Tissue tropism of bovine coronavirus may not be determined by viral genes

Tissue tropism of bovine coronavirus may not be determined by viral genes | 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 Short Report Tissue tropism of bovine coronavirus may not be determined by viral genes Keisuke Nakagawa, Aimi Yoshida, Keisuke Kuwata, Yuko Kitamura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8922291/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Bovine coronavirus (BCoV) causes respiratory and enteric diseases in cattle; however, the determinants of its tissue tropism remain unclear. In this study, we identified identical BCoV whole-genome sequences in tracheal and colonic samples obtained from a single adult cow that died from winter dysentery. After five passages in HRT-18 cells, virus isolation was successfully achieved only from the tracheal sample. Several amino acid substitutions were detected exclusively after isolation. These findings suggest that BCoV tissue tropism is not determined solely by viral genome sequences. It is important to analyze viral genomes directly from clinical samples prior to virus isolation. Figures Figure 1 Introduction Bovine coronavirus (BCoV) is an important pathogen of cattle that causes both respiratory and enteric diseases [ 1 ]. Infected animals shed virus in nasal secretions and feces, respectively. BCoV is associated with three major clinical syndromes: calf diarrhea; winter dysentery, characterized by hemorrhagic diarrhea, in adult cattle; and respiratory infections. BCoV is a viral pathogen that contributes to the bovine respiratory disease complex (BRDC). BRDC results in substantial economic losses to the cattle industry worldwide [ 2 ]; thus, it is important to understand the transmission routes and tissue tropism of its causative agents. BCoV is an enveloped, positive-sense single-stranded RNA virus belonging to the genus Betacoronavirus , subgenus Embecovirus [ 3 ]. The viral genome encodes several structural proteins, including spike (S), hemagglutinin esterase (HE), membrane (M), envelope (E), and nucleocapsid (N) proteins, as well as multiple non-structural proteins involved in viral replication and transcription [ 1 ]. Numerous molecular epidemiological studies have attempted to identify viral genetic determinants associated with respiratory or enteric tropism of BCoV [ 3 – 7 ]. However, the results remain controversial, and no consistent genetic markers that distinguish respiratory and enteric strains have been established [ 8 ]. A major limitation of previous studies is that viral genomes were typically compared between different animals, making it difficult to exclude host-related confounding factors. In the present study, we directly compared BCoV genomes detected in respiratory and intestinal tissues obtained from the same animal and examined the effects of virus isolation on viral genome sequences to investigate the relationship between viral genetics, tissue tropism, and cell culture adaptation. Materials and methods Sample collection In February 2022, a 2-year-old adult cow at a farm in Gifu Prefecture, Japan, died from winter dysentery. The animal was necropsied at the Gifu Prefectural Central Livestock Hygiene Service Center. At this time, a tracheal swab and diarrheic intestinal content (colonic sample) were collected. All samples were stored at − 80°C until further analysis. Virus isolation HRT-18 cells (human rectal adenocarcinoma cell line) were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. Ten percent (w/v) homogenates of tracheal swab and diarrheic samples in phosphate-buffered saline were inoculated onto HRT-18 cells. After incubation for 4–5 days, culture supernatants were collected and passaged onto fresh cells. Blind passages were performed a total of five times. Immunofluorescence assay Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated with a rabbit anti-BCoV N protein peptide antibody, followed by Alexa Fluor-conjugated secondary antibodies. Fluorescence signals were visualized using a KEYENCE BZ-X800 fluorescence microscope. Western blot analysis Western blot analysis HRT-18 cells infected with BCoV at a multiplicity of infection of 1 were lysed at 24 h post-infection and subjected to SDS-PAGE. BCoV N protein was detected using the same anti-BCoV N protein peptide antibody and horseradish peroxidase-conjugated secondary antibodies. Whole-genome sequencing and BLAST analysis Viral RNA was extracted from clinical samples and virus isolates, and complementary DNA was synthesized using random primers. Whole-genome sequencing was performed using next-generation sequencing combined with 5′- and 3′-RACE methods, as described previously [ 9 ]. BLAST analysis was performed using the NCBI database to assess nucleotide sequence identity against previously reported BCoV strains. Results In a previous study, we determined the whole-genome sequence of BCoV detected in diarrheic samples obtained from an