fc7f40d2f0ee.jpg"},{"id":86179817,"identity":"6e314389-1645-4a24-b2cd-21a345bc0e64","added_by":"auto","created_at":"2025-07-07 16:19:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1574335,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5980382/v1/75fa4572-5901-41bd-a9fc-56f2324597fc.pdf"},{"id":80796746,"identity":"1b7f6e4a-d0f5-4c51-b463-0c9f857cd939","added_by":"auto","created_at":"2025-04-17 07:42:40","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16204,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementtables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5980382/v1/e7b76ee0045d3f642391b7d4.xlsx"},{"id":80795386,"identity":"e74fc7f9-ee13-4310-b3a0-48e8eac4cea0","added_by":"auto","created_at":"2025-04-17 07:34:40","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":47077,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-5980382/v1/8efed1b74e6b53f531ccf808.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SARS-CoV-2 bioaerosol transmission in experimentally infected American mink","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSARS-CoV-2, a single-stranded RNA virus and the causative agent of the COVID-19 pandemic, is known to transmit among humans through the air via aerosols and droplets, and contaminated surfaces [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Since the emergence of the pandemic, the virus has been able to mutate and develop variants such as the Alpha, Beta and Delta variants. In this study, we focused on the more transmissible variant of SARS-CoV-2, BA.1 Omicron [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which has shown milder symptoms in humans [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides humans, SARS-CoV-2 has been shown to infect various animals, including American mink (\u003cem\u003eNeovison vison\u003c/em\u003e) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. American mink, though solitary in the wild, are co-housed in large numbers at fur farms, increasing the risk of virus transmission among the animals [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Such an environment serves as a reservoir for viral transmission [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and the accumulation of mutations, increasing the possibility of animal-to-human transmission [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to the open design of these farms, stray and wild animals, as well as birds, may encounter mink and their excreta, potentially carrying infections outside the farm [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCases of human-to-mink and mink-to-human transmission of SARS-CoV-2 have been documented [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, as a biosafety measure, most infected farms resorted to mass culling of millions of animals [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To prevent future crises, a thorough investigation on the sources and transmission of virus infections needs to be conducted to help inform the measures taken during future outbreaks.\u003c/p\u003e \u003cp\u003eSome studies have found SARS-CoV-2 on the surfaces of mink farms using PCR and animal bedding, but the presence of infectious viruses have not been detected [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, SARS-CoV-2 is known to be transmitted via aerosols among humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The virus was found to be viable in aerosols for up to 3 hours in laboratory conditions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] but detecting viable viruses from air samples in clinical and environmental settings has proven difficult [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Likely, this difficulty arises from the long delays between sample collection and culturing, as samples are often transported to distant laboratories for analysis.\u003c/p\u003e \u003cp\u003eThis manuscript is a part of multiple publications on a study of American mink experimentally infected with SARS-CoV-2. In a previous publication, we proved that the Omicron variant, despite being known for mild infections in humans, can also infect mink [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this manuscript, we focused on consistently capturing infectious viruses in aerosols collected around the experimentally infected mink, to provide evidence of aerosol transmission of SARS-CoV-2 at fur farms.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVirus stock, cells lines and cell media\u003c/h2\u003e \u003cp\u003eVirus stocks, cell lines, and cell media were used as previously published in Virtanen et al. (2022) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. SARS-CoV-2 ba.1 was acquired from the Finnish Institute of Health and Welfare ((original patient sample: hCoV-19/Finland/THL-202126660/2021, EPI_ISL_8768822 (Gisaid)). TMPRSS2-expressing VeroE6 (VE6T) cells were used for virus cultivation and were grown according to Rusanen et al. (2021) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental setup, animal infection and euthanasia\u003c/h3\u003e\n\u003cp\u003eThe mink were purchased from a commercial mink farm. Animals were acclimatized to the BSL3-laboratory room and custom-made enclosures for three days before infection. The enclosures were 84x76x58.4 cm metal bar cages, 10\u0026ndash;20 cm apart, with a 26.2x31.5x40 cm closed metal nest with an open bar cage roof, containing hay as nesting material. The laboratory room was 59 m\u003csup\u003e2\u003c/sup\u003e with ventilation flowing from left to right (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at a rate of approximately 242 l/s. Airflow in the room was turbulent or partially turbulent and could flow through animal enclosures. Animal waste was collected under the enclosures on a removable metal tray. Refer to animal enclosure setup in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eBefore infection, animals were anesthetized using 30 \u0026micro;l