Frontiers in Virology

MotivationInfluenza is an infectious disease associated with excess human deaths. For influenza A virus (IAV), the surface antigens hemagglutinin (HA) and neuraminidase (NA) define the specific subtype. The high mutation rate of IAV can make clinical testing and assigning subtype after sequencing challenging. Accurate subtype classification is important for tracking circulating IAV strains that may impact human health. ResultsWe analyzed a large IAV protein sequence dataset with known HA and NA subtypes. Using logistic regression and random forest approaches, we identified a small set of subtype-associated amino acids, which were then used to develop a subtype characterization method with near-optimal accuracy. Further analysis indicated that signal sequence peptide variation and indels in HA and NA explained the unique combination of subtype-associated amino acids.

Geminiviruses are plant DNA viruses that replicate in the nucleus of the host cell and rely on the host nucleocytoplasmic transport machinery to complete their infection cycle. While various geminiviral proteins have been reported to interact with plant transport factors, the contribution of nuclear pore complex components to geminivirus infection remains largely unexplored. Here, we identify nucleoporin 50a (NUP50a) as a previously unreported host factor that contributes to bhendi yellow vein mosaic virus (BYVMV) infection. Affinity purification coupled with mass spectrometry isolated NUP50a as a potential interactor of the BYVMV pathogenicity determinant C4, which was further validated by pull-down and co-immunoprecipitation assays. Yeast two-hybrid assays, bimolecular fluorescence complementation, and colocalization analysis demonstrated that BYVMV C4 directly associates with NUP50a predominantly in the nucleus. Virus-induced gene silencing of NbNUP50a significantly delayed symptom development and reduced viral DNA accumulation, suggesting that NUP50a is required for efficient BYVMV infection. Silencing NbNUP50a did not influence the subcellular localization of BYVMV C4, indicating that the role of NUP50a extends beyond determining C4 steady-state localization. Notably, NUP50a was found to associate with C4 proteins from three additional geminiviruses, supporting the possibility that targeting NUP50a represents a characteristic strategy among geminiviruses. Together, our findings provide evidence of a nuclear pore complex member involved in geminivirus pathogenesis. These results establish a framework for further study of the potential transport-dependent and/or transport-independent functions of NUP50a during viral infection.

Nipah is a virus borne zoonotic disease that mostly occurs in Southeast Asian countries. Nipah virus (NiV) infection is potentially lethal as it can cause fatal encephalitis, and as of now, there are no vaccines or therapeutic treatments for this disease. The highly pathogenic NiV belongs to the Paramyxovirus family and the natural host of NiV is fruit bats of the Pteropodidae family. Though NiV has not been detected in the US, rather recently the Camp Hill virus (CHV) in shrew species was identified in Camp Hill, Alabama. This virus is characteristically similar to Nipah, and belongs to the henipavirus genus that include the Nipah and Hendra viruses. NiV has two surface glycoproteins namely the attachment glycoprotein (NiVG) and the fusion glycoprotein (NiVF). Being associated with host cell attachment and fusion of viral membrane, these glycoproteins tend to facilitate the viral entry to the host cell. The present paper describes the structures of the aforesaid viral glycoproteins based on the available database of protein structures and examines if they have an evolutionary link to other viral glycoproteins. Certain similar glycoproteins found in different countries are also explored. The structure-function relationship for these two NiV glycoproteins are determined using time-based molecular dynamics (MD) simulation. The interfacial ligand-receptor interactions between NiV proteins with human receptor and monoclonal antibody, Fab 66, are examined. A primary motivation for these structural studies is that the results might potentially contribute to future therapeutic developments and drug designs to effectively combat NiV.