adult cow that died from winter dysentery [ 9 ]. In the present study, we determined the whole-genome sequence of BCoV from a tracheal swab collected from the same animal. Comparison of the sequences revealed that the BCoV genomes detected in tracheal and colonic samples were completely identical (Fig. 1 a). BLAST analysis demonstrated that the whole-genome sequence of this virus shared more than 99% nucleotide identity with previously reported BCoV strains (accession number, LC494186.1) detected in Japan, indicating that the virus analyzed in this study does not represent a genetically unusual or highly divergent variant. Virus isolation was subsequently attempted from both tracheal and colonic samples. Infectious BCoV was successfully isolated only from the tracheal sample after five passages in HRT-18 cells, whereas virus isolation from the colonic sample was unsuccessful. Productive infection of HRT-18 cells was confirmed by immunofluorescence staining and western blot analysis targeting the BCoV N protein (Fig. 1 b). Whole-genome sequencing of the isolated virus revealed one synonymous substitution in the S gene and several non-synonymous substitutions in the pp1b, HE, and S genes compared with the viral genomes detected directly from the clinical samples. Notably, all amino acid substitutions were detected only after virus isolation, suggesting that these mutations arose during cell culture adaptation (Fig. 1 a). Discussion In this study, we demonstrated that identical bovine coronavirus (BCoV) genomes were present in both respiratory and intestinal tissues of a single cow. This direct comparison within the same host provides evidence that viral genetic differences are not required to explain BCoV tissue distribution, indicating that tissue tropism is not determined solely by viral genome sequences. Consistent with this conclusion, BLAST analysis confirmed that the virus examined in this study is genetically close to previously reported Japanese BCoV strains, sharing more than 99% nucleotide identity at the whole-genome level. This finding indicates that the virus analyzed here does not represent a genetically unusual or divergent lineage. Therefore, the observed tissue distribution of BCoV cannot be attributed to infection with a distinct or atypical viral strain. We found that virus isolation resulted in the accumulation of amino acid substitutions in nsp12, HE, and S proteins, all of which were absent in viral genomes detected directly from clinical samples. These findings are consistent with a previous study by Zhang et al. [ 4 ], who demonstrated that during cell culture adaptation, an enteric BCoV strain accumulated mutations that rendered it genetically similar to a corresponding respiratory BCoV isolate obtained from the same animal. In the present study, several mutations identified after serial passages in HRT-18 cells closely corresponded to those reported by Zhang et al. as culture-adaptive changes. Notably, the two HE substitutions (V103I and N104D) were identical to the AH65-E → AH65-E-TC mutations described in their study. Similarly, the S protein substitutions (D28N, Q179K, and E965D) were located within regions where mutation accumulation was observed during in vitro adaptation. In addition, the ORF1ab substitutions (M4891I and N4894Y) were detected within the same genomic region (nt 14600–15000) previously reported to harbor clusters of adaptive mutations. Collectively, these observations strongly suggest that the mutations identified in our isolates represent cell culture-associated adaptation events rather than genetic determinants of tissue tropism in vivo. The amino acid substitutions detected in nsp12, HE, and S proteins during BCoV isolation are therefore likely to reflect viral adaptation to replication in HRT-18 cells. The two nsp12 substitutions (M4891I and N4894Y) are located within the thumb domain of the RNA-dependent RNA polymerase [ 10 ] and may influence local conformational flexibility or interactions with replication cofactors. The HE substitutions (V103I and N104D) occur near regions implicated in receptor binding and esterase activity [ 11 ] and may modify surface charge or receptor interactions in human-derived cells. Similarly, the S protein substitutions (D28N, Q179K, and E965D) affect residues within the S1 N-terminal domain and S2 region, which are involved in receptor recognition and membrane fusion [ 12 ]. Together, these changes may contribute to coordinated adaptation of viral entry, replication, and release processes, thereby facilitating more efficient propagation of BCoV in vitro. Although this study is based on a single case, the direct comparison of viral genomes derived from different tissues within the same animal minimizes host-related confounding factors. However, our findings do not exclude the possibility that viral genes influence tissue-specific pathogenicity under certain conditions. The recent development of reverse genetics