Molecular Characteristics of Bovine Viral Diarrhea Virus Strain Isolated from Commercial Foetal Bovine Serum

Molecular Characteristics of Bovine Viral Diarrhea Virus Strain Isolated from Commercial Foetal Bovine Serum | 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 Molecular Characteristics of Bovine Viral Diarrhea Virus Strain Isolated from Commercial Foetal Bovine Serum Juanjuan Pan, Jianfeng Jiang, Ruli Duan, Yueyi Dang, Weihao Yu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4489986/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 Bovine viral diarrhea virus (BVDV) is commonly detected in biological products such as vaccines and serum. In this study, we have detected BVDV in commercial foetal bovine serum. In order to determine whether the serum contained infectious virus or viral genes, we inoculated the serum sample into MDBK cells. After six passages, results of indirect immunofluorescence assay confirmed that the commercial foetal bovine serum was contaminated with an infectious strain of BVDV, designated as BI-2023. The complete genome sequence of this isolate was 12,273 nt. Subsequent sequence analysis revealed that the 5'UTR genes and the full genome of BI-2023 shared 98% and 94.74% nucleotideidentity, respectively, with the BVDV1b reference strain CC13B. This suggests that BI-2023 represents a new subtype within the BVDV1b lineage. Phylogenetic analysis of the 5'UTR and full genome sequences of BVDVs indicatedthat BI-2023 clusters closely with a known BVDV1blineage. These findings underscore the importance of screening commercial foetal bovine serum for adventitious viruses. Bovine viral diarrhoea virus Contaminating Commercial foetal bovine serum Figures Figure 1 Figure 2 Figure 3 Introduction Cell lines are essential for the production of biologics, requiring the addition of bovine serum to the culture medium to support cell growth. However, the presence of adventitious viruses or pathogenic factors in bovine serum can compromise the safety of these biologics [ 1 – 3 ]. Bovine serum is known to potentially harbor a range of adventitious viruses, including, bovine bluetongue virus (BTV), bovine adenovirus (BAV), bovine poliovirus (BPV), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), rabies virus (RV), parainfluenza virus type III (PIV III), and bovine infectious rhinotracheitis virus (IBRV), etc [ 1 – 11 ]. Among these viruses, BVDV is frequently reported in bovine serum [ 1 – 10 ]. BVDV belongs to the Flaviviridae family, the genus Pestivirus , and is the principal pathogen responsible for bovine viral diarrhea and mucosal disease in cattle. These conditions are characterized by symptoms such as fever, diarrhea, leukopenia, reduced milk production, reproductive disorders, and immune dysfunction [ 12 ]. BVDV is a single-stranded, positive-sense RNA virus with a genome approximately 12.3 kb in length [ 12 , 13 ]. According to the 5 'UTR gene sequence, the virus can be divided into BVDV-1, BVDV-2 and BVDV-3 genotypes. Within BVDV-1, there are at least 22 recognized gene subtypes (1a ~ 1v) [ 13 , 14 ]. Furthermore, BVDV can be categorized into two biotypes: cytopathogenic (CP) and non-cytopathogenic (NCP), which are distinguished by the virus's ability to cause cytopathic effects (CPE) in infected cells. In this study, we have successfully isolated a non-cytopathogenic BVDV from commercial foetal bovine serum. The molecular characteristics of this isolate indicated that it belongs to a novel subtype within the 1b genotype. This finding highlights the diversity of BVDV strains and underscores the importance of vigilant monitoring to ensure the safety of biological products derived from bovine sources. Materials and methods Cell Culture and Virus Isolation MDBK cells were provided by laboratory of Tu Changchun. The commercial foetal bovine serum was purchased from Biological Industries (LOT: 2153440) (Figure S1 ). For the experiment, the MDBK cells were inoculated with the commercial foetal bovine serum for 2 h at 37°C in a 5% CO 2 incubator. After this incubation period, the inoculum was removed and the cells were then cultured in DMEM supplemented with 2% foetal bovine serum (FBS). The cells were maintained under these conditions for 96 h, after which they were subjected to three freeze-thaw cycles to release the viruses. This process was followed by six more passages of the cells to further propagate the virus. Indirect Immunofluorescence Assay (IFA) MDBK cells were seeded in 96-well culture plates and allowed to grow until they covered 70–80% of the well surface. Each well was then inoculated with the virus solution prepared above, and cultured for 48 h. Subsequently, the cells were fixed using 4% formaldehyde for 30 min. After fixation, the cells were blocked with 1% BSA at room temperature for 30 min. This was followed by the addition of a 1:1000 dilution of BVDV monoclonal antibody (laboratory of Tu Changchun), and the plates were incubated for 1 h at 37°C. After washing with PBST, the cells were incubated with a 1:500 dilution of Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen) at 37°C for 2 h. Following another wash with PBST, the cells were examined under a fluorescence microscope (Zeiss). RNA Extraction