{"paper_id":"006016bc-3de0-4d5d-a3ef-a20601eabd62","body_text":"First Isolation and Molecular Characterization of the Bovine Leukemia Virus in the State of Pernambuco, Brazil | 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 First Isolation and Molecular Characterization of the Bovine Leukemia Virus in the State of Pernambuco, Brazil Jerlane Tarcilia Gomes Telles¹, Guilherme Valeriano Silva¹, Huber Rizzo¹, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7013947/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Enzootic Bovine Leukosis (EBL) is an infectious and contagious disease caused by the Bovine Leukemia Virus (BLV), which has a significant economic impact on livestock and a potential zoonotic risk under investigation. In Brazil, although reports of infection exist in several regions, studies on the genetic diversity of the virus are scarce, especially in the Northeast. This study describes, for the first time, the isolation of BLV in the state of Pernambuco, from a blood sample of a seropositive bovine, with laboratory confirmation by Nested-PCR, sequencing of the env gene (gp51), and phylogenetic analysis. The isolation was performed by inoculating leukocytes into MDBK cells, followed by cytopathic effect monitoring and viral DNA validation. Phylogenetic analysis of the env gene (gp51) demonstrated clustering within genotype 1, showing high nucleotide identity with reference isolates previously registered in GenBank. These findings advance our knowledge of BLV genetic diversity in Brazil and highlight the critical need for sustained genomic surveillance, especially considering the potential risk of zoonotic transmission. Viral genetics epidemiology veterinary medicine phylogeny One Health Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Enzootic Bovine Leukosis (EBL) is a mandatory notifiable infectious disease to the Ministry of Agriculture and Livestock (MAPA) and the World Organization for Animal Health (WOAH), with standardized diagnostic and control protocols detailed in the WOAH Terrestrial Manual (WOAH, 2018). This disease is caused by an oncogenic retrovirus, the Bovine Leukemia Virus (BLV), classified as Deltaretrovirus bovleu , genus Deltaretrovirus , family Retroviridae (ICTV, 2024). EBL has a significant negative economic impact on livestock production, causing direct losses such as reduced productivity and indirect losses such as trade restrictions on animals and derived products from infected areas (Marawan et al., 2021 ). Additionally, subclinical infection can affect productivity and reproductive performance, particularly in dairy herds (Szczotka et al., 2022). In Brazil, research on the genetic diversity of BLV is limited. In the state of Santa Catarina, five different genotypes were identified, highlighting the genetic heterogeneity of BLV in the region (Rodakiewicz et al., 2018 ). In São Paulo, genotypes 5 and 6 were detected in cattle herds (Gregory et al., 2017 ). Although there is an epidemiological study that reports the occurrence of BLV infection in Pernambuco (Santos et al., 2013), no investigations on the genetic diversity of the virus in this state have been conducted. The identification and genetic characterization of the virus are essential to understand its epidemiology, improve control and prevention strategies, and contribute to developing more effective vaccines and diagnostic methods. Therefore, this study aims to describe the first isolation and phylogenetic analysis of the env gene (gp51) of BLV in the state of Pernambuco, Brazil. MATERIAL AND METHODS The blood sample used for virus isolation and DNA sequencing was obtained from a farm located in the municipality of Abreu e Lima, Pernambuco, whose herd consisted of animals resulting from the crossbreeding of Senepol and Limousin breeds. In a previous study, 20 cattle aged between 7 and 72 months were evaluated, showing clinical signs compatible with EBL. Among these, a necropsy was performed on a calf that had been in permanent lateral recumbency for two days in an advanced state of cachexia. Lymph node fragments and lymphatic tissues from various organs were collected. The pathological findings were compatible with BLV infection (Melo et al., 2023). Serum samples from all animals were subjected to serology using the Agar Gel Immunodiffusion (AGID) technique with an in-house kit. One breeding bull stood out, showing a strong positive reaction in AGID and confirmation by PCR. This animal was selected for virus isolation and genetic sequencing. For this procedure, 4 mL of whole blood were collected by venipuncture into two tubes containing EDTA. The sample was centrifuged at 800×g for 20 minutes to separate the leukocyte layer. The leukocytes were carefully aspirated, washed with phosphate-buffered saline (PBS), and resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). MDBK cells were cultured in flasks containing the same medium supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, maintained at 37 °C in 5% CO₂ until reaching approximately 80% confluence. Parallel to the viral isolation, DNA extraction was performed directly from whole blood, followed by PCR, sequencing, and phylogenetic tree construction, comparing the sequence obtained from the isolated virus. Leukocytes were inoculated into MDBK cells and incubated at 37°C for two hours to allow viral adsorption. The medium was then removed and replaced with fresh RPMI-1640 supplemented with 2% FBS. The culture was maintained at 37°C with 5% CO₂ and monitored daily for cytopathic effects (CPE). The supernatant was collected every 72 hours and subjected to DNA extraction using the Wizard® SV Genomic DNA Purification System. A two-step Nested-PCR was performed using specific primers. The first reaction used primers env 5032 F (5´ TCT GTG CCA AGT CTC CCA GAT A 3´) and env 5608 R (5´ AAC AAC AAC CTC TGG GAA GGG T 3´). The second reaction used env 5099 F (5´ CCC ACA AGG GCG GCG CCG GTT T 3´) and env 5521 R (5´ GCG AGG CCG GGT CCA GAG CTG G 3´) (Fechner et al., 