systems for BCoV will enable future studies using genetically homogeneous viruses to more definitively address the contribution of viral genetic factors to tissue tropism and pathogenicity [ 13 ]. In this context, continuous epidemiological surveillance combined with careful genomic analysis of clinical samples will be essential for interpreting and validating such experimental findings. Declarations Author contributions Keisuke Nakagawa: Conceptualization,methodology, analysis, wrote the first draft, reviewed and edited final draft. Aimi Yoshida: Methodology, reviewed and edited final draft. Keisuke Kuwata: Resources, reviewed and edited final draft. Yuko Kitamura: Resources, reviewed and edited final draft. Funding This research was funded by research grants from Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (number 19K23706 and 23K05552) and a grant from the Mishima Kaiun Memorial Foundation. Data availability The complete genome sequences of strain GF2020 obtained from tracheal swab sample and isolate have been deposited in GenBank under accession numbers LC918946 and LC918947, respectively. The corresponding raw sequence data are available in the Sequence Read Archive (SRA) under accession numbers DRX326681 (BioSample SAMD00414418) Conflicts of Interest: The authors declare no conflict of interest. References Saif LJ (2010) Bovine respiratory coronavirus. Veterinary Clinics of North America - Food Animal Practice 26 Griffin D (1997) Economic impact associated with respiratory disease in beef cattle. Vet Clin North Am Food Anim Pract. https://doi.org/10.1016/S0749-0720(15)30302-9 . 13: Gorbalenya AE, Krupovic M, Mushegian A et al (2020) The new scope of virus taxonomy: partitioning the virosphere into 15 hierarchical ranks. Nat Microbiol 5 Zhang X, Hasoksuz M, Spiro D et al (2007) Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation. Virology 363. https://doi.org/10.1016/j.virol.2007.03.018 Hasoksuz M, Kayar A, Dodurka T, Ilgaz A (2005) Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in northwestern Turkey. Acta Vet Hung 53. https://doi.org/10.1556/AVet.53.2005.1.13 Hasoksuz M, Hoet AE, Loerch SC et al (2002) Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an Ohio feedlot. J Vet Diagn Invest 14. https://doi.org/10.1177/104063870201400406 Kanno T, Hatama S, Ishihara R, Uchida I (2007) Molecular analysis of the S glycoprotein gene of bovine coronaviruses isolated in Japan from 1999 to 2006. J Gen Virol 88. https://doi.org/10.1099/vir.0.82635-0 Suzuki T, Otake Y, Uchimoto S et al (2020) Genomic characterization and phylogenetic classification of bovine coronaviruses through whole genome sequence analysis. Viruses 12. https://doi.org/10.3390/v12020183 Nakagawa K, Kumano H, Kitamura Y et al (2021) Complete Genome Sequence of Bovine Coronavirus in Blood Diarrhea from Adult Cattle That Died from Winter Dysentery in Japan. Microbiol Resour Announc 10. https://doi.org/10.1128/mra.00807-21 te Velthuis AJW, Arnold JJ, Cameron CE et al (2009) The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent. Nucleic Acids Res 38. https://doi.org/10.1093/nar/gkp904 Zeng Q, Langereis MA, Van Vliet ALW et al (2008) Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution. Proc Natl Acad Sci U S A 105. https://doi.org/10.1073/pnas.0800502105 Belouzard S, Millet JK, Licitra BN, Whittaker GR (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4 Sugiura Y, Takahashi T, Ueno S et al (2025) Expression of spike and hemagglutinin-esterase proteins is necessary to recover infectious recombinant bovine coronavirus. J Virol 99. https://doi.org/10.1128/jvi.01027-25 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 20 Feb, 2026 Submission checks completed at journal 20 Feb, 2026 First submitted to journal 19 Feb, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8922291","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":597622188,"identity":"5896a077-7579-4ef2-9984-cd9d129708b3","order_by":0,"name":"Keisuke Nakagawa","email":"data:image/png;base64,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","orcid":"","institution":"Gifu University","correspondingAuthor":true,"prefix":"","firstName":"Keisuke","middleName":"","lastName":"Nakagawa","suffix":""},{"id":597622189,"identity":"a7a1d022-0852-4ad4-a690-4c22f526bc4e","order_by":1,"name":"Aimi Yoshida","email":"","orcid":"","institution":"Gifu University","correspondingAuthor":false,"prefix":"","firstName":"Aimi","middleName":"","lastName":"Yoshida","suffix":""},{"id":597622191,"identity":"58332aa7-3656-4d18-a9e3-b216e05d8e3a","order_by":2,"name":"Keisuke Kuwata","email":"","orcid":"","institution":"Gifu Prefectural Chuo Livestock Hygiene Service Center","correspondingAuthor":false,"prefix":"","firstName":"Keisuke","middleName":"","lastName":"Kuwata","suffix":""},{"id":597622192,"identity":"d4959b9e-53b9-426e-a317-e2c82a1d8eda","order_by":3,"name":"Yuko Kitamura","email":"","orcid":"","institution":"Gifu Prefectural Chuo Livestock Hygiene