and RT-PCR Viral RNA was extracted from 200 µL of commercial foetal bovine serum using a Geneaid kit according to the instructions provided in the kit. Reverse transcription (RT) was carried out using the PrimeScript II Reverse Transcriptase reagents from Takara, following the protocol provided by the manufacturer. This process resulted in the production of complementary DNA (cDNA) from the serum as described above. Complete genomic amplification and Sequencing PCR methods were developed to amplify the genomic sequence of BVDV using primer pairs listed in Table S1 . The amplification process utilized 2×TransStart® FastPfu Fly PCR SuperMix, with the following cycling conditions: an initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 20 s, annealing at 52°C for 20 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min (TransGen Biotech). The PCR products were then examined using 1% agarose gel electrophoresis. The positive PCR amplicons were ligated into a pESI-T vector (Yeasen Biotech) and used to transform E. coli DH5α Chemically Competent Cells (Weidi Biotech). (Weidi Biotech). Subsequently, three clones from each amplicon were selected for Sanger sequencing of the DNA (Sangon Biotech). Phylogenetic analyses Twenty reference strains of BVDV, covering various genotypes from Japan, the USA, Germany, and China, were obtained from GenBank. The detailed information (GenBank accession numbers) about the sequences used in this study has been provided in Fig. 2 . The sequences were thereafter analyzed by MegAlign software in Lasergene v7.1 and phylogenetic trees of all the different target sequences were generated using the maximum-likelihood method based on the Tamura–Nei model. The reliability of the tree topologies was also evaluated using 1,000 bootstrap replicates to ensure accuracy [ 15 ]. Results Virus isolation and identification BVDV-positive commercial foetal bovine serum was inoculated into MDBK cells, and the status of the cells was monitored daily. After six passages, no significant cytopathic effects were observed in the inoculated MDBK cells. However, the nucleic acid of BVDV was detected in the culture supernatant using RT-PCR. This finding suggests that the isolate present in the commercial foetal bovine serum was NCP type of BVDV, designated as BVDV BI-2023. The presence of BVDV BI-2023 in the commercial foetal bovine serum was further confirmed through an indirect immunofluorescence assay (Fig. 1 A, 1 B). Sequence analyses of 5’-UTR To identify the genotype of BI-2023, a phylogenetic analysis was conducted using the maximum likelihood method. This analysis involved the 5’-UTR sequence of BI-2023 and 20 BVDV reference strains. As illustrated in Fig. 2 , BI-2023 was grouped with CP7, CC13B, JL-1, and TJ2018 within the BVDV-1b sub-genotype cluster. A similar result was observed after analyzing the 5’-UTR sequence of BI-2023 alongside 20 published pestiviruses listed in Table S2 , confirming that BI-2023 belonged to the BVDV-1b sub-genotype lineage. Whole genome sequence comparisons and phylogenetic analyses To elucidate the molecular characteristics of the strain, the entire genome of BVDV BI-2023 was amplified and sequenced. The complete genome sequence, consisting of 12,273 nt, was registered under the accession number OR753412. The nucleotide sequence homology between BVDV BI-2023 and reference strains of BVDV ranged from 67.2–94.74%. Notably, BVDV BI-2023 and the subgenotype CC13B reference strain exhibited a 94.74% nt identity, classifying the isolate in this study as a novel BVDV-1b. Further multiple sequence alignments of BVDV BI-2023 isolated in the present study revealed nt identity of 91.5%-97%, 90.7%-93.6%, 92.4%-95.9%, 91.3%-94.4%, 90.5%-93.8%, 91.0%-93.3%, 92.2%-95.1%, 94.1%-96.6%, 93.4%-97.0%, 91.7%-95.2%, 90.7%-94.1%, and 93.6%-95.4%, for the Npro , Core , E rns , E1 , E2 , p7 , NS2 , NS3 , NS4A , NS4B , NS5A , and NS5B gene sequence, respectively, and 94.6%-97.0%, 92.3%-95.2%, 96.5%-98.7%, 93.3%-96.4%, 90.1%-92.2%, 91.4%-94.3%, 92.7%-95.9%, 99.1%-98.8%, 100%, 95.6%-96.8%, 91.3%-95.4%, and 96.4%-97.4% aa similar to the Npro, Core, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B protein sequences of available reference strains of BVDV1b reference strain CC13B, CP7, JL-1, and TJ2018 (accession numbers: KF772785, U63479, KF501393, and OR004812), respectively. Additionally, phylogenetic analysis based on the whole genome sequence of BVDV showed that BVDV BI-2023 was closely related to BVDV 1b reference strains CC13B, CP7, JL-1, and TJ2018. The analysis revealed that BVDV BI-2023 clusters within the lineage of BVDV1b, as shown in Fig. 3 . This relationship underscores the genetic similarity of BI-2023 to these well-characterized strains, confirming its classification within the BVDV1b subgenotype. Discussion Bovine serum has been a crucial component in cell culture for over 50 years, yet it remains significantly contaminated by adventitious viruses [ 1 – 11 ]. Several viruses have been detected in bovine serum or its biological products [ 1 – 11 ]. Among these, BVDV is the most prevalent in cattle and a common contaminant in bovine serum [ 1 – 11 ]. In this study, we have detected and isolated a Ncp-type BVDV from