1996). PCR was performed in a final volume of 25 µL, containing 12.5 µL of GoTaq Green Master Mix 2X, 9 µL of water, 0.75 µL of each primer, and 2 µL of DNA. Amplifications were performed under the following conditions: initial denaturation at 95°C for five minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for one minute, with a final extension at 72°C for five minutes. Amplified products were analyzed by electrophoresis on 2% agarose gel. The PCR product was purified using the ReliaPrep™ DNA Clean-Up and Concentration System (Promega, USA) and sent for sequencing on a Genetic Analyzer 3500 (Applied Biosystems) using the Sanger method. Forward and reverse sequences were edited with Staden Package 2.0.0b11 ( https://staden.sourceforge.net/ ) to generate a consensus sequence, which was then compared to sequences in GenBank using BLASTn. For phylogenetic analysis, the consensus sequence was aligned with 52 reference sequences using MUSCLE in MEGA11 (Tamura et al., 2021). The phylogenetic tree was inferred using the Maximum Likelihood method with the Kimura 2-parameter model (Kimura, 1980) and 1000 bootstrap replicates to estimate branch support. The initial tree for heuristic search was generated automatically using Neighbor-Joining and BioNJ algorithms based on pairwise distance matrices estimated with the Maximum Composite Likelihood approach. The topology with the highest log-likelihood was selected. Evolutionary rate variation among sites was modeled with a discrete Gamma distribution with six categories. The analysis included codon positions 1st, 2nd, 3rd, and non-coding regions. This study was approved by the Animal Use Ethics Committee (CEUA) of the Universidade Federal Rural de Pernambuco (UFRPE) under protocol number 4775270623 and registered in the SISGEN platform (Registration nº A090BA7). RESULTS AND DISCUSSION The BLV isolate was obtained from the leukocyte layer of blood from a seropositive animal. PCR performed on the culture supernatant was positive. The consensus sequence of the env (gp51) gene obtained was submitted to BLASTn similarity analysis. Among the 100 most similar sequences in GenBank, ten showed 100% identity with the analyzed isolate, while the others showed identity above 99.5% (Fig. 1 ), indicating high conservation of this genetic region. These findings suggest that the viral isolate identified in Pernambuco shares a highly similar genetic profile with previously reported isolates, reinforcing the genomic stability of the env gene. The significance of this study lies in the fact that, to date, there have been no reports of BLV isolation in the state of Pernambuco. However, various epidemiological studies have reported the presence of BLV infection in cattle herds in Pernambuco (Fig. 2 ). In Brazil, studies on BLV isolation remain limited. A study conducted at the Federal University of Minas Gerais (UFMG) in 2014 successfully isolated the virus from four bovine blood samples. Confirmation was achieved through PCR, and morphological characterization was conducted using transmission electron microscopy. However, that study did not include genetic sequencing or phylogenetic analysis, which hindered genotype identification. Furthermore, methodological and objective differences exist between the studies. Florentino (2014) utilized the Tb1Lu cell line derived from bat lung cells for antigen production for AGID, while the present study used bovine leukocytes cultured in MDBK cells (derived from bovine fetal kidney) with infection confirmation through Nested-PCR and subsequent sequencing and phylogenetic analysis. The cell lines used have relevant differences: Tb1Lu cells are naturally free from Bovine Viral Diarrhea Virus (BVDV), while MDBK cells are susceptible to BVDV contamination, requiring additional precautions. To rule out contamination, PCR for BVDV detection was performed on the culture, with negative results confirming the absence of contamination. The phylogenetic analysis based on the env gene sequences obtained from blood samples and the viral isolate demonstrated that the samples belong to genotype 1 – Group 1 (Fig. 3 ) consistent with those identified in the United States, Argentina, Uruguay, and other regions of Brazil. This finding corroborates a previous study conducted in Santa Catarina, which detected genotype 1 among other genotypes (2, 8, 9, and 10), highlighting the genetic heterogeneity of the virus in that region (Rodakiewicz et al., 2018 ). In the Brazilian Northeast, there were no previous reports of genotype 1 circulation, although, a study in Maranhão identified genotype 6 in cattle (Pereira et al., 2023 ). The difference between the genotype found in this study in Pernambuco and that found in Maranhão demonstrates the presence of genetic diversity in BLV in the Northeast, which may have implications for diagnosis, surveillance, and disease control. Genetic diversity significantly impacts BLV diagnosis. In molecular methods like PCR, amplification efficiency depends on the conservation of primer target regions. Nucleotide variations among genotypes can hinder primer binding, reduce sensitivity and lead to false negatives. Similarly, serological tests such as ELISA and AGID are affected by antigenic variability. Antigens derived from specific strains may fail to detect infections with genetically divergent strains due to epitope differences. Evidence of this phenomenon was demonstrated by Voss et al. ( 2021 ) when investigating human antibody responses to SARS-CoV-2 epitopes. They found that mutations in variants of concern altered immune recognition, reducing or preventing antibody binding and compromising serological tests. This highlights the importance of considering antigenic variability in designing diagnostic tools for BLV to avoid loss of sensitivity and detection failures in populations infected with genetically distinct variants. These diagnostic challenges directly affect control and prevention measures, as infected animals may remain undetected, facilitating the