{"paper_id":"3c787f04-ab20-49bf-b080-38e0fcd2c800","body_text":"Evolution of the 3′ Untranslated Region Enhances NS5B Binding During Cell Culture Adaptation of Classical Swine Fever Virus | 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 Research Article Evolution of the 3′ Untranslated Region Enhances NS5B Binding During Cell Culture Adaptation of Classical Swine Fever Virus Aman Kamboj, Tushar Gaikwad, Nachiket Atale, Praveen Kumar Gupta, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8668214/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 Classical swine fever virus (CSFV) adapts to cell culture through progressive genetic changes that enhance viral replication and fitness. Although adaptive mutations in viral proteins have been extensively characterized, the contribution of noncoding genomic regions to this process remains poorly understood. In this study, we investigated the evolutionary dynamics of the CSFV 3′ untranslated region (3′ UTR) during serial adaptation in PK-15 cells and examined its functional interaction with the viral RNA-dependent RNA polymerase NS5B. CSFV isolated from spleen tissue was serially passaged in PK-15 cells, resulting in enhanced viral replication and the emergence of cytopathic effects at later passages. Sequencing of the 3′ UTR across multiple passages revealed the accumulation of nucleotide substitutions, insertions, and recurrent deletions, indicating strong selective pressure on this regulatory region during in vitro adaptation. Phylogenetic analysis based on alignment-derived evolutionary distances demonstrated a passage-dependent divergence of 3′ UTR sequences, with early passages clustering closely, intermediate passages forming a distinct subclade, and late passages exhibiting increased evolutionary distances, consistent with a stepwise and non-linear adaptive trajectory. Molecular docking analyses showed a progressive increase in binding affinity between evolved 3′ UTR RNA variants and NS5B across successive passages. An intermediate passage variant (P20), occupying a transitional phylogenetic position, was selected for molecular dynamics simulation and formed a stable NS5B 3′ UTR complex under explicit solvent conditions. The complex maintained structural integrity throughout the simulation, characterized by sustained hydrogen bonding, low intermolecular distances, and limited conformational fluctuations. Collectively, these findings demonstrate that evolutionary remodeling of the CSFV 3′ UTR enhances its interaction with NS5B, stabilizes the viral replication complex, and contributes to efficient cell culture adaptation, highlighting the functional importance of noncoding RNA elements in viral evolution. Classical swine fever virus 3′ untranslated region (3′ UTR) Cell culture adaptation Viral evolution NS5B RNA-dependent RNA polymerase RNA–protein interactions Molecular dynamics simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Classical swine fever (CSF), also referred to as hog cholera, is a highly contagious viral disease of domestic pigs and wild boar that continues to pose a serious threat to the global pig industry. The disease is endemic in several regions of Asia, South and Central America, parts of Europe, and selected African and Caribbean countries (Edwards et al., 2000 ; World Organisation for Animal Health (WOAH), 2024 ). In India, CSF represents a major economic burden, particularly in the northeastern states where pig farming constitutes an important source of income for rural communities (Barman et al., 2016 ; Patil et al., 2018 ). Recurrent outbreaks result in substantial losses due to high morbidity and mortality, trade restrictions, and the costs associated with vaccination and control measures. A detailed understanding of the molecular mechanisms governing viral replication and adaptation is therefore essential for the development of effective prevention and control strategies, including vaccine design and molecular surveillance (Kamboj et al., 2024 ). CSF is caused by Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae (Fauquet et al., 2005 ). Related pestiviruses include bovine viral diarrhea virus (BVDV) and border disease virus (BDV) of sheep, which serve as established models for studying pestiviral replication and evolution. CSFV is an enveloped, positive-sense, single-stranded RNA virus with a genome of approximately 12.5 kb (Meyers & Thiel, 1996 ; Vanderhallen et al., 1999 ). The genome contains a single open reading frame (ORF) of about 11.7 kb that encodes a polyprotein of nearly 3,900 amino acids. This polyprotein is co- and post-translationally processed into four structural proteins (C, Erns, E1, and E2) and eight non-structural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which together coordinate viral assembly, genome replication, and modulation of host antiviral responses (Beer et al., 2015 ; Isken et al., 2004 ; Rios et al., 2017 ). Complete genome sequencing of Indian CSFV field isolates has further contributed to understanding the molecular diversity and evolutionary relationships of circulating strains (Kamboj et al., 2014 ). The viral ORF is flanked by highly structured 5 \\(\\:{\\prime\\:}\\) and 3 \\(\\:{\\prime\\:}\\) untranslated regions (UTRs) that play essential regulatory roles in the viral life cycle. The 5 \\(\\:{\\prime\\:}\\) UTR contains an internal ribosome entry site (IRES) that enables cap-independent initiation of translation, whereas the 3 \\(\\:{\\prime\\:}\\) UTR is involved in the initiation of RNA replication and in coordinating the balance between translation and replication (Hsu et al., 2014 ; Pankraz et al., 2005 ; Y. Xiao et al., 2004 ). In related pestiviruses, the 3 \\(\\:{\\prime\\:}\\) UTR has been shown to participate in long-range RNA interactions and to engage host and viral factors that regulate replication efficiency and viral fitness (Isken et al., 2004 ). Consequently, sequence variation or structural remodeling within the 3 \\(\\:{\\prime\\:}\\) UTR has the potential to substantially influence viral replication and pathogenicity. Among the viral non-structural proteins, NS5B functions as the RNA-dependent RNA polymerase (RdRp) and is indispensable for viral genome synthesis. NS5B interacts with both positive- and negative-sense RNA templates, with particular importance attributed to its interaction with the 3 \\(\\:{\\prime\\:}\\) UTR during initiation of RNA replication (Li et al., 2018 ; M. Xiao et al., 2004 ). Efficient and stable association between the 3 \\(\\:{\\prime\\:}\\) UTR and NS5B is therefore critical for productive infection and viral persistence. CSFV infections are typically non-cytopathic in cell culture, necessitating the use of molecular and ultrastructural techniques such as RT-PCR and transmission electron microscopy for virus detection and characterization (Blome et al., 2017 ; Ganges et al., 2020 ). Like other RNA viruses, CSFV exhibits substantial genetic diversity due to the absence of proofreading activity in its polymerase, resulting in a dynamic population of closely related variants, or quasispecies (Johnston et al., 2020 ; Martínez et al., 2012 ). During serial passage in cell culture, selective pressures favor variants with improved replication efficiency under in vitro conditions. While such adaptations can enhance viral growth, they may also alter virulence, antigenicity, and replication dynamics, with potential implications for vaccine development and diagnostic reliability (Hadsbjerg et al., 2016 ; S. W. Huang et al., 2012 ; Lamothe-Reyes et al., 2023 ; Wang et al., 2020 ). Advances in reverse genetics systems, including infectious cDNA clone construction, have facilitated detailed functional analyses of CSFV replication and adaptation mechanisms (Kamboj et al., 2015 ). Despite the recognized importance of the 3 \\(\\:{\\prime\\:}\\) UTR in pestiviral replication, relatively few studies have systematically examined how this region evolves during cell culture adaptation and how such changes influence RNA–polymerase interactions. To address this knowledge gap, the present study investigates the adaptation of CSFV to PK-15 cells with a specific focus on evolutionary changes within the 3 \\(\\:{}^{{\\prime\\:}}\\) untranslated region (3 \\(\\:{}^{{\\prime\\:}}\\) UTR). Genomic variations in the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR were systematically analyzed across serial passages to identify substitutions, insertions, and deletions associated with in vitro adaptation. In addition, phylogenetic analysis based on alignment-derived evolutionary distances was performed to resolve passage-dependent divergence patterns and to define transitional and late-stage adaptive lineages. Computational docking and molecular dynamics simulations were further employed to assess how adaptive remodeling of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR influences its interaction with the viral RNA-dependent RNA polymerase NS5B. By integrating experimental virology with phylogenetic, structural, and computational analyses, this study provides mechanistic insight into the role of 3 \\(\\:{}^{{\\prime\\:}}\\) UTR evolution in CSFV adaptation. Elucidating how noncoding regulatory RNA elements contribute to viral fitness and replication efficiency advances our understanding of pestiviral biology and may inform future strategies for vaccine design, antiviral development, and improved control of classical swine fever. Materials and Methods Cells and Virus An Indian field isolate of Classical swine fever virus (CSFV), designated CSFV/IVRI/VB0, was obtained from the spleen of a clinically infected pig. Virus isolation and adaptation were performed using porcine kidney (PK-15) cells (ATCC CCL-33). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) and an antibiotic–antimycotic mixture containing penicillin G (100 U/mL), streptomycin sulfate (100 \\(\\:\\mu\\:\\) g/mL), and amphotericin B (0.025 \\(\\:\\mu\\:\\) g/mL). Cultures were incubated at 37 \\(\\:{}^{\\circ\\:}\\) C in a humidified atmosphere with 5% CO \\(\\:{}_{2}\\) . Virus Isolation and Serial Passage Spleen tissue was processed to prepare a 10% (w/v) suspension in phosphate-buffered saline (PBS) and clarified by centrifugation at 15,000 rpm for 10 min at 4 \\(\\:{}^{\\circ\\:}\\) C. Confluent PK-15 monolayers were inoculated with 1 mL of clarified supernatant and incubated for 1 h to allow viral adsorption. The inoculum was then removed, fresh medium was added, and cultures were incubated under standard conditions. At four days post-infection, cells were subjected to a freeze–thaw cycle to release intracellular virus. The culture supernatant was used to infect fresh PK-15 monolayers. This procedure was repeated for 70 serial passages. Viral presence was confirmed at every tenth passage by RT-PCR. Transmission Electron Microscopy Virus particles were visualized using negative-staining transmission electron microscopy (TEM). Culture supernatants were filtered and fixed in 2% paraformaldehyde. Samples were adsorbed onto carbon-coated copper grids for 30–60 s, rinsed with deionized water, and negatively stained with phosphotungstic acid for 30 s. Grids were air-dried and examined under a transmission electron microscope to confirm virion morphology. RNA Extraction and Amplification of the 3UTR RNA Extraction and Amplification of the 3 \\(\\:{\\prime\\:}\\) UTR Total RNA was extracted from spleen tissue (passage 0) and from infected PK-15 cells at passages 1, 10, 20, 30, 40, 50, 60, and 70 using TRIzol™ reagent (Invitrogen, USA). RNA was resuspended in 20 \\(\\:\\mu\\:\\) L of nuclease-free water. Complementary DNA (cDNA) synthesis was carried out using the RevertAid Reverse Transcriptase kit (Thermo Scientific, USA). The full-length 3 \\(\\:{\\prime\\:}\\) UTR was amplified by two-step RT-PCR using self-designed primers. PCR amplification was performed using PfuUltra II Fusion HS DNA Polymerase (Agilent, USA). Amplified products were analyzed by 1% agarose gel electrophoresis. Cloning and Sequencing of the 