of Ketaminol (100 mg/ml, Intervet, Netherlands) and Domitor (1 mg/ml, Orion Pharma, Finland). Three male and two female mink were nasally infected with 200 \u0026micro;l of SARS-CoV-2 ba.1 stock (pfu\u0026thinsp;=\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e), as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Na\u0026iuml;ve recipient mink received PBS instead of the virus stock. Sedation was reversed using Revertor (5 mg/ml, Scanvet, Poland). One female mink had to be excluded from the study due to stress, resulting in only four animals in the female group. The nasally infected animals were euthanized in a CO\u003csub\u003e2\u003c/sub\u003e chamher on 7 dpi (days post infection), while the recipient males and females were euthanized at 10 and 11 dpi respectively. The experimental infection, sedation and euthanasia of the American mink are described in more detail in Virtanen et al. (2022) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperiment 1 consisted of five males while experiment 2 consisted of four females. The experiments were conducted successively. Reversing of the sedation of the animals indicated the starting point of the experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eThree air samples were collected simultaneously twice a day, before and after feeding, for 30 min using three 5 ml BioSamplers (SKC Inc., USA) positioned 30 cm from the enclosures, at the height of the animals. The samplers\u0026rsquo; inlets faced the table the enclosures were on (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All samplers were operated with a single air pump, with individual airflows monitored using Mass Flowmeter 3063-devices (TSI Inc., USA). BioSamplers were thoroughly disinfected and washed with ethanol and MilliQ-water between collections. Aerosols were collected into 5 ml of minimal essential eagle\u0026rsquo;s medium (MEM, Sigma-Aldrich, USA) using a flow rate of 12.5 LPM. Amphotericin B (Gibco, USA) was added to MEM for the female group after some samples from the male group had fungal contaminations. Due to dehydration, aerosol samples were refilled with fresh media to 3 ml before analysis.\u003c/p\u003e \u003cp\u003eAll animal and surface sample collection was done using Sigma Virocult\u0026reg;-swabs (MWE, UK). Saliva was collected as animals chewed on the swab, fecal samples collected from swabbing the feces, and surface samples collected using MEM dipped swabs swabbing a comprehensive surface area. Samples were collected in duplicates and post sampling the swabs were stored in 1ml of MEM awaiting further processing.\u003c/p\u003e \u003cp\u003eOne of the saliva and surface duplicates were transferred to cells for virus cultivation while the others were stored at -80\u0026deg;C until PCR analysis.\u003c/p\u003e\n\u003ch3\u003eVirus cultivation\u003c/h3\u003e\n\u003cp\u003e1 ml of samples were mixed with 2 ml of culture media and added to VE6T-cells on 6-well plates. Cells were incubated in 37\u0026deg;C for 9 days or until cytopathic effect (CPE) could be detected. A 140 \u0026micro;l sample was taken from wells with CPE for RNA extraction to confirm SARS-CoV-2 as the causing agent. Extracted RNA was analyzed using PCR.\u003c/p\u003e\n\u003ch3\u003ePCR\u003c/h3\u003e\n\u003cp\u003eSaliva and surface samples were extracted using the QIAamp Viral RNA Mini Kit (QIAGEN), while fecal and cell culture samples were extracted using QIAamp 96 Virus QIAcube HT kit (QIAGEN, off-board lysis). All samples were PCR tested for SARS-CoV-2 using Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit (NEB), targeting 2019-nCoV_N1 and 2019-nCoV_N2. Samples were positive (+) if they gave a signal with both probes, weak positive ((+)) if they only gave a signal with one probe, and negative (-) if they gave no signal. Cell culture samples were considered positive (+) if their Ct values were more than 5 cycles lower than the original non cell-cultured sample, possibly positive ((+)) if their Ct values were 1\u0026ndash;5 cycles lower, and negative (-) if the Ct values were similar or higher.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e Experimental procedures were approved by the Animal Experimental Board of Finland (ESAVI/33259) and carried out accordingly. This study is performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 Omicron transmission among mink\u003c/h2\u003e \u003cp\u003eInfectious viruses were detected from aerosol samples in both experiments. For the males, infectious viruses were detected mostly on the first 3 days post infection (dpi) while in the female group they were detected later, mostly on 5\u0026ndash;7 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCR positive aerosol samples were collected throughout the experiment from both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Most sampled surfaces tested PCR positive, however, infectious viruses were recovered only from one sample (Supplement Tables\u0026nbsp;1\u0026ndash;2). A few infectious samples were collected from animal saliva in both groups, however, most samples were culture negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the male group, infectious viruses were detected in the saliva of infected animals only during 1\u0026ndash;3 dpi, and in the recipient mink\u0026rsquo;s saliva already on 1 dpi. In the female group infectious viruses were observed on 1 and 7 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All saliva samples tested PCR positive for the infected mink in both groups. The male recipient mink saliva was consistently PCR positive from 3 dpi onwards, while only one of the female recipients was (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCR tests from infected mink feces were variably positive for both groups, however, more so in the male group. In the recipient mink feces, PCR positive results were observed only in the male group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies conducted in ferrets [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and hamsters [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] show the production of infectious SARS-CoV-2 containing aerosols, but this has not been demonstrated in American mink (\u003cem\u003eNeovison vison\u003c/em\u003e). Our study bridges that gap, as we show that American mink infected with SARS-CoV-2 BA.1 (Omicron) variant can transmit viral aerosols from infected to healthy animals in a laboratory setting. We were able to come up with better aerosol sampling methods to capture these live viruses, which were not possible in previous studies. In our work, infectious viruses were detected primarily in aerosol samples, indicating that virus transmission among animals is likely to occur when animal enclosures are in close proximity.