spread of the virus. Some studies suggest that different genotypes may be associated with variations in proviral load and disease progression, influencing immune response and the effectiveness of sanitary strategies (Polat et al., 2017 ). It is important to note that the Brazilian scientific literature on the genotypic characterization of circulating BLV remains limited. Consequently, there is insufficient data to accurately define the virus’s genetic diversity nationwide. Therefore, further genotypic studies in different Brazilian regions are essential to obtain a comprehensive map of BLV variability. This knowledge is crucial to support more effective control and eradication policies, improve diagnostic methods, and adapt them to the local epidemiological reality. Studies conducted in Brazil have raised increasing concerns about the zoonotic potential of EBL. In Rio Grande do Sul, researchers detected BLV DNA in human tumors, particularly breast cancer tissues. The viral sequences showed high similarity to genotype 1 (Canova et al., 2021 ), the same genotype identified in this study. However, this work did not confirm active viral replication, so zoonotic transmission has not been definitively established. International studies also raise concerns about zoonotic potential. Research in Turkey detected BLV DNA in raw milk and meat, along with antiviral antibodies in human serum samples, suggesting the possibility of foodborne transmission (Quadros et al., 2023 ). Regarding viral replication, a recent study in Argentina demonstrated that BLV could infect human mammary epithelial cells (MCF-10A), integrate into their DNA, and produce infectious viral particles. Moreover, the generated virus was capable of reinfecting bovine epithelial cells (MAC-T), showing cross-species replication capacity (Moran et al., 2025 ). Although this study was conducted in vitro and does not definitively confirm zoonotic behavior, the demonstration of viral replication in human cells strengthens the hypothesis that EBL may represent a zoonosis. Therefore, in-depth genotypic characterization is crucial to support effective One Health strategies for disease prevention and control. CONCLUSION The successful isolation of BLV in the state of Pernambuco marks a significant step forward in understanding the epidemiology of Enzootic Bovine Leukosis in Brazil. This is the first confirmed report of viral isolation in the state, validated through Nested-PCR, env gene sequencing, and phylogenetic analysis, which identified the presence of genotype 1 in the Northeast region. The characterization of local isolates provides valuable insights into the genetic diversity of BLV, supporting the development of more effective diagnostic tools, control measures, and prevention strategies tailored to regional contexts. Given the growing evidence of the virus's zoonotic potential, as highlighted in national and international studies, continued surveillance is essential. The methodological framework established in this study offers a robust foundation for future genomic monitoring initiatives, reinforcing the importance of integrating animal, human, and environmental health approaches under the One Health paradigm. Declarations ETHICS APPROVAL This study was approved by the Animal Use Ethics Committee (CEUA) of the Universidade Federal Rural de Pernambuco (UFRPE) under protocol number 4775270623. CONFLICT OF INTEREST The authors declare no conflict of interest. FUNDING The authors did not receive support from any organization for the submitted work. Author Contribution Collection and initial processing of biological material: J.T.G.T; H.R; E.S.S; S.A.N; R.C.C.M and J.W.P.JLaboratory tests: J.T.G.T; S.A.N; A.M.C; A.F.S and R.S.FMain writing: J.T.G.T; A.M.C; G.V.S; J.W.P.J; E.S.S; A.F.S and R.S.F and R.C.C.MAll authors reviewed and proofread the text of the article. Acknowledgement The authors would like to thank the CAPES program for providing the master's scholarship. References CANOVA, Raíssa et al. Bovine leukemia viral DNA found on human breast tissue is genetically related to the cattle virus. One Health , [S.l.], v. 13, p. 100252, 2021. DOI: 10.1016/j.onehlt.2021.100252. Acesso em: 27 mar. 2025 Fechner H. et al. (1996). Evaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle . Zentralbl Veterinarmed B. 43 (10):621–630. DOI: 10.1111/j.1439-0450.1996.tb00361.x. Acesso em: 15 mar. 2023 FLORENTINO, Gazielle Cossenzo. Leucose Enzoótica Bovina: Isolamento de Amostras Brasileiras do Vírus e Obtenção de Antígeno. 2015. Dissertação (Mestrado) – Universidade Federal de Minas Gerais, Belo Horizonte, 2015. Disponível em: https://repositorio.ufmg.br/handle/1843/BUBD-A9CFVN. Acesso em: 16 mar. 2025. GREGORY, Lilian et al. Bovine leukaemia virus genotypes 5 and 6 are circulating in cattle from the state of São Paulo, Brazil. Journal of Medical Microbiology , v. 66, n. 12, 2017. Disponível em: https://doi.org/10.1099/jmm.0.000639. Acesso em: 30 dez. 2024. INTERNATIONAL COMMITTEE ON TAXONOMY OF VIRUSES (ICTV). Deltaretrovirus bovleu . Disponível em: https://ictv.global/taxonomy/taxondetails?taxnode_id=202404998&taxon_name=Deltaretrovirus%20bovleu Acesso em: 20 maio 2025. KIMURA, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution , v. 16, p. 111–120, 1980. Acesso em: 21 maio 2025. MARAWAN, Marawan A. et al. Bovine Leukaemia Virus: Current Epidemiological Circumstance and Future Prospective. Viruses , v. 13, n. 11, p. 2167, 2021. DOI: 10.3390/v13112167. Disponível em: https://doi.org/10.3390/v13112167. Acesso em: 30 dez. 2024. MELO, Lúcio Esmeraldo Honório de. et al. Ocorrência e caracterização da leucose enzoótica dos bovinos em rebanho de corte criado na região Metropolitana do Recife, Pernambuco. In: XIV Congresso Brasileiro de Buiatria e V Congresso Nordestino de Buiatria, 2023, Recife. Revista Brasileira de Buiatria. Recife: Associação Pernambucana de Buiatria, 2023. v. 1. p. 431-431. MORAN, Pedro Edgardo et al. Infectivity and persistence of bovine leukemia virus in human breast cells: assessing a possible zoonotic link to cancer. Veterinary Research Communications , v. 49, p. 173, 2025. DOI: 10.1007/s11259-025-10738-4. Acesso em: 27 mar. 2025. NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION (NCBI). Basic Local Alignment Search Tool (BLAST) . Disponível em: https://blast.ncbi.nlm.nih.gov/Blast.cgi. Acesso em: 24 dez 2024 OMSA – ORGANIZAÇÃO MUNDIAL DA SAÚDE ANIMAL. Leucose Enzoótica Bovina ( Bovine Leukosis Enzootic ). Disponível em: https://www.woah.org/en/disease/enzootic-bovine-leukosis/. Acesso em: 20 nov. 2024. PEREIRA, José Gomes et al. Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil . Frontiers in Veterinary Science, [S.l.], v. 10, 13 jan. 2023. DOI: 10.3389/fvets.2022.1080994. Disponível em: https://www.frontiersin.org/articles/10.3389/fvets.2022.1080994/full. Acesso em: 27 maio 2025. POLAT M., Takeshima S.N., Aida Y. et al. (2017). Epidemiology and genetic diversity of bovine leukemia virus. Virology Journal, 14(1):209. DOI: 10.1186/s12985-017-0876-4. Acesso em: 8 maio 2025 QUADROS, Daniel Lazzari de et al. Oncogenic viral DNA related to human breast cancer found on cattle milk and meat. Comparative Immunology, Microbiology and Infectious Diseases , v. 101, p. 102053, 2023. DOI: 10.1016/j.cimid.2023.102053. Acesso em: 27 mar. 2025 RAMBAUT, A. FigTree v1.4.4: a graphical viewer of phylogenetic trees. 2018. Disponível em: http://tree.bio.ed.ac.uk/software/figtree/. Acesso em: 21 maio 2025. RODAKIEWICZ, Sheyla Michele et al. Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil. Arquivos do Instituto Biológico , v. 85, 2018. Disponível em: https://doi.org/10.1590/1808-1657000742016. Acesso em: 30 dez. 2024. SZCOTKA, Maria; KUŹMAK, Jacek. Expression of bovine leukaemia virus (BLV) gp51 protein in blood and milk cells of cows with leukosis. Journal of Veterinary Research , v. 66, n. 3, p. 337-345, 2022. Disponível em: https://doi.org/10.2478/jvetres-2022-0035. Acesso em: 30 dez. 2024. TAMURA, K.; STECHER, G.; KUMAR, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution , 2021. Disponível em: https://doi.org/10.1093/molbev/msab120. Acesso em: 21 maio 2025. VOSS, C. et al. Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern. Science Immunology , Washington, D.C., v. 6, n. 65, eabj1750, 2021. Disponível em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410046/. Acesso em: 6 abril 2025 WORLD ORGANISATION FOR ANIMAL HEALTH (OIE). Enzootic bovine leucosis. In: WORLD ORGANISATION FOR ANIMAL HEALTH (OIE). Terrestrial manual. Paris: OIE, 2018. p. 1113-1124. Disponível em: https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.04.09_EBL.pdf. Acesso em: 30 dez. 2024. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-7013947\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":489112453,\"identity\":\"375784e7-77ec-49f8-bb98-e3726d0c5269\",\"order_by\":0,\"name\":\"Jerlane Tarcilia Gomes 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2024\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7013947/v1/cfb7d85e8908dd60f18552e4.png\"},{\"id\":88048919,\"identity\":\"504c3b51-b30e-47be-8abe-e93f7be7ebe5\",\"added_by\":\"auto\",\"created_at\":\"2025-07-31 19:13:37\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":647726,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTimeline with studies on Enzootic Bovine Leukosis (EBL) in Pernambuco, highlighting prevalence and clinical records between 1991 and 2014 in different regions of the state.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7013947/v1/613c849e1506968221112d08.png\"},{\"id\":88049183,\"identity\":\"121720f7-3df0-4094-984f-d70a46b8c4a0\",\"added_by\":\"auto\",\"created_at\":\"2025-07-31 19:21:35\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":192238,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhylogenetic tree inferred by the Maximum Likelihood method, using Kimura's two-parameter model and 1000 bootstrap replicates, from 52 env gene sequences (351 positions). Node support is represented by a color scale: red indicates bootstrap value = 1.0; blue, bootstrap = 0.0. The samples highlighted in red (ID02_Blood and ID02_Lymphocyte) are grouped in G1 (Group 1), indicating a close phylogenetic relationship. The tree was visualized using FigTree software (Rambaut, 2018).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7013947/v1/853f20b2e870870e5a6a7f8c.png\"},{\"id\":89303150,\"identity\":\"ded80989-8862-450e-afdc-c1a934633221\",\"added_by\":\"auto\",\"created_at\":\"2025-08-18 14:47:30\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1443861,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7013947/v1/c2c90a43-01b8-4f8e-8c6e-9cceb510c279.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"First Isolation and Molecular Characterization of the Bovine Leukemia Virus in the State of Pernambuco, Brazil\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eEnzootic Bovine Leukosis (EBL) is a mandatory notifiable infectious disease to the Ministry of Agriculture and Livestock (MAPA) and the World Organization for Animal Health (WOAH), with standardized diagnostic and control protocols detailed in the WOAH Terrestrial Manual (WOAH, 2018). This disease is caused by an oncogenic retrovirus, the Bovine Leukemia Virus (BLV), classified as \\u003cem\\u003eDeltaretrovirus bovleu\\u003c/em\\u003e, genus \\u003cem\\u003eDeltaretrovirus\\u003c/em\\u003e, family \\u003cem\\u003eRetroviridae\\u003c/em\\u003e (ICTV, 2024). EBL has a significant negative economic impact on livestock production, causing direct losses such as reduced productivity and indirect losses such as trade restrictions on animals and derived products from infected areas (Marawan et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Additionally, subclinical infection can affect productivity and reproductive performance, particularly in dairy herds (Szczotka et al., 2022).