3UTR Cloning and Sequencing of the 3 \\(\\:{\\prime\\:}\\) UTR The 238 bp 3 \\(\\:{\\prime\\:}\\) UTR amplicons were gel-purified and cloned into the pJET1.2 blunt-end vector (Thermo Scientific, USA). Recombinant plasmids were transformed into Escherichia coli DH5 \\(\\:\\alpha\\:\\) competent cells and selected on ampicillin containing LB agar plates. Positive clones were confirmed by PCR using T7 promoter and insert-specific primers and subsequently sequenced by Sanger sequencing (Eurofins Genomics, Bengaluru, India). Sequence assembly and alignment were performed using Lasergene v6 and BioEdit software. Phylogenetic analysis of the CSFV 3 \\(\\:{}^{{\\prime\\:}}\\) UTR Phylogenetic analysis was conducted to assess the evolutionary relationships among Classical swine fever virus (CSFV) 3 \\(\\:{}^{{\\prime\\:}}\\) untranslated region (3 \\(\\:{}^{{\\prime\\:}}\\) UTR) sequences obtained across serial passages during cell culture adaptation. Full-length 3 \\(\\:{}^{{\\prime\\:}}\\) UTR sequences corresponding to passages 0, 1, 10, 20, 30, 40, 50, 60, and 70 were compiled for comparative analysis. Prior to alignment, sequences were standardized by converting all nucleotides to uppercase and removing non-canonical characters to retain only the standard A, C, G, and T bases. Pairwise global sequence alignments were performed using the PairwiseAligner module implemented in the Biopython library (Cock et al., 2009 ). A simple and uniform scoring scheme was applied, with a match score of 0 and penalties of \\(\\:-1\\) assigned for mismatches, gap opening, and gap extension, allowing unbiased estimation of sequence divergence. Evolutionary distances between sequence pairs were calculated as the proportion of total edit operations, including mismatches and gap events, relative to the overall alignment length. The resulting pairwise distances were organized into a lower triangular distance matrix and used to infer phylogenetic relationships using the Neighbor-Joining algorithm (Saitou & Nei, 1987 ). Tree construction was performed with the DistanceTreeConstructor module in Biopython, a distance-based approach widely applied for reconstructing evolutionary relationships among closely related viral sequences (Nei & Kumar, 2013 ). RNA Structure Prediction Secondary structures of the 3 \\(\\:{\\prime\\:}\\) UTR RNA sequences were predicted using RNAfold (ViennaRNA package) (Lorenz et al., 2011 ). The minimum free-energy (MFE) structures were selected and converted into three-dimensional RNA models using RNAComposer for structural and docking analyses (Popenda et al., 2012 ). Molecular Docking Molecular docking was performed to examine interactions between the CSFV 3 \\(\\:{\\prime\\:}\\) UTR RNA and the viral RNA-dependent RNA polymerase NS5B. The crystal structure of CSFV NS5B (PDB ID: 5YF5) was retrieved from the Protein Data Bank (Berman et al., 2000 ). Docking was conducted using HDOCKlite v1.0 in an ab initio mode, with NS5B as the receptor and the modeled 3 \\(\\:{\\prime\\:}\\) UTR RNA as the ligand (Yan et al., 2017 , 2020 ). Docked complexes were ranked based on docking scores. Structural visualization and interaction analysis were carried out using PyMOL (Schrödinger, LLC, 2023 ). Molecular Dynamics Simulation To evaluate the structural stability and dynamic behavior of the NS5B–3 \\(\\:{\\prime\\:}\\) UTR RNA complex, molecular dynamics (MD) simulations were performed for the selected passage 20 (P20) RNA–protein complex. System preparation was carried out using the CHARMM-GUI web server (Jo et al., 2008 ). The docked NS5B–3 \\(\\:{\\prime\\:}\\) UTR complex was parameterized using the CHARMM36m force field for proteins and nucleic acids (J. Huang & MacKerell, 2017 ). The system was solvated in an explicit TIP3P water box with a minimum buffer distance of 10 Å from the solute. Counterions (Na \\(\\:{}^{+}\\) and Cl \\(\\:{}^{-}\\) ) were added to neutralize the system and to achieve a physiological salt concentration of 0.15 M. All MD simulations were performed using GROMACS (version 2023) (Abraham et al., 2015 ). Energy minimization was carried out using the steepest descent algorithm until convergence. The system was equilibrated in two phases: NVT equilibration for 1 ns followed by NPT equilibration for 1 ns, maintaining the temperature at 300 K using the V-rescale thermostat and pressure at 1 bar using the Parrinello–Rahman barostat. A 100 ns production MD simulation was performed under periodic boundary conditions with a time step of 2 fs. Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method, and covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm. Trajectory analyses, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration, hydrogen bond occupancy, and interaction stability between NS5B and the 3 \\(\\:{\\prime\\:}\\) UTR RNA, were performed using built-in GROMACS tools and visualized using PyMOL (Schrödinger, LLC, 2023 ). Results Adaptation of CSFV in PK-15 Cells The CSFV field isolate was successfully propagated and adapted in PK-15 cells through serial passaging. During the initial passages, infected cell cultures did not exhibit any overt cytopathic effects, which is consistent with the known non-cytopathic nature of most classical swine fever virus strains. Up to the 30th passage, the PK-15 monolayer remained largely intact, with cellular morphology comparable to that of uninfected control cultures (Fig. 1 A). However, marked changes in cell morphology were observed at later passages. From passages beyond the 30th passage onward, progressive deterioration of the monolayer was evident, characterized by cell rounding, detachment, and eventual loss of monolayer integrity within 72 h post-infection (Fig. 1 B). The consistent appearance of these changes in successive passages suggests enhanced viral replication efficiency and improved adaptation of the virus to the PK-15 cell line during prolonged in vitro propagation. These observations indicate a transition from an initially non-cytopathic infection profile to a more pronounced cytopathic phenotype following extended serial passaging, reflecting adaptive changes acquired by the virus under cell culture conditions. Virus Detection by Transmission Electron Microscopy The presence of CSFV in infected PK-15 cell cultures was confirmed by negative-staining transmission electron microscopy (TEM). Examination of culture supernatants revealed the presence of virus-like particles exhibiting morphological characteristics consistent with members of the genus Pestivirus . The observed particles were predominantly spherical to pleomorphic in appearance and were surrounded by a lipid envelope derived from the host cell membrane. Virions measured approximately 50–70 nm in diameter and contained a centrally located electron-dense core of approximately 30 nm (Fig. 2 A,B). These dimensions and structural features are similar with previously reported ultrastructural characteristics of CSFV. The detection of intact virion particles by TEM provided direct visual confirmation of productive viral replication in PK-15 cells and supported the successful adaptation of the virus during serial passage. PCR Amplification and Cloning of the 3 \\(\\:{\\prime\\:}\\) UTR Two-step RT-PCR amplification of the CSFV 3 \\(\\:{\\prime\\:}\\) UTR from serially passaged virus samples was performed using a degenerate forward primer (5 \\(\\:{\\prime\\:}\\) -RCG CGG GYR ACC CGS GAT CTG RM-3 \\(\\:{\\prime\\:}\\) ) and a 3 \\(\\:{\\prime\\:}\\) UTR-specific reverse primer (5 \\(\\:{\\prime\\:}\\) -GGG CCG TTA GGA AAT TAC CTT AGT C-3 \\(\\:{\\prime\\:}\\) ). This strategy consistently produced a single, specific amplicon of 238 bp across all passages (Fig. 3 A), confirming successful amplification of the full-length 3 \\(\\:{\\prime\\:}\\) UTR and indicating the absence of major deletions affecting primer binding sites during in vitro adaptation. The amplified 3 \\(\\:{\\prime\\:}\\) UTR fragments were cloned into the pJET1.2 blunt-end vector, and recombinant clones were screened by PCR using the T7 promoter primer in combination with either the 3 \\(\\:{\\prime\\:}\\) UTR forward or reverse primer. As expected for non-directional blunt-end cloning, inserts were recovered in both orientations. Inserts from passages 1, 10, 20, and 40 were predominantly cloned in the forward orientation, whereas those from passages 0, 30, 50, 60, and 70 were recovered mainly in the reverse orientation (Fig. 3 B). These results confirm the successful amplification and cloning of the CSFV 3 \\(\\:{\\prime\\:}\\) UTR from all examined passages and provided high-quality templates for downstream sequencing and comparative genomic analyses. Sequencing Analysis of the 3 \\(\\:{\\prime\\:}\\) UTR Comparative sequencing analysis of the CSFV 3 \\(\\:{\\prime\\:}\\) untranslated region (3 \\(\\:{\\prime\\:}\\) UTR) across serial passages revealed the accumulation of nucleotide substitutions, insertions, and deletions during prolonged adaptation in PK-15 cells (Fig. 4 ). Sequence alignment of passage 0 through passage 70 demonstrated both conserved regions and passage-associated variations distributed along the length of the 3 \\(\\:{\\prime\\:}\\) UTR. Multiple nucleotide mismatches were consistently identified at positions 1, 8, 9, 22, 25, 29, 52, 62, 92, and 184 when compared with the original isolate. In addition to point mutations, a region of continuous deletions spanning approximately 4–10 nucleotides was observed between positions 65 and 75 across successive passages. This deletion hotspot was maintained throughout later passages, suggesting selective retention during cell culture adaptation. Furthermore, a single-nucleotide deletion at position 162 and a nucleotide insertion at position 226 were detected in all sequenced passages relative to the parental virus. The persistence of these changes across multiple passages indicates that they are likely tolerated or selectively neutral under in vitro growth conditions. Overall, the sequencing data demonstrate that the CSFV 3 \\(\\:{\\prime\\:}\\) UTR undergoes progressive yet structured genetic variation during serial passage, while retaining core sequence elements. These adaptive modifications may influence RNA secondary structure and regulatory interactions involved in viral replication. Phylogenetic analysis of the CSFV 3 \\(\\:{}^{{\\prime\\:}}\\) UTR Phylogenetic analysis of full-length CSFV 3 \\(\\:{}^{{\\prime\\:}}\\) untranslated region (3 \\(\\:{}^{{\\prime\\:}}\\) UTR) sequences derived from serial passages demonstrated a passage-dependent evolutionary divergence. Neighbor-joining analysis based on alignment-derived evolutionary distances revealed that early passages (P0, P1, P10, and P20) clustered closely, indicating limited sequence variation during the initial stages of cell culture adaptation(Fig. 8 ). Intermediate passages (P40, P50, and P60) formed a distinct subclade, reflecting the accumulation of adaptive nucleotide changes within the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR over continued passaging. In Later passages, particularly P30 and P70, segregated into a more distant branch characterized by substantially increased evolutionary distances relative to the parental virus, suggesting pronounced remodeling of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR during advanced adaptation. Notably, passage 20 occupied a transitional phylogenetic position between early conserved variants and more divergent late-passage sequences, consistent with the emergence of functionally relevant yet structurally stable adaptations. Overall, the phylogenetic topology supports a stepwise and non-linear evolutionary trajectory of the CSFV 3 \\(\\:{}^{{\\prime\\:}}\\) UTR under sustained in vitro selective pressure. Docking Studies Molecular docking was performed to evaluate interactions between the CSFV RNA-dependent RNA polymerase NS5B and 3 \\(\\:{\\prime\\:}\\) UTR RNA variants derived from serial passages during adaptation in PK-15 cells. Docking scores generated using HDOCKlite