commercial foetal bovine serum. However, other adventitious agents or pathogens that might be present in the serum were not accounted for, which could also impact the safety of biological products, underscoring the necessity for screening for adventitious viruses in commercial serum and related biological products prior to use. PCR is highly sensitive, the specificity of the primers used could limit the detection to particular strains of BVDV, potentially missing other variants or closely related viruses. Without adequate testing, biological products like commercial serum risk being contaminated by adventitious viruses, which can compromise experimental outcomes. Ncp-type BVDV, in particular, can establish persistent infections in laboratory-grown cells that are challenging to detect and have the potential to invalidate experiments and lead to trial failures. Techniques such as next-generation sequencing is effective for detecting such contaminants. Based on the comparison of 5'-UTR sequences, BVDV strains were classified into three genotypes, including BVDV-1, BVDV-2 and BVDV-3. Currently, the BVDV-1 genotype is further divided into 22 subgenotypes (1a-1v) [ 13 , 14 ]. Among BVDV, BVDV-1 strains are the most commonly detected, whereas BVDV-2 has been reported less frequently. In addition to nucleotide sequence differences, BVDV-2 is significantly more virulent than BVDV-1, often leading to severe acute infections and hemorrhagic syndromes [ 16 ]. BVDV-3, identified more recently, is akin to rinderpest virus and, similar to BVDV-1 and BVDV-2, is capable of causing respiratory and reproductive disorders as well as persistent infections [ 16 ]. In the present study, a new BVDV1b strain was identified in commercial foetal bovine serum. Next studies will screen the virus in different batches or sources of foetal bovine serum. Foetal bovine serum is an indispensable raw material in cell culture [ 1 – 3 ]. If the serum is contaminated with viruses, the resulting cell culture vaccines or biological products can also be contaminated [ 1 – 3 ]. Interestingly, prior studies have indicated that commercial bovine serum products from various regions in China are contaminated with at least one type of rinderpest virus [ 4 – 6 ]. In 1991, the National Animal Disease Center isolated 93 viruses from 190 batches of commercial foetal bovine serum [ 5 ]. Zhang et al. analyzed the genetic diversity of BVDV in commercial bovine serum and discovered that 3 out of 20 serum samples were highly homologous to pig BVDV, suggesting a significant presence of BVDV in bovine serum [ 4 ]. Contaminated serum can interfere with the diagnosis of viral infection, and using such contaminated serum to produce vaccines can lead to seroconversion or illness in vaccinated animals [ 4 – 6 ]. These findings underscore the frequent occurrence of BVDV contamination in cattle-derived materials. Presently, China permits the import of foetal bovine serum exclusively from countries free of BSE, such as Australia, New Zealand, and Uruguay. Nonetheless, the presence of American foetal bovine serum of unverified origin in the domestic market has brought uncertainty to the prevention and control of animal diseases in China [ 6 ]. In this study, commercial foetal bovine serum was imported from Biological Industries (LOT: 2153440), therefore, the laboratory must perform the detection of exogenous virus before using the foetal bovine serum. Conclusion In this study, a novel BVDV1b strain was identified in commercial foetal bovine serum through PCR amplification, culturing, and sequencing. This strain was classified as a NCP virus. It is imperative that regulatory agencies, vaccine manufacturers, and researchers take note of these findings. Additionally, this study highlights the need for improved detection technologies by quarantine departments and serum production companies to ensure the safety of biological products. Declarations Acknowledgments This study was supported by the major special science and technology project of Xinjiang Uyghur Autonomous Region (2023A02007-2), the National Natural Science Foundation of China (32060808) and the Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2022D01A167). Data availability The full genome sequence of BVDV BI-2023 generated in this study have been submitted to GenBank under accession no. OR753412. Conflict of interest The authors declare no conflict of interest to be disclosed for the present study. References Pastoret PP (2010) Human and animal vaccine contaminations. Biologicals 38(3):332–334 Gómez-Romero N, Velazquez-Salinas L, Ridpath JF, Verdugo-Rodríguez A, Basurto-Alcántara FJ (2021) Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and foetal bovine serum lots originating in Mexico. Arch Virol 166(7):1999–2003 Nuttal PA, Luther PD, Stott EJ (1977) Viral contamination of bovine foetal serum and cell cultures. Nature 266:835–837 Zhang Q, Li SQ, Li J, Xiong W, Wang QQ, Li CY, Huang ZR (2013) Detection of bovine viral diarrhoea virus contamination in imported foetal calf serum. Chin Anim Health Inspect 30(2):55–57 (In Chinese) Xia HY, Vijayaraghavan B, Belák S, Liu LH (2011) Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches. PLoS ONE 6(12):e28553 Wang