\\u003c/p\\u003e\\u003cp\\u003eIn Brazil, research on the genetic diversity of BLV is limited. In the state of Santa Catarina, five different genotypes were identified, highlighting the genetic heterogeneity of BLV in the region (Rodakiewicz et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). In S\\u0026atilde;o Paulo, genotypes 5 and 6 were detected in cattle herds (Gregory et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Although there is an epidemiological study that reports the occurrence of BLV infection in Pernambuco (Santos et al., 2013), no investigations on the genetic diversity of the virus in this state have been conducted. The identification and genetic characterization of the virus are essential to understand its epidemiology, improve control and prevention strategies, and contribute to developing more effective vaccines and diagnostic methods. Therefore, this study aims to describe the first isolation and phylogenetic analysis of the env gene (gp51) of BLV in the state of Pernambuco, Brazil.\\u003c/p\\u003e\"},{\"header\":\"MATERIAL AND METHODS\",\"content\":\"\\u003cp\\u003eThe blood sample used for virus isolation and DNA sequencing was obtained from a farm located in the municipality of Abreu e Lima, Pernambuco, whose herd consisted of animals resulting from the crossbreeding of Senepol and Limousin breeds. In a previous study, 20 cattle aged between 7 and 72 months were evaluated, showing clinical signs compatible with EBL. Among these, a necropsy was performed on a calf that had been in permanent lateral recumbency for two days in an advanced state of cachexia. Lymph node fragments and lymphatic tissues from various organs were collected. The pathological findings were compatible with BLV infection (Melo et al., 2023). Serum samples from all animals were subjected to serology using the Agar Gel Immunodiffusion (AGID) technique with an in-house kit. One breeding bull stood out, showing a strong positive reaction in AGID and confirmation by PCR. This animal was selected for virus isolation and genetic sequencing.\\u003c/p\\u003e\\n\\u003cp\\u003eFor this procedure, 4 mL of whole blood were collected by venipuncture into two tubes containing EDTA. The sample was centrifuged at 800\\u0026times;g for 20 minutes to separate the leukocyte layer. The leukocytes were carefully aspirated, washed with phosphate-buffered saline (PBS), and resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). MDBK cells were cultured in flasks containing the same medium supplemented with 100 U/mL penicillin and 100 \\u0026micro;g/mL streptomycin, maintained at 37 \\u0026deg;C in 5% CO₂ until reaching approximately 80% confluence. Parallel to the viral isolation, DNA extraction was performed directly from whole blood, followed by PCR, sequencing, and phylogenetic tree construction, comparing the sequence obtained from the isolated virus.\\u003c/p\\u003e\\n\\u003cp\\u003eLeukocytes were inoculated into MDBK cells and incubated at 37\\u0026deg;C for two hours to allow viral adsorption. The medium was then removed and replaced with fresh RPMI-1640 supplemented with 2% FBS. The culture was maintained at 37\\u0026deg;C with 5% CO₂ and monitored daily for cytopathic effects (CPE). The supernatant was collected every 72 hours and subjected to DNA extraction using the Wizard\\u0026reg; SV Genomic DNA Purification System. A two-step Nested-PCR was performed using specific primers. The first reaction used primers env 5032 F (5\\u0026acute; TCT GTG CCA AGT CTC CCA GAT A 3\\u0026acute;) and env 5608 R (5\\u0026acute; AAC AAC AAC CTC TGG GAA GGG T 3\\u0026acute;). The second reaction used env 5099 F (5\\u0026acute; CCC ACA AGG GCG GCG CCG GTT T 3\\u0026acute;) and env 5521 R (5\\u0026acute; GCG AGG CCG GGT CCA GAG CTG G 3\\u0026acute;) (Fechner et al., 1996).\\u003c/p\\u003e\\n\\u003cp\\u003ePCR was performed in a final volume of 25 \\u0026micro;L, containing 12.5 \\u0026micro;L of GoTaq Green Master Mix 2X, 9 \\u0026micro;L of water, 0.75 \\u0026micro;L of each primer, and 2 \\u0026micro;L of DNA. Amplifications were performed under the following conditions: initial denaturation at 95\\u0026deg;C for five minutes, followed by 40 cycles of denaturation at 94\\u0026deg;C for 30 seconds, annealing at 60\\u0026deg;C for 30 seconds, and extension at 72\\u0026deg;C for one minute, with a final extension at 72\\u0026deg;C for five minutes. Amplified products were analyzed by electrophoresis on 2% agarose gel. The PCR product was purified using the ReliaPrep\\u0026trade; DNA Clean-Up and Concentration System (Promega, USA) and sent for sequencing on a Genetic Analyzer 3500 (Applied Biosystems) using the Sanger method. Forward and reverse sequences were edited with Staden Package 2.0.0b11 (\\u003cu\\u003ehttps://staden.sourceforge.net/\\u003c/u\\u003e) to generate a consensus sequence, which was then compared to sequences in GenBank using BLASTn.\\u003c/p\\u003e\\n\\u003cp\\u003eFor phylogenetic analysis, the consensus sequence was aligned with 52 reference sequences using MUSCLE in MEGA11 (Tamura et al., 2021). The phylogenetic tree was inferred using the Maximum Likelihood method with the Kimura 2-parameter model (Kimura, 1980) and 1000 bootstrap replicates to estimate branch support.\\u003c/p\\u003e\\n\\u003cp\\u003eThe initial tree for heuristic search was generated automatically using Neighbor-Joining and BioNJ algorithms based on pairwise distance matrices estimated with the Maximum Composite Likelihood approach. The topology with the highest log-likelihood was selected. Evolutionary rate variation among sites was modeled with a discrete Gamma distribution with six categories. The analysis included codon positions 1st, 2nd, 3rd, and non-coding regions.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was approved by the Animal Use Ethics Committee (CEUA) of the Universidade Federal Rural de Pernambuco (UFRPE) under protocol number 4775270623 and registered in the SISGEN platform (Registration n\\u0026ordm; A090BA7).