v1.0 were used as a comparative measure of predicted RNA–protein binding affinity, with more negative values indicating stronger interactions. Analysis of docking scores revealed a progressive increase in binding affinity across successive passages (Fig. 5 ). The parental virus (passage 0) exhibited a relatively weak interaction with NS5B (approximately − 450), whereas later passages displayed markedly improved affinity, reaching values between − 1200 and − 1350. This trend suggests passage-dependent optimization of the 3 \\(\\:{\\prime\\:}\\) UTR for interaction with the viral polymerase during in vitro adaptation. Among all variants, passage 20 (P20) and passage 60 (P60) exhibited the strongest binding affinities and were selected as representative intermediates and late-stage adaptation variants, respectively, for detailed structural analysis. The P20 3 \\(\\:{\\prime\\:}\\) UTR RNA formed a stable complex with NS5B, occupying the polymerase catalytic cleft and establishing interactions with the palm, finger, and thumb subdomains (Fig. 6 ). The RNA was oriented along the positively charged RNA-binding channel, forming a balanced network of hydrogen bonds, cation–phosphate interactions, and hydrophobic contacts. This interaction pattern suggests efficient anchoring of the RNA template in a catalytically competent orientation, consistent with enhanced replication efficiency observed during intermediate stages of adaptation. Owing to its strong binding affinity and well-defined interaction geometry, the P20 complex was selected for subsequent molecular dynamics simulation to assess interaction stability under dynamic conditions. Molecular Dynamics Simulation of the NS5B–3 \\(\\:{\\prime\\:}\\) UTR (P20) Complex To evaluate the dynamic stability of the docked RNA–protein complex, a 100 ns molecular dynamics (MD) simulation was performed for CSFV NS5B in complex with the passage 20 (P20) 3 \\(\\:{\\prime\\:}\\) UTR RNA. Structural stability and interface persistence were assessed using RMSD, RMSF, radius of gyration (Rg), hydrogen bond occupancy, minimum intermolecular distance, and solvent-accessible surface area (SASA) (Abraham et al., 2015 ). The RMSD profile showed an initial increase during the early phase of the simulation, consistent with relaxation of the docked conformation, followed by stabilization after approximately 15–20 ns (Fig. 7 ). Thereafter, RMSD fluctuations remained within a moderate range, indicating that the complex reached a dynamically stable conformational state and remained intact over the remainder of the trajectory. The radius of gyration remained comparatively stable throughout the simulation, with only minor oscillations in total Rg and axial components (RgX, RgY, and RgZ) (Fig. 7 ). Suggests that the overall compactness of the NS5B–P20 complex was maintained during explicit-solvent MD. RMSF analysis indicated limited flexibility across most NS5B residues, with larger fluctuations confined mainly to terminal and loop regions (Fig. 7 ). Residues associated with the RNA-binding channel displayed comparatively low fluctuations, supporting stable engagement of the P20 RNA ligand within the binding cleft. Hydrogen bond analysis demonstrated sustained RNA–protein hydrogen bonding throughout the simulation (Fig. 7 ). While transient variations were observed, the overall hydrogen bond count remained substantial and increased during later stages of the trajectory, indicating progressive stabilization of the interface. The minimum distance between the RNA and NS5B remained low and stable over time (Fig. 7 ), indicating close contact without evidence of dissociation. SASA analysis showed moderate fluctuations without abrupt increases that would indicate destabilization or separation of the complex (Fig. 7 ). The overall SASA behavior is consistent with maintenance of a stable RNA–protein interface during simulation. Collectively, these MD analyses support that the NS5B–P20 3 \\(\\:{\\prime\\:}\\) UTR complex is dynamically stable over 100 ns in explicit solvent. The persistence of hydrogen bonds, stable minimum distance, limited residue-level fluctuations, and maintained compactness indicate a robust RNA–polymerase association for the P20 variant under the simulated conditions. Discussion Adaptation of Classical swine fever virus (CSFV) to cell culture represents a multistep evolutionary process shaped by selective pressures that favor efficient replication in a non-native cellular environment. In the present study, serial passaging of CSFV in PK-15 cells resulted in progressive phenotypic and molecular changes indicative of enhanced viral fitness. The transition from an initially non-cytopathic phenotype to extensive monolayer disruption at later passages reflects gradual optimization of virus–host interactions, consistent with pestiviral adaptation reported during prolonged in vitro propagation (Hadsbjerg et al., 2016 ; Johnston et al., 2020 ). Comprehensive sequencing analysis demonstrated that viral adaptation was accompanied by the accumulation of substitutions, insertions, and recurrent deletions within the 3 \\(\\:{}^{{\\prime\\:}}\\) untranslated region (3 \\(\\:{}^{{\\prime\\:}}\\) UTR). The consistent emergence and retention of deletions within a defined segment of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR across multiple passages suggests strong selective pressure acting on this regulatory region during cell culture adaptation. Although the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR does not encode protein, it plays a critical role in regulating RNA stability, long-range RNA interactions, and recruitment of the viral RNA-dependent RNA polymerase (RdRp), NS5B (Pankraz et al., 2005 ; Y. Xiao et al., 2004 ). Even subtle structural remodeling of this region can therefore exert profound effects on replication efficiency and viral fitness. Phylogenetic analysis further supported a passage-dependent evolutionary trajectory of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR, characterized by close clustering of early passages, divergence of intermediate variants, and increased evolutionary distances in late passages. This non-linear, stepwise pattern is consistent with quasispecies-driven adaptation, in which transitional variants emerge that balance structural stability with functional flexibility (Martínez et al., 2012 ). Notably, passage 20 (P20) occupied a transitional phylogenetic position, suggesting that key adaptive features can be established at intermediate stages rather than exclusively in late-passage viruses. Molecular docking analyses revealed a progressive increase in predicted binding affinity between evolved 3 \\(\\:{}^{{\\prime\\:}}\\) UTR RNA variants and the NS5B polymerase across serial passages. Early-passage RNAs exhibited relatively weak interactions, whereas intermediate and late-passage variants showed markedly stronger binding, supporting the hypothesis that adaptive remodeling of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR enhances its structural compatibility with NS5B. Similar functional importance of polymerase–UTR interactions has been reported for CSFV and related pestiviruses (Li et al., 2018 ; Y. Xiao et al., 2004 ). Molecular dynamics simulation of the NS5B–P20 3 \\(\\:{}^{{\\prime\\:}}\\) UTR complex provided dynamic validation of these observations. The complex remained stable throughout the 100 ns simulation, exhibiting sustained hydrogen bonding, low intermolecular distances, limited residue-level fluctuations within the RNA-binding cleft, and preserved overall compactness. These features indicate that adaptive mutations present in the P20 3 \\(\\:{}^{{\\prime\\:}}\\) UTR promote a robust yet flexible RNA–polymerase interaction under solvent-explicit conditions, consistent with structural and functional analyses of CSFV NS5B–RNA interactions (Li et al., 2018 ; Wang et al., 2020 ). Although later passage variants displayed stronger predicted docking affinities, maximal binding strength is not necessarily optimal for replication. Excessive stabilization of RNA–protein complexes can constrain conformational transitions required during initiation and elongation of RNA synthesis (Isken et al., 2004 ). Intermediate evolutionary states such as P20 may therefore represent an effective balance between binding stability and structural adaptability. Collectively, these findings support a model in which CSFV adaptation to PK-15 cells is driven, in part, by evolutionary remodeling of the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR that enhances its interaction with the NS5B polymerase. Adaptive nucleotide changes reshape RNA structure, strengthen RNA–polymerase binding, and stabilize the viral replication complex, thereby facilitating efficient replication under in vitro conditions. This study underscores the functional importance of noncoding RNA elements in viral evolution and identifies the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR–NS5B interface as a critical determinant of CSFV fitness and a potential target for antiviral intervention. Conclusion This study demonstrates that adaptation of Classical swine fever virus (CSFV) to PK-15 cells is driven by evolutionary remodeling of the 3 \\(\\:{}^{{\\prime\\:}}\\) untranslated region (3 \\(\\:{}^{{\\prime\\:}}\\) UTR) that enhances its functional interaction with the viral RNA-dependent RNA polymerase NS5B. Serial passaging selected for defined nucleotide substitutions, insertions, and recurrent deletions within the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR, indicating strong selective pressure acting on this noncoding regulatory region during in vitro adaptation. Integrated phylogenetic, docking, and molecular dynamics analyses revealed that these adaptive changes promote progressively stronger and more stable 3 \\(\\:{}^{{\\prime\\:}}\\) UTR–NS5B interactions. The evolved RNA variants exhibited improved structural compatibility with the polymerase, sustained hydrogen bonding, and limited conformational fluctuations, consistent with stabilization of the viral replication complex. Collectively, these findings highlight the critical role of 3 \\(\\:{}^{{\\prime\\:}}\\) UTR evolution in modulating CSFV replication efficiency, underscore the importance of RNA–protein interactions in viral fitness, and identify the 3 \\(\\:{}^{{\\prime\\:}}\\) UTR–NS5B interface as a potential target for antiviral intervention and rational vaccine design. Declarations Funding Not applicable Author Contribution A.K., conceived and designed the study and performed the majority of the experimental work. A.K. carried out virus isolation, serial passaging, and molecular characterization of the 3′ UTR. T.G. performed sequence analysis, phylogenetic analysis, and contributed to data interpretation. T.G and N.A. conducted molecular docking and molecular dynamics simulations and analyzed protein–RNA interaction data.P.K.G. and L.S.R. assisted in experimental design, provided technical support for virological and molecular assays, and contributed to data analysis. B.R.P. supported bioinformatics analysis and interpretation of evolutionary data. S.L. and M.S. assisted with laboratory experiments and data collection.R.C. supervised the overall study, provided critical intellectual input, coordinated the project, and finalized the manuscript.A.K., T.G., and N.A. wrote the initial draft of the manuscript. All authors reviewed, edited, and approved the final version of the manuscript. Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. Sequence data generated during this study are included in the article and/or its supplementary material. 