Service Center","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Kitamura","suffix":""}],"badges":[],"createdAt":"2026-02-20 04:39:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8922291/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8922291/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103613700,"identity":"631e4258-52d3-4095-b4f7-cb1f50fca413","added_by":"auto","created_at":"2026-02-27 16:21:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1037687,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8922291/v1/5d9e5af2c26fe472d21b3ed1.png"},{"id":103613703,"identity":"3ed759a1-6eaf-48b9-aac0-d99308e4154c","added_by":"auto","created_at":"2026-02-27 16:21:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1386795,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8922291/v1/8ca937eb-971c-4842-aba0-954dfeb7bc03.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tissue tropism of bovine coronavirus may not be determined by viral genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine coronavirus (BCoV) is an important pathogen of cattle that causes both respiratory and enteric diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Infected animals shed virus in nasal secretions and feces, respectively. BCoV is associated with three major clinical syndromes: calf diarrhea; winter dysentery, characterized by hemorrhagic diarrhea, in adult cattle; and respiratory infections. BCoV is a viral pathogen that contributes to the bovine respiratory disease complex (BRDC). BRDC results in substantial economic losses to the cattle industry worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]; thus, it is important to understand the transmission routes and tissue tropism of its causative agents.\u003c/p\u003e \u003cp\u003eBCoV is an enveloped, positive-sense single-stranded RNA virus belonging to the genus \u003cem\u003eBetacoronavirus\u003c/em\u003e, subgenus \u003cem\u003eEmbecovirus\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The viral genome encodes several structural proteins, including spike (S), hemagglutinin esterase (HE), membrane (M), envelope (E), and nucleocapsid (N) proteins, as well as multiple non-structural proteins involved in viral replication and transcription [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous molecular epidemiological studies have attempted to identify viral genetic determinants associated with respiratory or enteric tropism of BCoV [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the results remain controversial, and no consistent genetic markers that distinguish respiratory and enteric strains have been established [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA major limitation of previous studies is that viral genomes were typically compared between different animals, making it difficult to exclude host-related confounding factors. In the present study, we directly compared BCoV genomes detected in respiratory and intestinal tissues obtained from the same animal and examined the effects of virus isolation on viral genome sequences to investigate the relationship between viral genetics, tissue tropism, and cell culture adaptation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eIn February 2022, a 2-year-old adult cow at a farm in Gifu Prefecture, Japan, died from winter dysentery. The animal was necropsied at the Gifu Prefectural Central Livestock Hygiene Service Center. At this time, a tracheal swab and diarrheic intestinal content (colonic sample) were collected. All samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVirus isolation\u003c/h3\u003e\n\u003cp\u003eHRT-18 cells (human rectal adenocarcinoma cell line) were maintained in Dulbecco\u0026rsquo;s modified Eagle medium supplemented with 10% fetal bovine serum. Ten percent (w/v) homogenates of tracheal swab and diarrheic samples in phosphate-buffered saline were inoculated onto HRT-18 cells. After incubation for 4\u0026ndash;5 days, culture supernatants were collected and passaged onto fresh cells. Blind passages were performed a total of five times.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence assay\u003c/h3\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated with a rabbit anti-BCoV N protein peptide antibody, followed by Alexa Fluor-conjugated secondary antibodies. Fluorescence signals were visualized using a KEYENCE BZ-X800 fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot analysis\u003c/div\u003e \u003cp\u003eHRT-18 cells infected with BCoV at a multiplicity of infection of 1 were lysed at 24 h post-infection and subjected to SDS-PAGE. BCoV N protein was detected using the same anti-BCoV N protein peptide antibody and horseradish peroxidase-conjugated secondary antibodies.