\\u003c/p\\u003e\"},{\"header\":\"RESULTS AND DISCUSSION\",\"content\":\"\\u003cp\\u003eThe BLV isolate was obtained from the leukocyte layer of blood from a seropositive animal. PCR performed on the culture supernatant was positive. The consensus sequence of the env (gp51) gene obtained was submitted to BLASTn similarity analysis. Among the 100 most similar sequences in GenBank, ten showed 100% identity with the analyzed isolate, while the others showed identity above 99.5% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), indicating high conservation of this genetic region. These findings suggest that the viral isolate identified in Pernambuco shares a highly similar genetic profile with previously reported isolates, reinforcing the genomic stability of the env gene.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe significance of this study lies in the fact that, to date, there have been no reports of BLV isolation in the state of Pernambuco. However, various epidemiological studies have reported the presence of BLV infection in cattle herds in Pernambuco (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn Brazil, studies on BLV isolation remain limited. A study conducted at the Federal University of Minas Gerais (UFMG) in 2014 successfully isolated the virus from four bovine blood samples. Confirmation was achieved through PCR, and morphological characterization was conducted using transmission electron microscopy. However, that study did not include genetic sequencing or phylogenetic analysis, which hindered genotype identification. Furthermore, methodological and objective differences exist between the studies. Florentino (2014) utilized the Tb1Lu cell line derived from bat lung cells for antigen production for AGID, while the present study used bovine leukocytes cultured in MDBK cells (derived from bovine fetal kidney) with infection confirmation through Nested-PCR and subsequent sequencing and phylogenetic analysis. The cell lines used have relevant differences: Tb1Lu cells are naturally free from Bovine Viral Diarrhea Virus (BVDV), while MDBK cells are susceptible to BVDV contamination, requiring additional precautions. To rule out contamination, PCR for BVDV detection was performed on the culture, with negative results confirming the absence of contamination.\\u003c/p\\u003e\\u003cp\\u003eThe phylogenetic analysis based on the env gene sequences obtained from blood samples and the viral isolate demonstrated that the samples belong to genotype 1 \\u0026ndash; Group 1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) consistent with those identified in the United States, Argentina, Uruguay, and other regions of Brazil. This finding corroborates a previous study conducted in Santa Catarina, which detected genotype 1 among other genotypes (2, 8, 9, and 10), highlighting the genetic heterogeneity of the virus in that region (Rodakiewicz et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn the Brazilian Northeast, there were no previous reports of genotype 1 circulation, although, a study in Maranh\\u0026atilde;o identified genotype 6 in cattle (Pereira et al., \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The difference between the genotype found in this study in Pernambuco and that found in Maranh\\u0026atilde;o demonstrates the presence of genetic diversity in BLV in the Northeast, which may have implications for diagnosis, surveillance, and disease control. Genetic diversity significantly impacts BLV diagnosis. In molecular methods like PCR, amplification efficiency depends on the conservation of primer target regions. Nucleotide variations among genotypes can hinder primer binding, reduce sensitivity and lead to false negatives. Similarly, serological tests such as ELISA and AGID are affected by antigenic variability. Antigens derived from specific strains may fail to detect infections with genetically divergent strains due to epitope differences. Evidence of this phenomenon was demonstrated by Voss et al. (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) when investigating human antibody responses to SARS-CoV-2 epitopes. They found that mutations in variants of concern altered immune recognition, reducing or preventing antibody binding and compromising serological tests. This highlights the importance of considering antigenic variability in designing diagnostic tools for BLV to avoid loss of sensitivity and detection failures in populations infected with genetically distinct variants.\\u003c/p\\u003e\\u003cp\\u003eThese diagnostic challenges directly affect control and prevention measures, as infected animals may remain undetected, facilitating the spread of the virus. Some studies suggest that different genotypes may be associated with variations in proviral load and disease progression, influencing immune response and the effectiveness of sanitary strategies (Polat et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eIt is important to note that the Brazilian scientific literature on the genotypic characterization of circulating BLV remains limited. Consequently, there is insufficient data to accurately define the virus\\u0026rsquo;s genetic diversity nationwide. Therefore, further genotypic studies in different Brazilian regions are essential to obtain a comprehensive map of BLV variability. This knowledge is crucial to support more effective control and eradication policies, improve diagnostic methods, and adapt them to the local epidemiological reality. Studies conducted in Brazil have raised increasing concerns about the zoonotic potential of EBL. In Rio Grande do Sul, researchers detected BLV DNA in human tumors, particularly breast cancer tissues. The viral sequences showed high similarity to genotype 1 (Canova et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), the same genotype identified in this study. However, this work did not confirm active viral replication, so zoonotic transmission has not been definitively established.