Respiratory syncytial virus infection is a common cause of hospitalizations in infants and elderly patients. With the increased availability of vaccines and therapeutic antibodies, surveilling the sequence landscape of viral genomes is important to increase awareness of potentially neutralizing mutations. During the 2023-24 and 2024-25 RSV seasons, we collected unpaired nasopharyngeal swab specimens from patients presenting to Arizona healthcare systems with symptoms of influenza-like illness. Using either tiled am-plicon-based enrichment or a commercial oligo hybrid-enrichment panel and next generation sequencing, we obtained genome sequences for RSV and co-infecting viruses. We performed phylogenetic and genomic analyses to classify RSV genomes and detect mutations in the antigenic sites of the F protein and conserved central domain of the G protein. Phylogenetic analysis showed RSV-A strains belonged to the A.D. 1, A.D.2, A.D.3, and A.D. 5 subclades. RSV-B genomes belonged to the B.D.4.1, B.D.4.1.1, B.D.E.1, B.D.E.5, and B.D.E. 7 clades. We found 9 polymorphic sites located in the antigenic sites of the F protein in our RSV-A genomes and 18 polymorphic sites in our RSV-B genomes over the two-season surveillance period. We identified 7 polymorphic sites in our RSV-A genomes and 8 polymorphic sites in our RSV-B genomes that are in the conserved central domain of the G protein. We also discovered 3 (6.7%) and 27 (15.8%) incidences of viral coinfections in the 2023-24 and 2024-25 seasons, respectively. Viral genomic surveil-lance plays an important role in maintaining the efficacy of vaccine and therapeutic agents. Continued monitoring of circulating variants is an important component of public health.

Bunyaviricetes is a class of segmented negative strand RNA viruses (sNSV) that includes causative agents of severe zoonotic diseases resulting in hemorrhagic fever in humans and livestock. Hantaan virus (HTNV), family Hantaviridae, is an example of life-threatening bunyavirus transmitted by rodents which leads to Hantavirus hemorrhagic fever with renal syndrome in humans. Infection relies on the replication and transcription of their tripartite genomes through multifunctional RNA-dependent RNA polymerases (RdRp) in the context of ribonucleoproteins (RNPs) made of one viral RNA fragment, one RdRp and multiple copies of viral nucleoproteins. The RdRp, also known as L protein, performs replication by de novo initiation through a prime-and-realign mechanism while transcription initiates through a cap snatching mechanism, that engages a cap-binding domain and an endonuclease (EN) domain present in the L protein C and N terminal regions, respectively. In this work we show that HTNV L protein has a terminal nucleotidyl transferase (TNTase) activity over double-stranded RNA substrates (dsRNA) in vitro. We show that the TNTase activity is also present in other bunyaviral L proteins by comparing HTNV L protein activity with La Crosse (LACV, family Orthobunyaviridae) and Crimean Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) L proteins. Our results show that TNTase activity appears to be a general feature of Bunyaviruses and report the differences on substrate and nucleotide specificity for each family of virus.

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

Hospital-based wastewater surveillance may complement community and clinical surveillance data in important ways, and may be useful in jurisdictions without community-based wastewater surveillance. From May 2024-April 2026, we analyzed weekly samples (n=190) from three hospitals in New York City using digital PCR to evaluate the sensitivity, specificity, and positive predictive value (PPV) of wastewater viral detection against facility SARS-CoV-2 and influenza A/B inpatient caseloads. Sensitivity was 38-42% for SARS-CoV-2 and 36-49% for influenza A/B, while specificity exceeded 72% for all pathogens. During respiratory seasons, sensitivity reached 81% for SARS-CoV-2 and 81% for influenza A; both had 100% sensitivity during peak case weeks. Notably, off-peak influenza detections occurred in hospital wastewater at all three hospitals in summer 2024 without corresponding hospital case detection, suggesting the presence of undiagnosed cases. These findings underscore the potential utility of hospital-based wastewater monitoring for tracking respiratory virus activity.