\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing and BLAST analysis\u003c/h3\u003e\n\u003cp\u003eViral RNA was extracted from clinical samples and virus isolates, and complementary DNA was synthesized using random primers. Whole-genome sequencing was performed using next-generation sequencing combined with 5\u0026prime;- and 3\u0026prime;-RACE methods, as described previously [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. BLAST analysis was performed using the NCBI database to assess nucleotide sequence identity against previously reported BCoV strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn a previous study, we determined the whole-genome sequence of BCoV detected in diarrheic samples obtained from an adult cow that died from winter dysentery [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the present study, we determined the whole-genome sequence of BCoV from a tracheal swab collected from the same animal. Comparison of the sequences revealed that the BCoV genomes detected in tracheal and colonic samples were completely identical (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). BLAST analysis demonstrated that the whole-genome sequence of this virus shared more than 99% nucleotide identity with previously reported BCoV strains (accession number, LC494186.1) detected in Japan, indicating that the virus analyzed in this study does not represent a genetically unusual or highly divergent variant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVirus isolation was subsequently attempted from both tracheal and colonic samples. Infectious BCoV was successfully isolated only from the tracheal sample after five passages in HRT-18 cells, whereas virus isolation from the colonic sample was unsuccessful. Productive infection of HRT-18 cells was confirmed by immunofluorescence staining and western blot analysis targeting the BCoV N protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWhole-genome sequencing of the isolated virus revealed one synonymous substitution in the S gene and several non-synonymous substitutions in the pp1b, HE, and S genes compared with the viral genomes detected directly from the clinical samples. Notably, all amino acid substitutions were detected only after virus isolation, suggesting that these mutations arose during cell culture adaptation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that identical bovine coronavirus (BCoV) genomes were present in both respiratory and intestinal tissues of a single cow. This direct comparison within the same host provides evidence that viral genetic differences are not required to explain BCoV tissue distribution, indicating that tissue tropism is not determined solely by viral genome sequences. Consistent with this conclusion, BLAST analysis confirmed that the virus examined in this study is genetically close to previously reported Japanese BCoV strains, sharing more than 99% nucleotide identity at the whole-genome level. This finding indicates that the virus analyzed here does not represent a genetically unusual or divergent lineage. Therefore, the observed tissue distribution of BCoV cannot be attributed to infection with a distinct or atypical viral strain.\u003c/p\u003e \u003cp\u003eWe found that virus isolation resulted in the accumulation of amino acid substitutions in nsp12, HE, and S proteins, all of which were absent in viral genomes detected directly from clinical samples. These findings are consistent with a previous study by Zhang et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], who demonstrated that during cell culture adaptation, an enteric BCoV strain accumulated mutations that rendered it genetically similar to a corresponding respiratory BCoV isolate obtained from the same animal. In the present study, several mutations identified after serial passages in HRT-18 cells closely corresponded to those reported by Zhang et al. as culture-adaptive changes. Notably, the two HE substitutions (V103I and N104D) were identical to the AH65-E \u0026rarr; AH65-E-TC mutations described in their study. Similarly, the S protein substitutions (D28N, Q179K, and E965D) were located within regions where mutation accumulation was observed during in vitro adaptation. In addition, the ORF1ab substitutions (M4891I and N4894Y) were detected within the same genomic region (nt 14600\u0026ndash;15000) previously reported to harbor clusters of adaptive mutations. Collectively, these observations strongly suggest that the mutations identified in our isolates represent cell culture-associated adaptation events rather than genetic determinants of tissue tropism in vivo.\u003c/p\u003e \u003cp\u003eThe amino acid substitutions detected in nsp12, HE, and S proteins during BCoV isolation are therefore likely to reflect viral adaptation to replication in HRT-18 cells. The two nsp12 substitutions (M4891I and N4894Y) are located within the thumb domain of the RNA-dependent RNA polymerase [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and may influence local conformational flexibility or interactions with replication cofactors. The HE substitutions (V103I and N104D) occur near regions implicated in receptor binding and esterase activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and may modify surface charge or receptor interactions in human-derived cells. Similarly, the S protein substitutions (D28N, Q179K, and E965D) affect residues within the S1 N-terminal domain and S2 region, which are involved in receptor recognition and membrane fusion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Together, these changes may contribute to coordinated adaptation of viral entry, replication, and release processes, thereby facilitating more efficient propagation of BCoV in vitro.