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Virus Res 289:198151. https://doi.org/10.1016/j.virusres.2020.198151 Hadsbjerg J, Friis MB, Fahnøe U, Nielsen J, Belsham G, Rasmussen TB (2016) Sequence adaptations during growth of rescued classical swine fever viruses in cell culture and within infected pigs. Vet Microbiol 192:123–134. https://doi.org/10.1016/j.vetmic.2016.07.004 Hsu WL, Chen CL, Huang SW, Wu CC, Chen IH et al (2014) The untranslated regions of classic swine fever virus RNA trigger apoptosis. PLoS ONE 9(2):e88863. https://doi.org/10.1371/journal.pone.0088863 Huang J, MacKerell AD (2017) CHARMM36m: An improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71–73 Huang SW, Chan MY, Hsu WL, Huang CC, Tsai CH (2012) The 3 \\:{}^{{\\prime\\:}} -terminal hexamer sequence of classical swine fever virus RNA plays a role in negatively regulating the IRES-mediated translation. PLoS ONE 7(3):e33764. https://doi.org/10.1371/journal.pone.0033764 Isken O, Grassmann CW, Behrens SE (2004) Complex signals in the 3 \\:{}^{{\\prime\\:}} nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA. RNA 10(10):1637–1652 Jo S, Kim T, Iyer VG, Im W (2008) CHARMM-GUI: A web-based graphical user interface for CHARMM. J Comput Chem 29(11):1859–1865 Johnston CM, Fahnøe U, Lohse L, Bukh J, Belsham GJ, Rasmussen TB (2020) Analysis of virus population profiles within pigs infected with virulent classical swine fever viruses. J Virol 94(19):e01119–e01120. https://doi.org/10.1128/JVI.01119-20 Kamboj A, Dumka S, Saxena MK, Singh Y, Kaur BP, Silva SJR, da, Kumar S (2024) A comprehensive review of our understanding and challenges of viral vaccines against swine pathogens. Viruses 16(6):833. https://doi.org/10.3390/v16060833 Kamboj A, Patel CL, Chaturvedi VK, Saini M, Gupta PK (2014) Complete genome sequence of an indian field isolate of classical swine fever virus belonging to subgenotype 1.1. Genome Announcements 2(5):e00886–e00814. https://doi.org/10.1128/genomeA.00886-14 Kamboj A, Saini M, Rajan LS, Patel CL, Chaturvedi VK, Gupta PK (2015) Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination. J Virol Methods 226:60–66. https://doi.org/10.1016/j.jviromet.2015.10.006 Lamothe-Reyes Y, Figueroa M, Sánchez O (2023) Host cell factors involved in classical swine fever virus entry. Vet Res 54(1):115. https://doi.org/10.1186/s13567-023-01238-x Li W, Wu B, Soca WA, An L (2018) Crystal structure of classical swine fever virus NS5B reveals a novel n-terminal domain. J Virol 92(14):e00324–e00318. https://doi.org/10.1128/JVI.00324-18 Lorenz R, Bernhart SH, Höner C et al (2011) ViennaRNA package 2.0. Algorithms for Molecular Biology , 6 , 26 Martínez MA, Martrus G, Capel E, Parera M, Franco S, Nevot M (2012) Quasispecies dynamics of RNA viruses. In Viruses: Essential agents of life (pp. 21–42). Springer. https://doi.org/10.1007/978-94-007-4899-6_2 Meyers G, Thiel HJ (1996) Molecular characterization of pestiviruses. Adv Virus Res 47:53–118 Nei M, Kumar S (2013) Molecular evolution and phylogenetics. Oxford University Press Pankraz A, Thiel HJ, Behrens SE (2005) Genetic analysis of the pestivirus 3 \\:{}^{{\\prime\\:}} nontranslated region: Dissection of functional elements influencing translation and RNA replication. J Virol 79(2):1059–1073 Patil SS, Suresh KP, Saha S, Prajapati A, Hemadri D, Roy P (2018) Meta-analysis of classical swine fever prevalence in pigs in india: A 5-year study. Veterinary World 11(3):297–303. https://doi.org/10.14202/vetworld.2018.297-303 Popenda M, Szachniuk M, Antczak M et al (2012) RNAComposer: A tool for RNA 3D structure prediction. Nucleic Acids Res 40(W1):W385–W390 Rios L, Coronado L, Naranjo-Feliciano D, Martínez-Pérez O, Perera CL, Hernandez-Alvarez L, de Arce Díaz, Núñez H, Ganges JI, L., Pérez LJ (2017) Deciphering the emergence, genetic diversity and evolution of classical swine fever virus. Sci Rep 7(1):17887. https://doi.org/10.1038/s41598-017-18196-y Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Schrödinger LLC (2023) The PyMOL molecular graphics system Vanderhallen H, Mittelholzer C, Hofmann MA (1999) Classical swine fever virus isotypes: Correlation between genetic and antigenic properties. Virus Res 64(1):27–37 Wang M, Liniger M, Muñoz-González S, Bohórquez JA, Hinojosa Y, Gerber M, López-Soria S, Rosell R, Ruggli N, Ganges L (2020) A polyuridine insertion in the 3 \\:{}^{{\\prime\\:}} untranslated region of classical swine fever virus activates immunity and reduces viral virulence in piglets. J Virol 94(2):e01214–e01219. https://doi.org/10.1128/JVI.01214-19 World Organisation for Animal Health (WOAH) (2024) Terrestrial animal health code, Chap. 15.2 . https://www.woah.org/file/eng/Health_standards/tahc/2024/chapitre_csf.pdf Xiao M, Gao J, Wang W et al (2004) Specific interaction between the classical swine fever virus NS5B protein and the viral genome. Eur J Biochem 271(19):3888–3896. https://doi.org/10.1111/j.1432-1033.2004.04325.x Xiao Y, Davidson D, Liu D (2004) Analysis of the 3 \\:{}^{{\\prime\\:}} untranslated region of pestivirus genome and its function in viral replication. Virol J 1:21 Yan Y, Tao H, He J, Huang S-Y (2020) The HDOCK server for integrated protein–protein docking. Nat Protoc. https://doi.org/10.1038/s41596-020-0312-x Yan Y, Zhang D, Zhou P, Li B, Huang S-Y (2017) HDOCK: A web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy. Nucleic Acids Res 45(W1):W365–W373 Additional Declarations No competing interests reported. Supplementary Files supplementaryfiles.docx GraphicalAbstract.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-8668214\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":597108741,\"identity\":\"b0115589-158d-4129-8244-c961bd5d55d2\",\"order_by\":0,\"name\":\"Aman Kamboj\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Veterinary and Animal Sciences, G. B. Pant University of Agriculture and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Aman\",\"middleName\":\"\",\"lastName\":\"Kamboj\",\"suffix\":\"\"},{\"id\":597108743,\"identity\":\"fb46a2ce-d7d5-499c-8154-b5f49ad59c6a\",\"order_by\":1,\"name\":\"Tushar Gaikwad\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Institute of Science, Dr. Homi Bhabha State University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tushar\",\"middleName\":\"\",\"lastName\":\"Gaikwad\",\"suffix\":\"\"},{\"id\":597108744,\"identity\":\"54e3275f-daa9-4343-85f7-3f89e1104ab6\",\"order_by\":2,\"name\":\"Nachiket Atale\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Institute of Science, Dr. Homi Bhabha State University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nachiket\",\"middleName\":\"\",\"lastName\":\"Atale\",\"suffix\":\"\"},{\"id\":597108746,\"identity\":\"ce9fed20-4ddf-4cfc-9b16-38e7a0234fbf\",\"order_by\":3,\"name\":\"Praveen Kumar Gupta\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"ICAR-Indian Veterinary Research Institute (ICAR-IVRI)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Praveen\",\"middleName\":\"Kumar\",\"lastName\":\"Gupta\",\"suffix\":\"\"},{\"id\":597108764,\"identity\":\"25d80ace-562e-4b06-8792-8682343544ab\",\"order_by\":4,\"name\":\"Lekshmi S Rajan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"ICAR-National Institute of Virology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lekshmi\",\"middleName\":\"S\",\"lastName\":\"Rajan\",\"suffix\":\"\"},{\"id\":597108769,\"identity\":\"e93e68c8-f8dc-433e-9206-f4cc686a4d86\",\"order_by\":5,\"name\":\"Bikash Ranjan Prusty\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Nanaji Deshmukh Veterinary Science University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bikash\",\"middleName\":\"Ranjan\",\"lastName\":\"Prusty\",\"suffix\":\"\"},{\"id\":597108771,\"identity\":\"3052436f-136d-4ee3-824f-133995ec5b05\",\"order_by\":6,\"name\":\"Mohini Saini\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"ICAR-Indian Veterinary Research Institute (ICAR-IVRI)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mohini\",\"middleName\":\"\",\"lastName\":\"Saini\",\"suffix\":\"\"},{\"id\":597108775,\"identity\":\"594f61e2-daef-40e4-8558-116a4415f92e\",\"order_by\":7,\"name\":\"Rajendra Choure\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYHCCBAYGNhDNA+bJscHE2bArx9RiTIwWBhQtiQ2EXGXefuDh44Kye/IM7GcPPubdY5Pex3/46QaGGjsGPmnsumXOJCQbzzhXbNjAk5dszPMsLbdNIs3sBsOxZAY2mQNYtUgwJKRJ87YlMDZI8JhJ8xw4DNTCw3aDge0AA5tEAnYt/A/SfwO12EO1/E9n4z8D1PIPjxaJhDRmoJZEqJYDCWwMOWw3GNvwaXmQLM1zLiG5jSfH2HDOgWRDsF8S+5J5cDssJ/EzT1mCbT/7GcMHbw7Yycv3H35248M3Ozn5Gdi1AKMDIoEacQmwaMIK2A/glhsFo2AUjIJRAAIAMEhPwA/oFAMAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"The Institute of Science, Dr. Homi Bhabha State University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Rajendra\",\"middleName\":\"\",\"lastName\":\"Choure\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-01-22 10:08:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8668214/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8668214/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":103515081,\"identity\":\"cebd6c27-5714-4cd5-a6d1-edd019b27d9b\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:22:40\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":676823,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdaptation of CSFV in PK-15 cells. (A) Uninfected PK-15 cells showing normal monolayer morphology. (B) PK-15 cells infected with CSFV at a later passage, showing loss of monolayer integrity and extensive cellular detachment.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/27ad6b6d2300b9a4906f4a02.png\"},{\"id\":103515075,\"identity\":\"06a7833d-c938-4e55-b035-2118638aceaa\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:22:39\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":292429,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTransmission electron micrographs of CSFV particles obtained from infected PK-15 cell culture supernatants following negative staining. (A) Representative field showing multiple enveloped CSFV virions (scale bar: 1 μm). (B) Higher magnification image of a single CSFV virion displaying an electron-dense core and surrounding lipid envelope.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/72d085ef4dc91d075a7d8f92.png\"},{\"id\":103515358,\"identity\":\"f85d8ac0-14ed-4049-b163-8cbd8bdfe560\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:23:26\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":232638,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePCR amplification and cloning analysis of the CSFV 3'UTR. (A) Agarose gel electrophoresis showing the specific 238 bp 3'UTR amplicon generated by two-step RT-PCR (Lane M: 1 kb plus DNA ladder; Lane 1: 3'UTR PCR product). (B) Confirmation of insert orientation in recombinant pJET1.2 plasmids by PCR. Upper panel: amplification using T7 promoter primer and 3'UTR forward primer. Lower panel: amplification using T7 promoter primer and 3'UTR reverse primer. Lanes correspond to passages 0, 1, 10, 20, 30, 40, 50, 60, and 70, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/e0218efdf258e68b3d091456.png\"},{\"id\":103516788,\"identity\":\"a813bbad-c9c6-4467-a47e-3bb29f264f7e\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:29:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":332073,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMultiple sequence alignment of the CSFV 3'UTR derived from passages 0 to 70. Conserved regions, nucleotide substitutions, insertions, and deletions are highlighted, illustrating passage-associated genomic variation during cell culture adaptation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/69562cb5354e763c7d4a9f2a.png\"},{\"id\":103515347,\"identity\":\"7f374a59-fe88-4160-86ba-60eae6c39514\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:23:20\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34912,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePredicted binding affinity scores between CSFV NS5B (PDB ID: 5YF5) and 3'UTR RNAs derived from different passages during cell culture adaptation. Lower values indicate stronger RNA–protein interactions.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/e24a93ed7c2133c1b6823ae1.png\"},{\"id\":103515162,\"identity\":\"1243b13a-12f5-445f-9a98-6ca6e7dfb30e\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:22:53\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":365869,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDocking complex of CSFV NS5B with the 3'UTR RNA from passage 20. (A) Surface representation showing RNA engagement within the polymerase catalytic cleft. (B) Electrostatic surface highlighting positively charged regions involved in RNA binding.