JH, Li S, He WR, Zhao Q, Qiu HJ (2016) Isolation and identification of bovine viral diarrhea virus from imported foetal bovine serum. Chin J Biol 29(3):308–311 (In Chinese) Bolin SR, Matthews PJ, Ridpath JF (1991) Methods for detection and frequency of contamination of foetal calf serum with bovine viral diarrhoea virus and antibodies against bovine viral diarrhoea virus. J Vet Diagn Invest 3(3):199–203 Bolin SR, Ridpath JF (1998) Prevalence of bovine viral diarrhoea virus genotypes and antibody against those viral genotypes in foetal bovine serum. J Vet Diagn Invest 10:135–139 Makoschey B, van Gelder PT, Keijsers V, Goovaerts D (2003) Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production. Biologicals 31(3):203–208 Zabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL (2000) Contamination of bovine foetal serum with bovine viral diarrhea virus. Rev Argent Microbiol 32(1):27–32 Xia H, Vijayaraghavan B, Belák S, Liu L (2011) Detection and identifcation of the atypical bovine pestiviruses in comercial foetal serum batches. PLoS ONE 6(12):1–3 Chi SS, Chen S, Jia WJ, He YJ, Ren LZ, Wang XL (2022) Non-structural proteins of bovine viral diarrhea virus. Virus Genes 58(6):491–500 Zhang SQ, Tan B, Liu ZY, Wang C, Guo L, Sun L, Cheng SP (2019) Isolation identification and pathogenicity analysis of bovine viral diarrhea virus JLS01 strain from porcine. Chin Anim Husband Vet Med 46(2):557–564 Mucellini CI, Júnior JVJS, de Oliveira PSB, Weiblen R, Flores EF (2023) Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Virus Genes 59(6):836–844 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874 Riitho V, Strong R, Larska M, Graham SP, Steinbach F (2020) Bovine pestivirus heterogeneity and its potential impact on vaccination and diagnosis. Viruses 12(10):1134 Additional Declarations No competing interests reported. Supplementary Files FigureS1.jpg TableS1.docx 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. 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-4489986","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":312372023,"identity":"20f98272-8329-4625-9944-2ec1a545701b","order_by":0,"name":"Juanjuan Pan","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Juanjuan","middleName":"","lastName":"Pan","suffix":""},{"id":312372024,"identity":"7e09b6df-00cd-416e-a5cc-3067eaf6b12a","order_by":1,"name":"Jianfeng Jiang","email":"","orcid":"","institution":"Jilin 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10:00:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4489986/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4489986/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58053895,"identity":"02d21622-bf05-4d20-adcd-32ee81adcdba","added_by":"auto","created_at":"2024-06-10 13:40:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7579243,"visible":true,"origin":"","legend":"\u003cp\u003eIndirect immunofluorescence assay for detecting BVDV.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/dc82386542510a05f26fa1cb.png"},{"id":58053894,"identity":"d043a7e8-51be-477e-a647-7c1ff76ea23e","added_by":"auto","created_at":"2024-06-10 13:40:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":758703,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of the 5'-UTR nucleotide sequences of BVDV. Fixed circles represent the BI-2023 strain isolated in this study.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/7ac3c4b8d940e93b932a543c.png"},{"id":58053896,"identity":"6f46f15f-90e6-48a6-8f72-cfaf8fa198b2","added_by":"auto","created_at":"2024-06-10 13:40:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":845322,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on the complete genomic sequences of BVDV. Fixed circles indicate the BI-2023 isolated in this study.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/98bff863e4caebe00df031f9.png"},{"id":58840794,"identity":"e75e5da1-e7c2-4ddf-8cac-d2009a6139c3","added_by":"auto","created_at":"2024-06-21 22:47:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10059390,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/98abb85d-f89f-40ff-a7db-7ad365c42242.pdf"},{"id":58053898,"identity":"2796e57e-00c2-4a55-a964-ca04284f41cf","added_by":"auto","created_at":"2024-06-10 13:40:16","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":183548,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/e52278973029fda98f64683b.jpg"},{"id":58053897,"identity":"ce9fd4d2-212c-4323-94f1-05bec3983d23","added_by":"auto","created_at":"2024-06-10 13:40:16","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13487,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4489986/v1/d8398dee523bc5d6954e6815.