\u003c/p\u003e \u003cp\u003eMost culture positive samples were obtained with aerosol collectors, suggesting that aerosol transmission is the main mode of transmission for the Omicron variant when animals are in separate enclosures. However, culture positive surface- and saliva samples were also detected, making surface and contact transmission also possible routes for infection.\u003c/p\u003e \u003cp\u003ePrevious studies on viral bioaerosol transmission have had difficulties in detecting infectious viruses from bioaerosols [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] or have only focused on RNA detection [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, while filter collectors have been the most used device for SARS-CoV-2 air sampling [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], liquid-based collectors have been found to retain virus infectivity much better [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Here, we successfully detected infectious viruses in multiple bioaerosol samples by using 5 ml BioSamplers and culturing the samples immediately after collection.\u003c/p\u003e \u003cp\u003eA pattern was observed in the aerosol sample results, where both groups of mink had a three-day period when most of the culture positive samples were collected. Interestingly, this period happened in the first 1\u0026ndash;3 dpi in the male group and later at 5\u0026ndash;8 dpi in the female group. It is likely that the recipient male mink were infected already on 1 dpi, resulting in an earlier peak in culture positive aerosol samples. Additionally, SARS-CoV-2 has been reported to have more severe symptoms in males [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which could be a reason for earlier infection and detection of virus-containing aerosols. However, as this experiment only consisted of two groups of animals, more studies are needed to confirm these differences. Still, the three-day period observed here is much shorter than reported in humans [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, as not all culture positive samples were PCR positive from the original sample, the virus concentrations in aerosols were likely low. A previous study has also shown that viral loads on surfaces and air can be quite low [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile the SARS-CoV-2 Omicron variant has been shown to survive on surfaces for multiple days [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we observed infectious viruses in only one surface sample. By contrast, most surface samples were PCR positive, indicating high environmental contamination, however the longevity of virus viability appears to be brief. Additionally, it is important to acknowledge that the initial titer of the virus affects its survival [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, viruses originating from aerosols or droplets might not survive as long on surfaces compared to \u003cem\u003ein vitro\u003c/em\u003e experiments in which higher titers of infectious particles are used.\u003c/p\u003e \u003cp\u003eSARS-CoV-2 was also variably found in the feces of both infected and recipient mink with PCR. Previous studies have suggested that infected mink feces could spread SARS-CoV-2 to other wildlife and farm animals, such as cats [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], foxes, and other animals [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], if accessible. Although birds have not been observed to be susceptible to SARS-CoV-2 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], they could still transport the virus from the feces to other housing units or even other farms. As the animals often defecate through their enclosures onto the ground below, it is accessible by wild animals. This area should be restricted to prevent the spreading of viruses. Additionally, dust in mink sheds has previously been shown to contain SARS-CoV-2 RNA [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], providing another possible route for the viruses to spread at the farms and outside them. Viral RNA has also been detected in outside air of recently infected mink farms, however only near the entrance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, antibodies against SARS-CoV-2 have also been found in escaped mink around mink farms [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], though the infections could have occurred before escaping.\u003c/p\u003e \u003cp\u003eOverall, more PCR positive samples were collected during the male group (63% vs. 45%, Supplement tables 1\u0026ndash;2), suggesting that male mink have higher virus shedding than female mink. However, this difference was not observed in viable virus detections (13% vs. 15%, Supplement tables 1\u0026ndash;2). Similar findings regarding viral RNA shedding have been made in humans [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], however, more studies are needed with better infectious virus detection methods to understand whether there is also a difference in infectious virus shedding between males and females.