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/d82c959a45e979e798d9ab1f.png\"},{\"id\":103515463,\"identity\":\"2a7641bb-3081-46d0-8f65-b4dd58d6b4dc\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:23:46\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":367018,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMolecular dynamics analysis of the NS5B–P20 3'UTR RNA complex over a 100 ns simulation. (a) RMSD shows initial relaxation followed by stabilization. (b) RMSF indicates flexibility mainly in terminal and loop regions, with a rigid RNA-binding interface. (c) Radius of gyration remains stable, indicating overall compactness. (d) Intermolecular hydrogen bonds persist throughout the simulation. (e) Minimum intermolecular distance remains low, confirming sustained RNA–protein contact.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/d7fb496102bcdf706dc7bbdf.png\"},{\"id\":103514915,\"identity\":\"b0b76137-77be-4eda-9db3-3b7fa1248f46\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:22:22\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":56136,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeighbor-joining phylogenetic tree inferred from full-length CSFV 3'UTR RNA sequences obtained across serial passages during PK-15 cell culture adaptation (P0, P1, P10, P20, P30, P40, P50, P60, and P70). Branch lengths represent alignment-based evolutionary distances.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/bf3ca30ba6b67af7f46a6d0c.png\"},{\"id\":106098516,\"identity\":\"dad7b6ba-db05-449a-9be5-6079b18c1c46\",\"added_by\":\"auto\",\"created_at\":\"2026-04-03 12:03:18\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2916978,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/ce9b96c1-e194-489b-86bb-094761f934ac.pdf\"},{\"id\":103515195,\"identity\":\"30d8ebdb-cc88-4b33-babb-aad0dd70760a\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:23:03\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":193886,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"supplementaryfiles.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/c6c1763a653b0918b85fd331.docx\"},{\"id\":103515343,\"identity\":\"f1f16137-3a8d-43a4-8880-18f3c83e906d\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 14:23:18\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":365358,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"GraphicalAbstract.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8668214/v1/d394aed49e145f264efa721e.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Evolution of the 3′ Untranslated Region Enhances NS5B Binding During Cell Culture Adaptation of Classical Swine Fever Virus\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eClassical swine fever (CSF), also referred to as hog cholera, is a highly contagious viral disease of domestic pigs and wild boar that continues to pose a serious threat to the global pig industry. The disease is endemic in several regions of Asia, South and Central America, parts of Europe, and selected African and Caribbean countries (Edwards et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e; World Organisation for Animal Health (WOAH), \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In India, CSF represents a major economic burden, particularly in the northeastern states where pig farming constitutes an important source of income for rural communities (Barman et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Patil et al., \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Recurrent outbreaks result in substantial losses due to high morbidity and mortality, trade restrictions, and the costs associated with vaccination and control measures. A detailed understanding of the molecular mechanisms governing viral replication and adaptation is therefore essential for the development of effective prevention and control strategies, including vaccine design and molecular surveillance (Kamboj et al., \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eCSF is caused by Classical swine fever virus (CSFV), a member of the genus \\u003cem\\u003ePestivirus\\u003c/em\\u003e within the family \\u003cem\\u003eFlaviviridae\\u003c/em\\u003e (Fauquet et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). Related pestiviruses include bovine viral diarrhea virus (BVDV) and border disease virus (BDV) of sheep, which serve as established models for studying pestiviral replication and evolution. CSFV is an enveloped, positive-sense, single-stranded RNA virus with a genome of approximately 12.5 kb (Meyers \\u0026amp; Thiel, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Vanderhallen et al., \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e). The genome contains a single open reading frame (ORF) of about 11.7 kb that encodes a polyprotein of nearly 3,900 amino acids. This polyprotein is co- and post-translationally processed into four structural proteins (C, Erns, E1, and E2) and eight non-structural proteins (N\\u003csup\\u003epro\\u003c/sup\\u003e, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which together coordinate viral assembly, genome replication, and modulation of host antiviral responses (Beer et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Isken et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Rios et al., \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Complete genome sequencing of Indian CSFV field isolates has further contributed to understanding the molecular diversity and evolutionary relationships of circulating strains (Kamboj et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe viral ORF is flanked by highly structured 5\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e and 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated regions (UTRs) that play essential regulatory roles in the viral life cycle. The 5\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR contains an internal ribosome entry site (IRES) that enables cap-independent initiation of translation, whereas the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR is involved in the initiation of RNA replication and in coordinating the balance between translation and replication (Hsu et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Pankraz et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Y. Xiao et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). In related pestiviruses, the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR has been shown to participate in long-range RNA interactions and to engage host and viral factors that regulate replication efficiency and viral fitness (Isken et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Consequently, sequence variation or structural remodeling within the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR has the potential to substantially influence viral replication and pathogenicity.\\u003c/p\\u003e \\u003cp\\u003eAmong the viral non-structural proteins, NS5B functions as the RNA-dependent RNA polymerase (RdRp) and is indispensable for viral genome synthesis. NS5B interacts with both positive- and negative-sense RNA templates, with particular importance attributed to its interaction with the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR during initiation of RNA replication (Li et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; M. Xiao et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Efficient and stable association between the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR and NS5B is therefore critical for productive infection and viral persistence. CSFV infections are typically non-cytopathic in cell culture, necessitating the use of molecular and ultrastructural techniques such as RT-PCR and transmission electron microscopy for virus detection and characterization (Blome et al., \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Ganges et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eLike other RNA viruses, CSFV exhibits substantial genetic diversity due to the absence of proofreading activity in its polymerase, resulting in a dynamic population of closely related variants, or quasispecies (Johnston et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Mart\\u0026iacute;nez et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). During serial passage in cell culture, selective pressures favor variants with improved replication efficiency under \\u003cem\\u003ein vitro\\u003c/em\\u003e conditions. While such adaptations can enhance viral growth, they may also alter virulence, antigenicity, and replication dynamics, with potential implications for vaccine development and diagnostic reliability (Hadsbjerg et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; S. W. Huang et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Lamothe-Reyes et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Wang et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Advances in reverse genetics systems, including infectious cDNA clone construction, have facilitated detailed functional analyses of CSFV replication and adaptation mechanisms (Kamboj et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Despite the recognized importance of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR in pestiviral replication, relatively few studies have systematically examined how this region evolves during cell culture adaptation and how such changes influence RNA\\u0026ndash;polymerase interactions.\\u003c/p\\u003e \\u003cp\\u003eTo address this knowledge gap, the present study investigates the adaptation of CSFV to PK-15 cells with a specific focus on evolutionary changes within the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR). Genomic variations in the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR were systematically analyzed across serial passages to identify substitutions, insertions, and deletions associated with in vitro adaptation. In addition, phylogenetic analysis based on alignment-derived evolutionary distances was performed to resolve passage-dependent divergence patterns and to define transitional and late-stage adaptive lineages. Computational docking and molecular dynamics simulations were further employed to assess how adaptive remodeling of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR influences its interaction with the viral RNA-dependent RNA polymerase NS5B.\\u003c/p\\u003e \\u003cp\\u003eBy integrating experimental virology with phylogenetic, structural, and computational analyses, this study provides mechanistic insight into the role of 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR evolution in CSFV adaptation. Elucidating how noncoding regulatory RNA elements contribute to viral fitness and replication efficiency advances our understanding of pestiviral biology and may inform future strategies for vaccine design, antiviral development, and improved control of classical swine fever.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCells and Virus\\u003c/h2\\u003e \\u003cp\\u003eAn Indian field isolate of Classical swine fever virus (CSFV), designated CSFV/IVRI/VB0, was obtained from the spleen of a clinically infected pig. Virus isolation and adaptation were performed using porcine kidney (PK-15) cells (ATCC CCL-33). Cells were maintained in Dulbecco\\u0026rsquo;s Modified Eagle Medium (DMEM; HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) and an antibiotic\\u0026ndash;antimycotic mixture containing penicillin G (100 U/mL), streptomycin sulfate (100 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\mu\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003eg/mL), and amphotericin B (0.025 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\mu\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003eg/mL). Cultures were incubated at 37 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{\\\\circ\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eC in a humidified atmosphere with 5% CO\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}_{2}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eVirus Isolation and Serial Passage\\u003c/h3\\u003e\\n\\u003cp\\u003eSpleen tissue was processed to prepare a 10% (w/v) suspension in phosphate-buffered saline (PBS) and clarified by centrifugation at 15,000 rpm for 10 min at 4 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{\\\\circ\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eC. Confluent PK-15 monolayers were inoculated with 1 mL of clarified supernatant and incubated for 1 h to allow viral adsorption. The inoculum was then removed, fresh medium was added, and cultures were incubated under standard conditions.