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Characteristics of Bovine Viral Diarrhea Virus Strain Isolated from Commercial Foetal Bovine Serum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCell lines are essential for the production of biologics, requiring the addition of bovine serum to the culture medium to support cell growth. However, the presence of adventitious viruses or pathogenic factors in bovine serum can compromise the safety of these biologics [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Bovine serum is known to potentially harbor a range of adventitious viruses, including, bovine bluetongue virus (BTV), bovine adenovirus (BAV), bovine poliovirus (BPV), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), rabies virus (RV), parainfluenza virus type III (PIV III), and bovine infectious rhinotracheitis virus (IBRV), etc [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these viruses, BVDV is frequently reported in bovine serum [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBVDV belongs to the \u003cem\u003eFlaviviridae\u003c/em\u003e family, the genus \u003cem\u003ePestivirus\u003c/em\u003e, and is the principal pathogen responsible for bovine viral diarrhea and mucosal disease in cattle. These conditions are characterized by symptoms such as fever, diarrhea, leukopenia, reduced milk production, reproductive disorders, and immune dysfunction [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. BVDV is a single-stranded, positive-sense RNA virus with a genome approximately 12.3 kb in length [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. According to the 5 'UTR gene sequence, the virus can be divided into BVDV-1, BVDV-2 and BVDV-3 genotypes. Within BVDV-1, there are at least 22 recognized gene subtypes (1a\u0026thinsp;~\u0026thinsp;1v) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, BVDV can be categorized into two biotypes: cytopathogenic (CP) and non-cytopathogenic (NCP), which are distinguished by the virus's ability to cause cytopathic effects (CPE) in infected cells.\u003c/p\u003e \u003cp\u003eIn this study, we have successfully isolated a non-cytopathogenic BVDV from commercial foetal bovine serum. The molecular characteristics of this isolate indicated that it belongs to a novel subtype within the 1b genotype. This finding highlights the diversity of BVDV strains and underscores the importance of vigilant monitoring to ensure the safety of biological products derived from bovine sources.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture and Virus Isolation\u003c/h2\u003e \u003cp\u003eMDBK cells were provided by laboratory of Tu Changchun. The commercial foetal bovine serum was purchased from Biological Industries (LOT: 2153440) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the experiment, the MDBK cells were inoculated with the commercial foetal bovine serum for 2 h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After this incubation period, the inoculum was removed and the cells were then cultured in DMEM supplemented with 2% foetal bovine serum (FBS). The cells were maintained under these conditions for 96 h, after which they were subjected to three freeze-thaw cycles to release the viruses. This process was followed by six more passages of the cells to further propagate the virus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIndirect Immunofluorescence Assay (IFA)\u003c/h2\u003e \u003cp\u003eMDBK cells were seeded in 96-well culture plates and allowed to grow until they covered 70\u0026ndash;80% of the well surface. Each well was then inoculated with the virus solution prepared above, and cultured for 48 h. Subsequently, the cells were fixed using 4% formaldehyde for 30 min. After fixation, the cells were blocked with 1% BSA at room temperature for 30 min. This was followed by the addition of a 1:1000 dilution of BVDV monoclonal antibody (laboratory of Tu Changchun), and the plates were incubated for 1 h at 37\u0026deg;C. After washing with PBST, the cells were incubated with a 1:500 dilution of Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen) at 37\u0026deg;C for 2 h. Following another wash with PBST, the cells were examined under a fluorescence microscope (Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and RT-PCR\u003c/h2\u003e \u003cp\u003eViral RNA was extracted from 200 \u0026micro;L of commercial foetal bovine serum using a Geneaid kit according to the instructions provided in the kit. Reverse transcription (RT) was carried out using the PrimeScript II Reverse Transcriptase reagents from Takara, following the protocol provided by the manufacturer. This process resulted in the production of complementary DNA (cDNA) from the serum as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eComplete genomic amplification and Sequencing\u003c/h2\u003e \u003cp\u003ePCR methods were developed to amplify the genomic sequence of BVDV using primer pairs listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The amplification process utilized \u003cem\u003e2\u0026times;TransStart\u0026reg; FastPfu Fly\u003c/em\u003e PCR SuperMix, with the following cycling conditions: an initial denaturation at 95\u0026deg;C for 2 min, followed by 35 cycles of denaturation at 95\u0026deg;C for 20 s, annealing at 52\u0026deg;C for 20 s, and extension at 72\u0026deg;C for 30 s, with a final extension at 72\u0026deg;C for 5 min (TransGen Biotech). The PCR products were then examined using 1% agarose gel electrophoresis. The positive PCR amplicons were ligated into a pESI-T vector (Yeasen Biotech) and used to transform \u003cem\u003eE. coli\u003c/em\u003e DH5α Chemically Competent Cells (Weidi Biotech). (Weidi Biotech). Subsequently, three clones from each amplicon were selected for Sanger sequencing of the DNA (Sangon Biotech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e \u003cp\u003eTwenty reference strains of BVDV, covering various genotypes from Japan, the USA, Germany, and China, were obtained from GenBank. The detailed information (GenBank accession numbers) about the sequences used in this study has been provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The sequences were thereafter analyzed by MegAlign software in Lasergene v7.1 and phylogenetic trees of all the different target sequences were generated using the maximum-likelihood method based on the Tamura\u0026ndash;Nei model. The reliability of the tree topologies was also evaluated using 1,000 bootstrap replicates to ensure accuracy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVirus isolation and identification\u003c/h2\u003e \u003cp\u003eBVDV-positive commercial foetal bovine serum was inoculated into MDBK cells, and the status of the cells was monitored daily. After six passages, no significant cytopathic effects were observed in the inoculated MDBK cells. However, the nucleic acid of BVDV was detected in the culture supernatant using RT-PCR. This finding suggests that the isolate present in the commercial foetal bovine serum was NCP type of BVDV, designated as BVDV BI-2023. The presence of BVDV BI-2023 in the commercial foetal bovine serum was further confirmed through an indirect immunofluorescence assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSequence analyses of 5\u0026rsquo;-UTR\u003c/h2\u003e \u003cp\u003eTo identify the genotype of BI-2023, a phylogenetic analysis was conducted using the maximum likelihood method. This analysis involved the 5\u0026rsquo;-UTR sequence of BI-2023 and 20 BVDV reference strains. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, BI-2023 was grouped with CP7, CC13B, JL-1, and TJ2018 within the BVDV-1b sub-genotype cluster. A similar result was observed after analyzing the 5\u0026rsquo;-UTR sequence of BI-2023 alongside 20 published pestiviruses listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, confirming that BI-2023 belonged to the BVDV-1b sub-genotype lineage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequence comparisons and phylogenetic analyses\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular characteristics of the strain, the entire genome of BVDV BI-2023 was amplified and sequenced. The complete genome sequence, consisting of 12,273 nt, was registered under the accession number OR753412. The nucleotide sequence homology between BVDV BI-2023 and reference strains of BVDV ranged from 67.2\u0026ndash;94.74%. Notably, BVDV BI-2023 and the subgenotype CC13B reference strain exhibited a 94.74% nt identity, classifying the isolate in this study as a novel BVDV-1b. Further multiple sequence alignments of BVDV BI-2023 isolated in the present study revealed nt identity of 91.5%-97%, 90.7%-93.6%, 92.4%-95.9%, 91.3%-94.4%, 90.5%-93.8%, 91.0%-93.3%, 92.2%-95.1%, 94.1%-96.6%, 93.4%-97.0%, 91.7%-95.2%, 90.7%-94.1%, and 93.6%-95.4%, for the \u003cem\u003eNpro\u003c/em\u003e, \u003cem\u003eCore\u003c/em\u003e, \u003cem\u003eE\u003c/em\u003e\u003csup\u003e\u003cem\u003erns\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eE1\u003c/em\u003e, \u003cem\u003eE2\u003c/em\u003e, \u003cem\u003ep7\u003c/em\u003e, \u003cem\u003eNS2\u003c/em\u003e, \u003cem\u003eNS3\u003c/em\u003e, \u003cem\u003eNS4A\u003c/em\u003e, \u003cem\u003eNS4B\u003c/em\u003e, \u003cem\u003eNS5A\u003c/em\u003e, and \u003cem\u003eNS5B\u003c/em\u003e gene sequence, respectively, and 94.6%-97.0%, 92.3%-95.2%, 96.5%-98.7%, 93.3%-96.4%, 90.1%-92.2%, 91.4%-94.3%, 92.7%-95.9%, 99.1%-98.8%, 100%, 95.6%-96.8%, 91.3%-95.4%, and 96.4%-97.4% aa similar to the Npro, Core, E\u003csup\u003erns\u003c/sup\u003e, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B protein sequences of available reference strains of BVDV1b reference strain CC13B, CP7, JL-1, and TJ2018 (accession numbers: KF772785, U63479, KF501393, and OR004812), respectively.\u003c/p\u003e \u003cp\u003eAdditionally, phylogenetic analysis based on the whole genome sequence of BVDV showed that BVDV BI-2023 was closely related to BVDV 1b reference strains CC13B, CP7, JL-1, and TJ2018. The analysis revealed that BVDV BI-2023 clusters within the lineage of BVDV1b, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. This relationship underscores the genetic similarity of BI-2023 to these well-characterized strains, confirming its classification within the BVDV1b subgenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBovine serum has been a crucial component in cell culture for over 50 years, yet it remains significantly contaminated by adventitious viruses [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several viruses have been detected in bovine serum or its biological products [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these, BVDV is the most prevalent in cattle and a common contaminant in bovine serum [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, we have detected and isolated a Ncp-type BVDV from commercial foetal bovine serum. However, other adventitious agents or pathogens that might be present in the serum were not accounted for, which could also impact the safety of biological products, underscoring the necessity for screening for adventitious viruses in commercial serum and related biological products prior to use.