\u003c/p\u003e \u003cp\u003eAlthough this study is based on a single case, the direct comparison of viral genomes derived from different tissues within the same animal minimizes host-related confounding factors. However, our findings do not exclude the possibility that viral genes influence tissue-specific pathogenicity under certain conditions. The recent development of reverse genetics systems for BCoV will enable future studies using genetically homogeneous viruses to more definitively address the contribution of viral genetic factors to tissue tropism and pathogenicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this context, continuous epidemiological surveillance combined with careful genomic analysis of clinical samples will be essential for interpreting and validating such experimental findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKeisuke Nakagawa: \u003c/strong\u003eConceptualization,methodology, analysis, wrote the first draft, reviewed and edited final draft.\u003cstrong\u003e Aimi Yoshida:\u003c/strong\u003e Methodology, reviewed and edited final draft. \u003cstrong\u003eKeisuke Kuwata:\u003c/strong\u003e Resources, reviewed and edited final draft. \u003cstrong\u003eYuko Kitamura:\u003c/strong\u003e Resources, reviewed and edited final draft.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by research grants from Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (number 19K23706 and 23K05552) and a grant from the Mishima Kaiun Memorial Foundation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequences of strain GF2020 obtained from tracheal swab sample and isolate have been deposited in GenBank under accession numbers LC918946 and LC918947, respectively. The corresponding raw sequence data are available in the Sequence Read Archive (SRA) under accession numbers DRX326681 (BioSample SAMD00414418)\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest: \u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaif LJ (2010) Bovine respiratory coronavirus. Veterinary Clinics of North America - Food Animal Practice 26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffin D (1997) Economic impact associated with respiratory disease in beef cattle. 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Viruses 4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugiura Y, Takahashi T, Ueno S et al (2025) Expression of spike and hemagglutinin-esterase proteins is necessary to recover infectious recombinant bovine coronavirus. J Virol 99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jvi.01027-25\u003c/span\u003e\u003cspan address=\"10.1128/jvi.01027-25\" 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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8922291/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8922291/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine coronavirus (BCoV) causes respiratory and enteric diseases in cattle; however, the determinants of its tissue tropism remain unclear. In this study, we identified identical BCoV whole-genome sequences in tracheal and colonic samples obtained from a single adult cow that died from winter dysentery. After five passages in HRT-18 cells, virus isolation was successfully achieved only from the tracheal sample. Several amino acid substitutions were detected exclusively after isolation. These findings suggest that BCoV tissue tropism is not determined solely by viral genome sequences. It is important to analyze viral genomes directly from clinical samples prior to virus isolation.\u003c/p\u003e","manuscriptTitle":"Tissue tropism of bovine coronavirus may not be determined by viral genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 16:21:32","doi":"10.21203/rs.3.rs-8922291/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-02-25T13:03:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T11:34:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T11:34:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2026-02-20T04:21:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"22366682-0076-414b-b69e-54414b4d14a5","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T16:21:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 16:21:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8922291","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8922291","identity":"rs-8922291","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}