Worldwide, physical habitats and biological communities are being reshaped by wildfire. Such changes have obvious potential to alter pathogen ecology, yet few studies, outside of those focused on ectoparasites, have tested the effects of wildfire on pathogen prevalence in wildlife. Even fewer have focused on the impacts of strategies for post-fire remediation on pathogen circulation. Here, we investigated the effect of wildfire and wildfire mitigation on the prevalence and viral load of Sin Nombre virus (SNV) infection in its reservoir host, the western deer mouse, using a study design of matched burned, unburned, and post-burn reseeded sites in northern New Mexico. In total, we screened 411 individual deer mice for SNV via RT-qPCR and analyzed the effect of wildfire and wildfire mitigation on relative mouse abundance, individual infection probability, site-level prevalence and viral load. Relative abundance was highest at reseeded sites, and model selection indicated that habitat structure, particularly the gradient from closed canopy to open-herbaceous habitat, was consistently associated with increased relative abundance. Similarly, SNV prevalence was significantly higher in reseeded sites than either burned or unburned sites but did not differ in burned versus unburned sites. Viral load did not differ between burn history types, and zero-inflated gamma hurdle models did not identify any strong predictors of viral load. Serendipitously, we were also able to investigate the impacts of an El Nino Southern Oscillation (ENSO) cycle on patterns of infection in a subsample of sites that were studied during and one year after an ENSO year and found that SNV prevalence increased significantly post-ENSO. Both reseeding and ENSO deliver resource pulses that can support increases in mouse density and thereby enhance SNV transmission. The impacts of post-fire management practices on SNV prevalence in its reservoir host that were revealed in this study should be considered when implementing such strategies and when utilizing treated areas.

Positive-sense RNA viruses extensively exploit host membrane trafficking pathways to establish intracellular replication organelles required for efficient viral replication. However, the contribution of endosomal trafficking proteins to infection by Drosophila C virus (DCV), a natural dicistrovirus of Drosophila melanogaster, remains poorly understood. Here, we investigated the role of Myopic (Mop), a conserved ESCRT-associated endosomal trafficking protein, during DCV infection. Knockdown of mop in cultured S2 cells significantly increased DCV RNA and viral protein accumulation without affecting viral binding or entry, indicating that Mop restricts DCV replication at a post-entry stage. In addition, depletion of mop altered the intracellular distribution of viral proteins, suggesting that Mop may influence the organization of DCV-associated intracellular membrane compartments. Unexpectedly, fat body-specific knockdown of mop in adult flies produced the opposite phenotype, resulting in reduced viral accumulation and prolonged survival following systemic DCV infection. This antiviral phenotype was not associated with enhanced activation of the STING or JAK-STAT pathways. Instead, transcriptomic and proteomic analyses revealed extensive remodeling of gene and protein expression, including altered abundance of proteins associated with autophagy and RNA interference. Together, our findings identify Mop as a previously unrecognized regulator of DCV infection and reveal distinct cell-autonomous and systemic functions of this conserved trafficking protein. These results highlight the importance of endosomal trafficking in antiviral defense and demonstrate that host membrane trafficking factors can exert fundamentally different effects on virus infection at the cellular and organismal levels.

Antibodies exhibit species-specific sequence and structural features that influence their antigen-recognition properties. Although several studies have investigated porcine antibodies, their repertoire and structural characteristics remain less well characterized than those of several other mammalian species. In this study, we analyzed public porcine heavy-chain repertoire sequencing data together with available antibody structural data to identify characteristic features of porcine antibodies. We found several residues enriched in porcine antibody framework regions, particularly at the base of heavy-chain complementarity-determining region 3 (CDR-H3). In particular, Arg101 and Glu123 were closely positioned in available structures and may influence CDR-H3 conformation at its base, whereas Pro120 may help constrain local backbone conformation. We also observed non-canonical cysteine usage in both framework region 1 and CDR-H3, which may contribute to structural diversity in the porcine repertoire. Finally, we evaluated the humanization potential of a porcine antibody using a human antibody language model and found that human-likeness increased after model-guided substitutions, although the resulting sequences did not exceed the T20 score threshold. Overall, these results indicate that porcine antibodies possess distinct sequence and structural features that may influence CDR-H3 properties and should be considered in future antibody analysis and engineering.