\u003c/p\u003e \u003cp\u003eOur study demonstrated that American mink infected with the SARS-CoV-2 BA.1 variant produce aerosols containing infectious virus particles, capable of spreading the virus to nearby animals. We also found that culturing aerosol samples immediately after collection significantly improves the detection of viable viruses, making diagnostics for both infected animals and humans more accurate. To prevent animal suffering and avoid mink farms becoming reservoirs for respiratory viruses, enhanced preventive measures and surveillance are essential. However, these measures can only be effective if the various modes of transmission, including aerosol spread, are thoroughly researched and understood.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jari Elemo, Mari Elemo, and other animal caretakers for handling the animals and assessing their health, and Esa Pohjalainen, Sanna M\u0026auml;ki, Tiina Sihvonen, Johanna Rintam\u0026auml;ki, Hanna Valtonen, Marika Sk\u0026ouml;n, Larissa Laine,and Elina V\u0026auml;is\u0026auml;nen for technical assistance. We thank Kati Kuipers, Anne Kujanp\u0026auml;\u0026auml;, Laura V\u0026auml;h\u0026auml;l\u0026auml;, and the Finnish Centre for Laboratory Animal Pathology (FCLAP) for expert technical help, as well as Johanna Korpela and Jussi Peura from Finnish Fur Breeders Association and Jan Segervall and Maarit Mohaibes from the Kannus Research Farm Luova Ltd. for providing the animals. We also thank E3 Excellence in Pandemic Response and Enterprise Solutions co-innovation project and all its parties.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental procedures were approved by the Animal Experimental Board of Finland (ESAVI/33259).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Academy of Finland (grant no. 336490, 339510), VEO\u0026ndash;European Union\u0026rsquo;s Horizon 2020 (grant no. 874735), Business Finland E3 (4917/31/2021), Finnish Institute for Health and Welfare, and the Jane and Aatos Erkko Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003cbr\u003e\u003c/strong\u003eWe report no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on a reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSantarpia, J. 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Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: Retrospective cohort study. \u003cem\u003eBMJ\u003c/em\u003e, \u003cem\u003e369\u003c/em\u003e, m1443. https://doi.org/10.1136/bmj.m1443.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShastri, A. et al. \u003cem\u003eDelayed clearance of SARS-CoV2 in male compared to female patients: High ACE2 expression in testes suggests possible existence of gender-specific viral reservoirs\u003c/em\u003e (s. 2020.04.16.20060566). medRxiv. (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2020.04.16.20060566\u003c/span\u003e\u003cspan address=\"10.1101/2020.04.16.20060566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5980382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5980382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe SARS-CoV-2 BA.1 (Omicron) variant, which emerged in late 2021, is more transmissible than earlier variants but causes milder symptoms in humans. Mink farms, where animals are housed in close quarters, present a high risk for virus transmission and mutation, necessitating strict control measures due to documented cases of mink-to-human and human-to-mink transmission. Hence, we aimed to detect viable airborne SARS-CoV-2 using BioSampler-air collectors and to investigate aerosol transmission between groups of American mink (\u003cem\u003eNeovison vison\u003c/em\u003e). Two groups (male and female) were infected with the BA.1 variant, and samples were collected from aerosols, saliva, feces, and surfaces. The results indicated that infectious viruses were predominantly detected in aerosol samples over a three-day period in both groups. Surface, saliva, and fecal samples also showed potential for virus transmission. Notably, infectious viruses were cultivated from aerosol samples, confirming aerosol transmission among American mink. This study highlights the importance of immediate sample culturing to improve infectious virus detection and emphasizes the need for enhanced preventive measures on mink farms to mitigate the spread of viruses.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 bioaerosol transmission in experimentally infected American mink","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 07:34:36","doi":"10.21203/rs.3.rs-5980382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-14T04:17:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-10T04:31:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-21T15:57:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163674673896891890841926923164675390374","date":"2025-04-14T13:49:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140184842848969913763662264618384164407","date":"2025-04-14T13:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-14T12:58:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-12T09:07:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-01T11:43:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48fe53a7-c446-4a3e-b899-20217ffaf963","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47134865,"name":"Biological sciences/Microbiology/Virology/Sars cov 2"},{"id":47134866,"name":"Biological sciences/Microbiology/Virology/Viral transmission"}],"tags":[],"updatedAt":"2025-07-07T16:11:42+00:00","versionOfRecord":{"articleIdentity":"rs-5980382","link":"https://doi.org/10.1038/s41598-025-08111-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:58:03","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-17 07:34:36","video":"","vorDoi":"10.1038/s41598-025-08111-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08111-1","workflowStages":[]},"version":"v1","identity":"rs-5980382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5980382","identity":"rs-5980382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}