\\u003c/p\\u003e \\u003cp\\u003eAt four days post-infection, cells were subjected to a freeze\\u0026ndash;thaw cycle to release intracellular virus. The culture supernatant was used to infect fresh PK-15 monolayers. This procedure was repeated for 70 serial passages. Viral presence was confirmed at every tenth passage by RT-PCR.\\u003c/p\\u003e\\n\\u003ch3\\u003eTransmission Electron Microscopy\\u003c/h3\\u003e\\n\\u003cp\\u003eVirus particles were visualized using negative-staining transmission electron microscopy (TEM). Culture supernatants were filtered and fixed in 2% paraformaldehyde. Samples were adsorbed onto carbon-coated copper grids for 30\\u0026ndash;60 s, rinsed with deionized water, and negatively stained with phosphotungstic acid for 30 s. Grids were air-dried and examined under a transmission electron microscope to confirm virion morphology.\\u003c/p\\u003e\\n\\u003ch3\\u003eRNA Extraction and Amplification of the 3UTR\\u003c/h3\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003eRNA Extraction and Amplification of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/div\\u003e \\u003cp\\u003eTotal RNA was extracted from spleen tissue (passage 0) and from infected PK-15 cells at passages 1, 10, 20, 30, 40, 50, 60, and 70 using TRIzol\\u0026trade; reagent (Invitrogen, USA). RNA was resuspended in 20 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\mu\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003eL of nuclease-free water.\\u003c/p\\u003e \\u003cp\\u003eComplementary DNA (cDNA) synthesis was carried out using the RevertAid Reverse Transcriptase kit (Thermo Scientific, USA). The full-length 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR was amplified by two-step RT-PCR using self-designed primers. PCR amplification was performed using PfuUltra II Fusion HS DNA Polymerase (Agilent, USA). Amplified products were analyzed by 1% agarose gel electrophoresis.\\u003c/p\\u003e\\n\\u003ch3\\u003eCloning and Sequencing of the 3UTR\\u003c/h3\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003eCloning and Sequencing of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/div\\u003e \\u003cp\\u003eThe 238 bp 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR amplicons were gel-purified and cloned into the pJET1.2 blunt-end vector (Thermo Scientific, USA). Recombinant plasmids were transformed into \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e DH5\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\alpha\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003e competent cells and selected on ampicillin containing LB agar plates. Positive clones were confirmed by PCR using T7 promoter and insert-specific primers and subsequently sequenced by Sanger sequencing (Eurofins Genomics, Bengaluru, India). Sequence assembly and alignment were performed using Lasergene v6 and BioEdit software.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhylogenetic analysis of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/h2\\u003e \\u003cp\\u003ePhylogenetic analysis was conducted to assess the evolutionary relationships among Classical swine fever virus (CSFV) 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR) sequences obtained across serial passages during cell culture adaptation. Full-length 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR sequences corresponding to passages 0, 1, 10, 20, 30, 40, 50, 60, and 70 were compiled for comparative analysis.\\u003c/p\\u003e \\u003cp\\u003ePrior to alignment, sequences were standardized by converting all nucleotides to uppercase and removing non-canonical characters to retain only the standard A, C, G, and T bases. Pairwise global sequence alignments were performed using the PairwiseAligner module implemented in the Biopython library (Cock et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). A simple and uniform scoring scheme was applied, with a match score of 0 and penalties of \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:-1\\\\)\\u003c/span\\u003e\\u003c/span\\u003e assigned for mismatches, gap opening, and gap extension, allowing unbiased estimation of sequence divergence.\\u003c/p\\u003e \\u003cp\\u003eEvolutionary distances between sequence pairs were calculated as the proportion of total edit operations, including mismatches and gap events, relative to the overall alignment length. The resulting pairwise distances were organized into a lower triangular distance matrix and used to infer phylogenetic relationships using the Neighbor-Joining algorithm (Saitou \\u0026amp; Nei, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e1987\\u003c/span\\u003e). Tree construction was performed with the DistanceTreeConstructor module in Biopython, a distance-based approach widely applied for reconstructing evolutionary relationships among closely related viral sequences (Nei \\u0026amp; Kumar, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eRNA Structure Prediction\\u003c/h3\\u003e\\n\\u003cp\\u003eSecondary structures of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA sequences were predicted using RNAfold (ViennaRNA package) (Lorenz et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The minimum free-energy (MFE) structures were selected and converted into three-dimensional RNA models using RNAComposer for structural and docking analyses (Popenda et al., \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eMolecular Docking\\u003c/h3\\u003e\\n\\u003cp\\u003eMolecular docking was performed to examine interactions between the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA and the viral RNA-dependent RNA polymerase NS5B. The crystal structure of CSFV NS5B (PDB ID: 5YF5) was retrieved from the Protein Data Bank (Berman et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e). Docking was conducted using HDOCKlite v1.0 in an \\u003cem\\u003eab initio\\u003c/em\\u003e mode, with NS5B as the receptor and the modeled 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA as the ligand (Yan et al., \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Docked complexes were ranked based on docking scores. Structural visualization and interaction analysis were carried out using PyMOL (Schr\\u0026ouml;dinger, LLC, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular Dynamics Simulation\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the structural stability and dynamic behavior of the NS5B\\u0026ndash;3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA complex, molecular dynamics (MD) simulations were performed for the selected passage 20 (P20) RNA\\u0026ndash;protein complex. System preparation was carried out using the CHARMM-GUI web server (Jo et al., \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). The docked NS5B\\u0026ndash;3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR complex was parameterized using the CHARMM36m force field for proteins and nucleic acids (J. Huang \\u0026amp; MacKerell, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). The system was solvated in an explicit TIP3P water box with a minimum buffer distance of 10 \\u0026Aring; from the solute. Counterions (Na\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{+}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e and Cl\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{-}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e) were added to neutralize the system and to achieve a physiological salt concentration of 0.15 M. All MD simulations were performed using GROMACS (version 2023) (Abraham et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Energy minimization was carried out using the steepest descent algorithm until convergence. The system was equilibrated in two phases: NVT equilibration for 1 ns followed by NPT equilibration for 1 ns, maintaining the temperature at 300 K using the V-rescale thermostat and pressure at 1 bar using the Parrinello\\u0026ndash;Rahman barostat. A 100 ns production MD simulation was performed under periodic boundary conditions with a time step of 2 fs. Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method, and covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm. Trajectory analyses, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration, hydrogen bond occupancy, and interaction stability between NS5B and the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA, were performed using built-in GROMACS tools and visualized using PyMOL (Schr\\u0026ouml;dinger, LLC, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAdaptation of CSFV in PK-15 Cells\\u003c/h2\\u003e \\u003cp\\u003eThe CSFV field isolate was successfully propagated and adapted in PK-15 cells through serial passaging. During the initial passages, infected cell cultures did not exhibit any overt cytopathic effects, which is consistent with the known non-cytopathic nature of most classical swine fever virus strains. Up to the 30th passage, the PK-15 monolayer remained largely intact, with cellular morphology comparable to that of uninfected control cultures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). However, marked changes in cell morphology were observed at later passages. From passages beyond the 30th passage onward, progressive deterioration of the monolayer was evident, characterized by cell rounding, detachment, and eventual loss of monolayer integrity within 72 h post-infection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). The consistent appearance of these changes in successive passages suggests enhanced viral replication efficiency and improved adaptation of the virus to the PK-15 cell line during prolonged \\u003cem\\u003ein vitro\\u003c/em\\u003e propagation. These observations indicate a transition from an initially non-cytopathic infection profile to a more pronounced cytopathic phenotype following extended serial passaging, reflecting adaptive changes acquired by the virus under cell culture conditions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eVirus Detection by Transmission Electron Microscopy\\u003c/h2\\u003e \\u003cp\\u003eThe presence of CSFV in infected PK-15 cell cultures was confirmed by negative-staining transmission electron microscopy (TEM). Examination of culture supernatants revealed the presence of virus-like particles exhibiting morphological characteristics consistent with members of the genus \\u003cem\\u003ePestivirus\\u003c/em\\u003e. The observed particles were predominantly spherical to pleomorphic in appearance and were surrounded by a lipid envelope derived from the host cell membrane. Virions measured approximately 50\\u0026ndash;70 nm in diameter and contained a centrally located electron-dense core of approximately 30 nm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA,B). These dimensions and structural features are similar with previously reported ultrastructural characteristics of CSFV. The detection of intact virion particles by TEM provided direct visual confirmation of productive viral replication in PK-15 cells and supported the successful adaptation of the virus during serial passage.