\u003c/p\u003e \u003cp\u003ePCR is highly sensitive, the specificity of the primers used could limit the detection to particular strains of BVDV, potentially missing other variants or closely related viruses. Without adequate testing, biological products like commercial serum risk being contaminated by adventitious viruses, which can compromise experimental outcomes. Ncp-type BVDV, in particular, can establish persistent infections in laboratory-grown cells that are challenging to detect and have the potential to invalidate experiments and lead to trial failures. Techniques such as next-generation sequencing is effective for detecting such contaminants.\u003c/p\u003e \u003cp\u003eBased on the comparison of 5'-UTR sequences, BVDV strains were classified into three genotypes, including BVDV-1, BVDV-2 and BVDV-3. Currently, the BVDV-1 genotype is further divided into 22 subgenotypes (1a-1v) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among BVDV, BVDV-1 strains are the most commonly detected, whereas BVDV-2 has been reported less frequently. In addition to nucleotide sequence differences, BVDV-2 is significantly more virulent than BVDV-1, often leading to severe acute infections and hemorrhagic syndromes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. BVDV-3, identified more recently, is akin to rinderpest virus and, similar to BVDV-1 and BVDV-2, is capable of causing respiratory and reproductive disorders as well as persistent infections [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the present study, a new BVDV1b strain was identified in commercial foetal bovine serum. Next studies will screen the virus in different batches or sources of foetal bovine serum.\u003c/p\u003e \u003cp\u003eFoetal bovine serum is an indispensable raw material in cell culture [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. If the serum is contaminated with viruses, the resulting cell culture vaccines or biological products can also be contaminated [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Interestingly, prior studies have indicated that commercial bovine serum products from various regions in China are contaminated with at least one type of rinderpest virus [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In 1991, the National Animal Disease Center isolated 93 viruses from 190 batches of commercial foetal bovine serum [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Zhang et al. analyzed the genetic diversity of BVDV in commercial bovine serum and discovered that 3 out of 20 serum samples were highly homologous to pig BVDV, suggesting a significant presence of BVDV in bovine serum [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Contaminated serum can interfere with the diagnosis of viral infection, and using such contaminated serum to produce vaccines can lead to seroconversion or illness in vaccinated animals [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These findings underscore the frequent occurrence of BVDV contamination in cattle-derived materials.\u003c/p\u003e \u003cp\u003ePresently, China permits the import of foetal bovine serum exclusively from countries free of BSE, such as Australia, New Zealand, and Uruguay. Nonetheless, the presence of American foetal bovine serum of unverified origin in the domestic market has brought uncertainty to the prevention and control of animal diseases in China [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, commercial foetal bovine serum was imported from Biological Industries (LOT: 2153440), therefore, the laboratory must perform the detection of exogenous virus before using the foetal bovine serum.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a novel BVDV1b strain was identified in commercial foetal bovine serum through PCR amplification, culturing, and sequencing. This strain was classified as a NCP virus. It is imperative that regulatory agencies, vaccine manufacturers, and researchers take note of these findings. Additionally, this study highlights the need for improved detection technologies by quarantine departments and serum production companies to ensure the safety of biological products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the\u0026nbsp;major special science and technology project\u0026nbsp;of\u0026nbsp;Xinjiang Uyghur Autonomous Region\u0026nbsp;(2023A02007-2),\u0026nbsp;the National Natural Science Foundation of China (32060808) and the Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2022D01A167).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe full genome sequence of BVDV BI-2023\u0026nbsp;generated in this study have been submitted to GenBank under accession no.\u0026nbsp;OR753412.