\\u003c/p\\u003e\\u003cp\\u003eInternational studies also raise concerns about zoonotic potential. Research in Turkey detected BLV DNA in raw milk and meat, along with antiviral antibodies in human serum samples, suggesting the possibility of foodborne transmission (Quadros et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eRegarding viral replication, a recent study in Argentina demonstrated that BLV could infect human mammary epithelial cells (MCF-10A), integrate into their DNA, and produce infectious viral particles. Moreover, the generated virus was capable of reinfecting bovine epithelial cells (MAC-T), showing cross-species replication capacity (Moran et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). Although this study was conducted \\u003cem\\u003ein vitro\\u003c/em\\u003e and does not definitively confirm zoonotic behavior, the demonstration of viral replication in human cells strengthens the hypothesis that EBL may represent a zoonosis. Therefore, in-depth genotypic characterization is crucial to support effective One Health strategies for disease prevention and control.\\u003c/p\\u003e\"},{\"header\":\"CONCLUSION\",\"content\":\"\\u003cp\\u003eThe successful isolation of BLV in the state of Pernambuco marks a significant step forward in understanding the epidemiology of Enzootic Bovine Leukosis in Brazil. This is the first confirmed report of viral isolation in the state, validated through Nested-PCR, env gene sequencing, and phylogenetic analysis, which identified the presence of genotype 1 in the Northeast region. The characterization of local isolates provides valuable insights into the genetic diversity of BLV, supporting the development of more effective diagnostic tools, control measures, and prevention strategies tailored to regional contexts. Given the growing evidence of the virus's zoonotic potential, as highlighted in national and international studies, continued surveillance is essential. The methodological framework established in this study offers a robust foundation for future genomic monitoring initiatives, reinforcing the importance of integrating animal, human, and environmental health approaches under the One Health paradigm.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eETHICS APPROVAL\\u003c/h2\\u003e\\n\\u003cp\\u003eThis study was approved by the Animal Use Ethics Committee (CEUA) of the Universidade Federal Rural de Pernambuco (UFRPE) under protocol number 4775270623.\\u003c/p\\u003e\\n\\u003ch2\\u003eCONFLICT OF INTEREST\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare no conflict of interest.\\u003c/p\\u003e\\n\\u003ch2\\u003eFUNDING\\u0026nbsp;\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors did not receive support from any organization for the submitted work.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\n\\u003cp\\u003eCollection and initial processing of biological material: J.T.G.T; H.R; E.S.S; S.A.N; R.C.C.M and J.W.P.JLaboratory tests: J.T.G.T; S.A.N; A.M.C; A.F.S and R.S.FMain writing: J.T.G.T; A.M.C; G.V.S; J.W.P.J; E.S.S; A.F.S and R.S.F and R.C.C.MAll authors reviewed and proofread the text of the article.\\u003c/p\\u003e\\n\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors would like to thank the CAPES program for providing the master\\u0026apos;s scholarship.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eCANOVA, Ra\\u0026iacute;ssa et al. Bovine leukemia viral DNA found on human breast tissue is genetically related to the cattle virus. \\u003cem\\u003eOne Health\\u003c/em\\u003e, [S.l.], v. 13, p. 100252, 2021. DOI: 10.1016/j.onehlt.2021.100252. Acesso em: 27 mar. 2025\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cstrong\\u003eFechner H. et al.\\u003c/strong\\u003e (1996). \\u003cem\\u003eEvaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle\\u003c/em\\u003e. Zentralbl Veterinarmed B. \\u003cstrong\\u003e43\\u003c/strong\\u003e(10):621\\u0026ndash;630. DOI: 10.1111/j.1439-0450.1996.tb00361.x. Acesso em: 15 mar. 2023\\u003c/li\\u003e\\n\\u003cli\\u003eFLORENTINO, Gazielle Cossenzo. Leucose Enzo\\u0026oacute;tica Bovina: Isolamento de Amostras Brasileiras do V\\u0026iacute;rus e Obten\\u0026ccedil;\\u0026atilde;o de Ant\\u0026iacute;geno. 2015. Disserta\\u0026ccedil;\\u0026atilde;o (Mestrado) \\u0026ndash; Universidade Federal de Minas Gerais, Belo Horizonte, 2015. Dispon\\u0026iacute;vel em: https://repositorio.ufmg.br/handle/1843/BUBD-A9CFVN. Acesso em: 16 mar. 2025.\\u003c/li\\u003e\\n\\u003cli\\u003eGREGORY, Lilian et al. Bovine leukaemia virus genotypes 5 and 6 are circulating in cattle from the state of S\\u0026atilde;o Paulo, Brazil. \\u003cem\\u003eJournal of Medical Microbiology\\u003c/em\\u003e, v. 66, n. 12, 2017. Dispon\\u0026iacute;vel em: https://doi.org/10.1099/jmm.0.000639. Acesso em: 30 dez. 2024.\\u003c/li\\u003e\\n\\u003cli\\u003eINTERNATIONAL COMMITTEE ON TAXONOMY OF VIRUSES (ICTV). \\u003cem\\u003eDeltaretrovirus bovleu\\u003c/em\\u003e. Dispon\\u0026iacute;vel em: https://ictv.global/taxonomy/taxondetails?taxnode_id=202404998\\u0026amp;taxon_name=Deltaretrovirus%20bovleu Acesso em: 20 maio 2025.\\u003c/li\\u003e\\n\\u003cli\\u003eKIMURA, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. \\u003cem\\u003eJournal of Molecular Evolution\\u003c/em\\u003e, v. 16, p. 111\\u0026ndash;120, 1980. Acesso em: 21 maio 2025.\\u003c/li\\u003e\\n\\u003cli\\u003eMARAWAN, Marawan A. et al. Bovine Leukaemia Virus: Current Epidemiological Circumstance and Future Prospective. \\u003cem\\u003eViruses\\u003c/em\\u003e, v. 13, n. 11, p. 2167, 2021. DOI: 10.3390/v13112167. Dispon\\u0026iacute;vel em: https://doi.org/10.3390/v13112167. Acesso em: 30 dez. 2024.\\u003c/li\\u003e\\n\\u003cli\\u003eMELO, L\\u0026uacute;cio Esmeraldo Hon\\u0026oacute;rio de. et al. Ocorr\\u0026ecirc;ncia e caracteriza\\u0026ccedil;\\u0026atilde;o da leucose enzo\\u0026oacute;tica dos bovinos em rebanho de corte criado na regi\\u0026atilde;o Metropolitana do Recife, Pernambuco. In: XIV Congresso Brasileiro de Buiatria e V Congresso Nordestino de Buiatria, 2023, Recife. Revista Brasileira de Buiatria. Recife: Associa\\u0026ccedil;\\u0026atilde;o Pernambucana de Buiatria, 2023. v. 1. p. 431-431.