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePCR Amplification and Cloning of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/h2\\u003e \\u003cp\\u003eTwo-step RT-PCR amplification of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR from serially passaged virus samples was performed using a degenerate forward primer (5\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e-RCG CGG GYR ACC CGS GAT CTG RM-3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e) and a 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR-specific reverse primer (5\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e-GGG CCG TTA GGA AAT TAC CTT AGT C-3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e). This strategy consistently produced a single, specific amplicon of 238 bp across all passages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), confirming successful amplification of the full-length 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR and indicating the absence of major deletions affecting primer binding sites during in vitro adaptation. The amplified 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR fragments were cloned into the pJET1.2 blunt-end vector, and recombinant clones were screened by PCR using the T7 promoter primer in combination with either the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR forward or reverse primer. As expected for non-directional blunt-end cloning, inserts were recovered in both orientations. Inserts from passages 1, 10, 20, and 40 were predominantly cloned in the forward orientation, whereas those from passages 0, 30, 50, 60, and 70 were recovered mainly in the reverse orientation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). These results confirm the successful amplification and cloning of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR from all examined passages and provided high-quality templates for downstream sequencing and comparative genomic analyses.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSequencing Analysis of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/h2\\u003e \\u003cp\\u003eComparative sequencing analysis of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR) across serial passages revealed the accumulation of nucleotide substitutions, insertions, and deletions during prolonged adaptation in PK-15 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Sequence alignment of passage 0 through passage 70 demonstrated both conserved regions and passage-associated variations distributed along the length of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR. Multiple nucleotide mismatches were consistently identified at positions 1, 8, 9, 22, 25, 29, 52, 62, 92, and 184 when compared with the original isolate. In addition to point mutations, a region of continuous deletions spanning approximately 4\\u0026ndash;10 nucleotides was observed between positions 65 and 75 across successive passages. This deletion hotspot was maintained throughout later passages, suggesting selective retention during cell culture adaptation. Furthermore, a single-nucleotide deletion at position 162 and a nucleotide insertion at position 226 were detected in all sequenced passages relative to the parental virus. The persistence of these changes across multiple passages indicates that they are likely tolerated or selectively neutral under \\u003cem\\u003ein vitro\\u003c/em\\u003e growth conditions. Overall, the sequencing data demonstrate that the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR undergoes progressive yet structured genetic variation during serial passage, while retaining core sequence elements. These adaptive modifications may influence RNA secondary structure and regulatory interactions involved in viral replication.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePhylogenetic analysis of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u003c/h2\\u003e \\u003cp\\u003ePhylogenetic analysis of full-length CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR) sequences derived from serial passages demonstrated a passage-dependent evolutionary divergence. Neighbor-joining analysis based on alignment-derived evolutionary distances revealed that early passages (P0, P1, P10, and P20) clustered closely, indicating limited sequence variation during the initial stages of cell culture adaptation(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). Intermediate passages (P40, P50, and P60) formed a distinct subclade, reflecting the accumulation of adaptive nucleotide changes within the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR over continued passaging.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn Later passages, particularly P30 and P70, segregated into a more distant branch characterized by substantially increased evolutionary distances relative to the parental virus, suggesting pronounced remodeling of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR during advanced adaptation. Notably, passage 20 occupied a transitional phylogenetic position between early conserved variants and more divergent late-passage sequences, consistent with the emergence of functionally relevant yet structurally stable adaptations. Overall, the phylogenetic topology supports a stepwise and non-linear evolutionary trajectory of the CSFV 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR under sustained in vitro selective pressure.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDocking Studies\\u003c/h2\\u003e \\u003cp\\u003eMolecular docking was performed to evaluate interactions between the CSFV RNA-dependent RNA polymerase NS5B and 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA variants derived from serial passages during adaptation in PK-15 cells. Docking scores generated using HDOCKlite v1.0 were used as a comparative measure of predicted RNA\\u0026ndash;protein binding affinity, with more negative values indicating stronger interactions. Analysis of docking scores revealed a progressive increase in binding affinity across successive passages (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The parental virus (passage 0) exhibited a relatively weak interaction with NS5B (approximately \\u0026minus;\\u0026thinsp;450), whereas later passages displayed markedly improved affinity, reaching values between \\u0026minus;\\u0026thinsp;1200 and \\u0026minus;\\u0026thinsp;1350. This trend suggests passage-dependent optimization of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR for interaction with the viral polymerase during \\u003cem\\u003ein vitro\\u003c/em\\u003e adaptation. Among all variants, passage 20 (P20) and passage 60 (P60) exhibited the strongest binding affinities and were selected as representative intermediates and late-stage adaptation variants, respectively, for detailed structural analysis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe P20 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA formed a stable complex with NS5B, occupying the polymerase catalytic cleft and establishing interactions with the palm, finger, and thumb subdomains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). The RNA was oriented along the positively charged RNA-binding channel, forming a balanced network of hydrogen bonds, cation\\u0026ndash;phosphate interactions, and hydrophobic contacts. This interaction pattern suggests efficient anchoring of the RNA template in a catalytically competent orientation, consistent with enhanced replication efficiency observed during intermediate stages of adaptation. Owing to its strong binding affinity and well-defined interaction geometry, the P20 complex was selected for subsequent molecular dynamics simulation to assess interaction stability under dynamic conditions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular Dynamics Simulation of the NS5B\\u0026ndash;3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR (P20) Complex\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the dynamic stability of the docked RNA\\u0026ndash;protein complex, a 100 ns molecular dynamics (MD) simulation was performed for CSFV NS5B in complex with the passage 20 (P20) 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA. Structural stability and interface persistence were assessed using RMSD, RMSF, radius of gyration (Rg), hydrogen bond occupancy, minimum intermolecular distance, and solvent-accessible surface area (SASA) (Abraham et al., \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe RMSD profile showed an initial increase during the early phase of the simulation, consistent with relaxation of the docked conformation, followed by stabilization after approximately 15\\u0026ndash;20 ns (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Thereafter, RMSD fluctuations remained within a moderate range, indicating that the complex reached a dynamically stable conformational state and remained intact over the remainder of the trajectory. The radius of gyration remained comparatively stable throughout the simulation, with only minor oscillations in total Rg and axial components (RgX, RgY, and RgZ) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Suggests that the overall compactness of the NS5B\\u0026ndash;P20 complex was maintained during explicit-solvent MD.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eRMSF analysis indicated limited flexibility across most NS5B residues, with larger fluctuations confined mainly to terminal and loop regions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Residues associated with the RNA-binding channel displayed comparatively low fluctuations, supporting stable engagement of the P20 RNA ligand within the binding cleft. Hydrogen bond analysis demonstrated sustained RNA\\u0026ndash;protein hydrogen bonding throughout the simulation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). While transient variations were observed, the overall hydrogen bond count remained substantial and increased during later stages of the trajectory, indicating progressive stabilization of the interface.\\u003c/p\\u003e \\u003cp\\u003eThe minimum distance between the RNA and NS5B remained low and stable over time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e), indicating close contact without evidence of dissociation. SASA analysis showed moderate fluctuations without abrupt increases that would indicate destabilization or separation of the complex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). The overall SASA behavior is consistent with maintenance of a stable RNA\\u0026ndash;protein interface during simulation. Collectively, these MD analyses support that the NS5B\\u0026ndash;P20 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{\\\\prime\\\\:}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR complex is dynamically stable over 100 ns in explicit solvent. The persistence of hydrogen bonds, stable minimum distance, limited residue-level fluctuations, and maintained compactness indicate a robust RNA\\u0026ndash;polymerase association for the P20 variant under the simulated conditions.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAdaptation of Classical swine fever virus (CSFV) to cell culture represents a multistep evolutionary process shaped by selective pressures that favor efficient replication in a non-native cellular environment. In the present study, serial passaging of CSFV in PK-15 cells resulted in progressive phenotypic and molecular changes indicative of enhanced viral fitness. The transition from an initially non-cytopathic phenotype to extensive monolayer disruption at later passages reflects gradual optimization of virus\\u0026ndash;host interactions, consistent with pestiviral adaptation reported during prolonged in vitro propagation (Hadsbjerg et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Johnston et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eComprehensive sequencing analysis demonstrated that viral adaptation was accompanied by the accumulation of substitutions, insertions, and recurrent deletions within the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR). The consistent emergence and retention of deletions within a defined segment of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR across multiple passages suggests strong selective pressure acting on this regulatory region during cell culture adaptation. Although the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR does not encode protein, it plays a critical role in regulating RNA stability, long-range RNA interactions, and recruitment of the viral RNA-dependent RNA polymerase (RdRp), NS5B (Pankraz et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Y. Xiao et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Even subtle structural remodeling of this region can therefore exert profound effects on replication efficiency and viral fitness.