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest to be disclosed for the present study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePastoret PP (2010) Human and animal vaccine contaminations. Biologicals 38(3):332\u0026ndash;334\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026oacute;mez-Romero N, Velazquez-Salinas L, Ridpath JF, Verdugo-Rodr\u0026iacute;guez A, Basurto-Alc\u0026aacute;ntara FJ (2021) Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and foetal bovine serum lots originating in Mexico. Arch Virol 166(7):1999\u0026ndash;2003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuttal PA, Luther PD, Stott EJ (1977) Viral contamination of bovine foetal serum and cell cultures. Nature 266:835\u0026ndash;837\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Li SQ, Li J, Xiong W, Wang QQ, Li CY, Huang ZR (2013) Detection of bovine viral diarrhoea virus contamination in imported foetal calf serum. Chin Anim Health Inspect 30(2):55\u0026ndash;57 (In Chinese)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia HY, Vijayaraghavan B, Bel\u0026aacute;k S, Liu LH (2011) Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches. PLoS ONE 6(12):e28553\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang JH, Li S, He WR, Zhao Q, Qiu HJ (2016) Isolation and identification of bovine viral diarrhea virus from imported foetal bovine serum. Chin J Biol 29(3):308\u0026ndash;311 (In Chinese)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolin SR, Matthews PJ, Ridpath JF (1991) Methods for detection and frequency of contamination of foetal calf serum with bovine viral diarrhoea virus and antibodies against bovine viral diarrhoea virus. J Vet Diagn Invest 3(3):199\u0026ndash;203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolin SR, Ridpath JF (1998) Prevalence of bovine viral diarrhoea virus genotypes and antibody against those viral genotypes in foetal bovine serum. J Vet Diagn Invest 10:135\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakoschey B, van Gelder PT, Keijsers V, Goovaerts D (2003) Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production. Biologicals 31(3):203\u0026ndash;208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL (2000) Contamination of bovine foetal serum with bovine viral diarrhea virus. Rev Argent Microbiol 32(1):27\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia H, Vijayaraghavan B, Bel\u0026aacute;k S, Liu L (2011) Detection and identifcation of the atypical bovine pestiviruses in comercial foetal serum batches. PLoS ONE 6(12):1\u0026ndash;3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChi SS, Chen S, Jia WJ, He YJ, Ren LZ, Wang XL (2022) Non-structural proteins of bovine viral diarrhea virus. Virus Genes 58(6):491\u0026ndash;500\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang SQ, Tan B, Liu ZY, Wang C, Guo L, Sun L, Cheng SP (2019) Isolation identification and pathogenicity analysis of bovine viral diarrhea virus JLS01 strain from porcine. Chin Anim Husband Vet Med 46(2):557\u0026ndash;564\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMucellini CI, J\u0026uacute;nior JVJS, de Oliveira PSB, Weiblen R, Flores EF (2023) Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Virus Genes 59(6):836\u0026ndash;844\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870\u0026ndash;1874\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiitho V, Strong R, Larska M, Graham SP, Steinbach F (2020) Bovine pestivirus heterogeneity and its potential impact on vaccination and diagnosis. Viruses 12(10):1134\u003c/span\u003e\u003c/li\u003e\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":"[email protected]","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":"Bovine viral diarrhoea virus, Contaminating, Commercial foetal bovine serum","lastPublishedDoi":"10.21203/rs.3.rs-4489986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4489986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBovine viral diarrhea virus (BVDV) is commonly detected in biological products such as vaccines and serum. In this study, we have detected BVDV in commercial foetal bovine serum. In order to determine whether the serum contained infectious virus or viral genes, we inoculated the serum sample into MDBK cells. After six passages, results of indirect immunofluorescence assay confirmed that the commercial foetal bovine serum was contaminated with an infectious strain of BVDV, designated as BI-2023. The complete genome sequence of this isolate was 12,273 nt. Subsequent sequence analysis revealed that the 5'UTR genes and the full genome of BI-2023 shared 98% and 94.74% nucleotideidentity, respectively, with the BVDV1b reference strain CC13B. This suggests that BI-2023 represents a new subtype within the BVDV1b lineage. Phylogenetic analysis of the 5'UTR and full genome sequences of BVDVs indicatedthat BI-2023 clusters closely with a known BVDV1blineage. These findings underscore the importance of screening commercial foetal bovine serum for adventitious viruses.\u003c/p\u003e","manuscriptTitle":"Molecular Characteristics of Bovine Viral Diarrhea Virus Strain Isolated from Commercial Foetal Bovine Serum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 13:40:11","doi":"10.21203/rs.3.rs-4489986/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"6625dce3-2473-4309-a69d-edce74719388","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T01:39:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 13:40:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4489986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4489986","identity":"rs-4489986","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}