\\u003c/li\\u003e\\n\\u003cli\\u003eMORAN, Pedro Edgardo et al. Infectivity and persistence of bovine leukemia virus in human breast cells: assessing a possible zoonotic link to cancer. \\u003cem\\u003eVeterinary Research Communications\\u003c/em\\u003e, v. 49, p. 173, 2025. DOI: 10.1007/s11259-025-10738-4. Acesso em: 27 mar. 2025.\\u003c/li\\u003e\\n\\u003cli\\u003eNATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION (NCBI). \\u003cstrong\\u003eBasic Local Alignment Search Tool (BLAST)\\u003c/strong\\u003e. Dispon\\u0026iacute;vel em: https://blast.ncbi.nlm.nih.gov/Blast.cgi. Acesso em: 24 dez 2024\\u003c/li\\u003e\\n\\u003cli\\u003eOMSA \\u0026ndash; ORGANIZA\\u0026Ccedil;\\u0026Atilde;O MUNDIAL DA SA\\u0026Uacute;DE ANIMAL. Leucose Enzo\\u0026oacute;tica Bovina (\\u003cem\\u003eBovine Leukosis Enzootic\\u003c/em\\u003e). Dispon\\u0026iacute;vel em: https://www.woah.org/en/disease/enzootic-bovine-leukosis/. Acesso em: 20 nov. 2024.\\u003c/li\\u003e\\n\\u003cli\\u003ePEREIRA, Jos\\u0026eacute; Gomes et al. \\u003cem\\u003eDiagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil\\u003c/em\\u003e. Frontiers in Veterinary Science, [S.l.], v. 10, 13 jan. 2023. DOI: 10.3389/fvets.2022.1080994. Dispon\\u0026iacute;vel em: https://www.frontiersin.org/articles/10.3389/fvets.2022.1080994/full. Acesso em: 27 maio 2025.\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cstrong\\u003ePOLAT M., Takeshima S.N., Aida Y. et al. (2017). Epidemiology and genetic diversity of bovine leukemia virus. Virology Journal, 14(1):209. DOI: 10.1186/s12985-017-0876-4. Acesso em: 8 maio 2025\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003eQUADROS, Daniel Lazzari de et al. Oncogenic viral DNA related to human breast cancer found on cattle milk and meat. \\u003cem\\u003eComparative Immunology, Microbiology and Infectious Diseases\\u003c/em\\u003e, v. 101, p. 102053, 2023. DOI: 10.1016/j.cimid.2023.102053. Acesso em: 27 mar. 2025\\u003c/li\\u003e\\n\\u003cli\\u003eRAMBAUT, A. FigTree v1.4.4: a graphical viewer of phylogenetic trees. 2018. Dispon\\u0026iacute;vel em: http://tree.bio.ed.ac.uk/software/figtree/. Acesso em: 21 maio 2025.\\u003c/li\\u003e\\n\\u003cli\\u003eRODAKIEWICZ, Sheyla Michele et al. Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil. \\u003cem\\u003eArquivos do Instituto Biol\\u0026oacute;gico\\u003c/em\\u003e, v. 85, 2018. Dispon\\u0026iacute;vel em: https://doi.org/10.1590/1808-1657000742016. Acesso em: 30 dez. 2024.\\u003c/li\\u003e\\n\\u003cli\\u003eSZCOTKA, Maria; KUŹMAK, Jacek. Expression of bovine leukaemia virus (BLV) gp51 protein in blood and milk cells of cows with leukosis. \\u003cem\\u003eJournal of Veterinary Research\\u003c/em\\u003e, v. 66, n. 3, p. 337-345, 2022. Dispon\\u0026iacute;vel em: https://doi.org/10.2478/jvetres-2022-0035. Acesso em: 30 dez. 2024.\\u003c/li\\u003e\\n\\u003cli\\u003eTAMURA, K.; STECHER, G.; KUMAR, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. \\u003cem\\u003eMolecular Biology and Evolution\\u003c/em\\u003e, 2021. Dispon\\u0026iacute;vel em: https://doi.org/10.1093/molbev/msab120. Acesso em: 21 maio 2025.\\u003c/li\\u003e\\n\\u003cli\\u003e\\u003cstrong\\u003eVOSS, C.\\u003c/strong\\u003e et al. Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern. \\u003cem\\u003eScience Immunology\\u003c/em\\u003e, Washington, D.C., v. 6, n. 65, eabj1750, 2021. Dispon\\u0026iacute;vel em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410046/. Acesso em: 6 abril 2025\\u003c/li\\u003e\\n\\u003cli\\u003eWORLD ORGANISATION FOR ANIMAL HEALTH (OIE). Enzootic bovine leucosis. In: WORLD ORGANISATION FOR ANIMAL HEALTH (OIE). \\u003cem\\u003eTerrestrial manual.\\u003c/em\\u003e Paris: OIE, 2018. p. 1113-1124. Dispon\\u0026iacute;vel em: https://www.woah.org/fileadmin/Home/fr/Health_standards/tahm/3.04.09_EBL.pdf. Acesso em: 30 dez. 2024.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Viral genetics, epidemiology, veterinary medicine, phylogeny, One Health\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7013947/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7013947/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eEnzootic Bovine Leukosis (EBL) is an infectious and contagious disease caused by the Bovine Leukemia Virus (BLV), which has a significant economic impact on livestock and a potential zoonotic risk under investigation. In Brazil, although reports of infection exist in several regions, studies on the genetic diversity of the virus are scarce, especially in the Northeast. This study describes, for the first time, the isolation of BLV in the state of Pernambuco, from a blood sample of a seropositive bovine, with laboratory confirmation by Nested-PCR, sequencing of the env gene (gp51), and phylogenetic analysis. The isolation was performed by inoculating leukocytes into MDBK cells, followed by cytopathic effect monitoring and viral DNA validation. Phylogenetic analysis of the env gene (gp51) demonstrated clustering within genotype 1, showing high nucleotide identity with reference isolates previously registered in GenBank. These findings advance our knowledge of BLV genetic diversity in Brazil and highlight the critical need for sustained genomic surveillance, especially considering the potential risk of zoonotic transmission.\\u003c/p\\u003e\",\"manuscriptTitle\":\"First Isolation and Molecular Characterization of the Bovine Leukemia Virus in the State of Pernambuco, Brazil\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-07-31 19:13:30\",\"doi\":\"10.21203/rs.3.rs-7013947/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":1}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"966e9d72-580f-407e-a949-aa3a6a8d0f87\",\"owner\":[],\"postedDate\":\"July 31st, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-08-18T14:39:16+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-07-31 19:13:30\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7013947\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7013947\",\"identity\":\"rs-7013947\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}