\\u003c/p\\u003e \\u003cp\\u003ePhylogenetic analysis further supported a passage-dependent evolutionary trajectory of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR, characterized by close clustering of early passages, divergence of intermediate variants, and increased evolutionary distances in late passages. This non-linear, stepwise pattern is consistent with quasispecies-driven adaptation, in which transitional variants emerge that balance structural stability with functional flexibility (Mart\\u0026iacute;nez et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Notably, passage 20 (P20) occupied a transitional phylogenetic position, suggesting that key adaptive features can be established at intermediate stages rather than exclusively in late-passage viruses.\\u003c/p\\u003e \\u003cp\\u003eMolecular docking analyses revealed a progressive increase in predicted binding affinity between evolved 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR RNA variants and the NS5B polymerase across serial passages. Early-passage RNAs exhibited relatively weak interactions, whereas intermediate and late-passage variants showed markedly stronger binding, supporting the hypothesis that adaptive remodeling of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR enhances its structural compatibility with NS5B. Similar functional importance of polymerase\\u0026ndash;UTR interactions has been reported for CSFV and related pestiviruses (Li et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Y. Xiao et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMolecular dynamics simulation of the NS5B\\u0026ndash;P20 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR complex provided dynamic validation of these observations. The complex remained stable throughout the 100 ns simulation, exhibiting sustained hydrogen bonding, low intermolecular distances, limited residue-level fluctuations within the RNA-binding cleft, and preserved overall compactness. These features indicate that adaptive mutations present in the P20 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR promote a robust yet flexible RNA\\u0026ndash;polymerase interaction under solvent-explicit conditions, consistent with structural and functional analyses of CSFV NS5B\\u0026ndash;RNA interactions (Li et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Wang et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAlthough later passage variants displayed stronger predicted docking affinities, maximal binding strength is not necessarily optimal for replication. Excessive stabilization of RNA\\u0026ndash;protein complexes can constrain conformational transitions required during initiation and elongation of RNA synthesis (Isken et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Intermediate evolutionary states such as P20 may therefore represent an effective balance between binding stability and structural adaptability.\\u003c/p\\u003e \\u003cp\\u003eCollectively, these findings support a model in which CSFV adaptation to PK-15 cells is driven, in part, by evolutionary remodeling of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR that enhances its interaction with the NS5B polymerase. Adaptive nucleotide changes reshape RNA structure, strengthen RNA\\u0026ndash;polymerase binding, and stabilize the viral replication complex, thereby facilitating efficient replication under in vitro conditions. This study underscores the functional importance of noncoding RNA elements in viral evolution and identifies the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u0026ndash;NS5B interface as a critical determinant of CSFV fitness and a potential target for antiviral intervention.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThis study demonstrates that adaptation of Classical swine fever virus (CSFV) to PK-15 cells is driven by evolutionary remodeling of the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e untranslated region (3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR) that enhances its functional interaction with the viral RNA-dependent RNA polymerase NS5B. Serial passaging selected for defined nucleotide substitutions, insertions, and recurrent deletions within the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR, indicating strong selective pressure acting on this noncoding regulatory region during in vitro adaptation.\\u003c/p\\u003e \\u003cp\\u003eIntegrated phylogenetic, docking, and molecular dynamics analyses revealed that these adaptive changes promote progressively stronger and more stable 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u0026ndash;NS5B interactions. The evolved RNA variants exhibited improved structural compatibility with the polymerase, sustained hydrogen bonding, and limited conformational fluctuations, consistent with stabilization of the viral replication complex. Collectively, these findings highlight the critical role of 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR evolution in modulating CSFV replication efficiency, underscore the importance of RNA\\u0026ndash;protein interactions in viral fitness, and identify the 3\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{}^{{\\\\prime\\\\:}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eUTR\\u0026ndash;NS5B interface as a potential target for antiviral intervention and rational vaccine design.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eNot applicable\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eA.K., conceived and designed the study and performed the majority of the experimental work. A.K. carried out virus isolation, serial passaging, and molecular characterization of the 3\\u0026prime; UTR. T.G. performed sequence analysis, phylogenetic analysis, and contributed to data interpretation. T.G and N.A. conducted molecular docking and molecular dynamics simulations and analyzed protein\\u0026ndash;RNA interaction data.P.K.G. and L.S.R. assisted in experimental design, provided technical support for virological and molecular assays, and contributed to data analysis. B.R.P. supported bioinformatics analysis and interpretation of evolutionary data. S.L. and M.S. assisted with laboratory experiments and data collection.R.C. supervised the overall study, provided critical intellectual input, coordinated the project, and finalized the manuscript.A.K., T.G., and N.A. wrote the initial draft of the manuscript. All authors reviewed, edited, and approved the final version of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request. Sequence data generated during this study are included in the article and/or its supplementary material.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAbraham MJ, Murtola T, Schulz R et al (2015) GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. \\u003cem\\u003eSoftwareX\\u003c/em\\u003e, \\u003cem\\u003e1\\u0026ndash;2\\u003c/em\\u003e, 19\\u0026ndash;25\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarman NN, Bora DP, Khatoon E, Mandal S, Rakshit A, Rajbongshi G, Depner K, Chakraborty A, Kumar S (2016) Classical swine fever in wild hog: Report of its prevalence in northeast india. 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Virol J 1:21\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYan Y, Tao H, He J, Huang S-Y (2020) The HDOCK server for integrated protein\\u0026ndash;protein docking. Nat Protoc. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1038/s41596-020-0312-x\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/s41596-020-0312-x\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYan Y, Zhang D, Zhou P, Li B, Huang S-Y (2017) HDOCK: A web server for protein\\u0026ndash;protein and protein\\u0026ndash;DNA/RNA docking based on a hybrid strategy. Nucleic Acids Res 45(W1):W365\\u0026ndash;W373\\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\":\"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\":\"Classical swine fever virus, 3′ untranslated region (3′ UTR), Cell culture adaptation, Viral evolution, NS5B RNA-dependent RNA polymerase, RNA–protein interactions, Molecular dynamics simulation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8668214/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8668214/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eClassical swine fever virus (CSFV) adapts to cell culture through progressive genetic changes that enhance viral replication and fitness. Although adaptive mutations in viral proteins have been extensively characterized, the contribution of noncoding genomic regions to this process remains poorly understood. In this study, we investigated the evolutionary dynamics of the CSFV 3\\u0026prime; untranslated region (3\\u0026prime; UTR) during serial adaptation in PK-15 cells and examined its functional interaction with the viral RNA-dependent RNA polymerase NS5B. CSFV isolated from spleen tissue was serially passaged in PK-15 cells, resulting in enhanced viral replication and the emergence of cytopathic effects at later passages. Sequencing of the 3\\u0026prime; UTR across multiple passages revealed the accumulation of nucleotide substitutions, insertions, and recurrent deletions, indicating strong selective pressure on this regulatory region during in vitro adaptation. Phylogenetic analysis based on alignment-derived evolutionary distances demonstrated a passage-dependent divergence of 3\\u0026prime; UTR sequences, with early passages clustering closely, intermediate passages forming a distinct subclade, and late passages exhibiting increased evolutionary distances, consistent with a stepwise and non-linear adaptive trajectory. Molecular docking analyses showed a progressive increase in binding affinity between evolved 3\\u0026prime; UTR RNA variants and NS5B across successive passages. An intermediate passage variant (P20), occupying a transitional phylogenetic position, was selected for molecular dynamics simulation and formed a stable NS5B 3\\u0026prime; UTR complex under explicit solvent conditions. The complex maintained structural integrity throughout the simulation, characterized by sustained hydrogen bonding, low intermolecular distances, and limited conformational fluctuations. Collectively, these findings demonstrate that evolutionary remodeling of the CSFV 3\\u0026prime; UTR enhances its interaction with NS5B, stabilizes the viral replication complex, and contributes to efficient cell culture adaptation, highlighting the functional importance of noncoding RNA elements in viral evolution.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Evolution of the 3′ Untranslated Region Enhances NS5B Binding During Cell Culture Adaptation of Classical Swine Fever Virus\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-26 13:49:41\",\"doi\":\"10.21203/rs.3.rs-8668214/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"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\":\"4cd4e0ca-0e6b-4f36-8c37-340bc513a5e0\",\"owner\":[],\"postedDate\":\"February 26th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-03T12:02:01+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-26 13:49:41\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8668214\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8668214\",\"identity\":\"rs-8668214\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}