H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33{o}C, 37{o}C, and 39{o}C - temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37{o}C and 39{o}C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33{o}C. While H5N1 D1.1 also showed restricted replication at 37{o}C and 39{o}C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09.

High Plains wheat mosaic virus (HPWMoV) is a wheat and maize-infecting virus of phytosanitary concern due to its potential for seed transmission. Recent phytosanitary restrictions have required sweet corn seed lots to test negative for HPWMoV prior to import into certain countries. To inform the design of more sensitive and broad-spectrum diagnostic primers for seed health testing and phytosanitary certification, we performed deep sequencing of HPWMoV-positive tissue collected from fields in two major sweet corn seed production regions in the Pacific Northwest, the Columbia Basin and Treasure Valley. Virus-like particle enrichment prior to Illumina sequencing facilitated near complete genome coverage (>95%) for the 21 HPWMoV isolates sequenced. De novo assembly of the eight viral genome segments revealed high levels of diversity for each segment, with at least two variants identified for each RNA and three variants for RNA3, RNA6, and RNA8. Within each sample, only one variant per RNA segment was usually present, with the notable exception of RNA3, sorting each isolate into what we designated type A and type B isolates. All but one previously sequenced HPWMoV isolate can be sorted into these two types. Two samples contained at least two variants for every RNA, totaling 17 genome segments, potentially representing a co-infection of type A and type B isolates. Despite this variability, we successfully designed two primer and probe sets for reverse transcription-quantitative polymerase chain reactions (RT-qPCR) that detected all 20 isolates tested in a duplex diagnostic assay, making the assay suitable for seed health testing for HPWMoV.

Background: Neutrophils migrate to the respiratory epithelium during rhinovirus (RV) infection, but whether RV directly activates neutrophils or whether activation arises from epithelial responses remains unclear. Objective: To determine whether RV directly activates human neutrophils or indirectly activates them through RV infected airway epithelial cells. Methods: Human neutrophils were exposed to purified RV, and activation was assessed by myeloperoxidase activity, CD11b/CD66b expression, and reactive oxygen species (ROS) production. Neutrophils were co-cultured with RV-infected air liquid interface (ALI) bronchial epithelium evaluated for effects on epithelial integrity. At 48 to 72 hours post-infection, we used live-cell imaging to measure ROS responses and neutrophil-tracking assays to measure migration velocity and ROS responses to basolateral supernatants, clarified apical washes, and apical debris from RV-infected epithelial cells. Plaque assays were used to assess for RV infectivity after incubation with apical neutrophils. Results: RV did not directly activate neutrophils. Adding neutrophils to the apical surface of airway epithelial cells increased neutrophil ROS production at 48 to 72 hrs post-infection but did not alter epithelial cytotoxicity. Conversely, basolateral supernatants from infected cells enhanced neutrophil migration but not ROS, consistent with cytokine-mediated priming. Clarified apical washes had no effect on migration or ROS. By contrast, apical debris from infected cultures triggered a robust ROS response. Basolateral fractions from medium control or RV infected cultures did not induce ROS. Infectivity is not impacted by apical neutrophil presence. Conclusion: RV does not directly activate neutrophils. Instead, RV infected epithelial cells release basolateral factors that enhance neutrophil migration, while apical debris from infected cells drives ROS activation. These findings indicate that epithelial injury, not RV itself, is the dominant trigger of neutrophil activation during infection.

Kyasanur Forest Disease Virus (KFDV) NS5 methyltransferase (MTase) protein is the essential enzyme that is involved in the cap methylation of viral RNA, viral replication, and immune evasion, and therefore it is an important protein of interest for antiviral research and drug design. In the present work, we successfully resolved the three-dimensional crystal structures of KFDV NS5 MTase co-crystallised with SAH and GTP at resolutions of 2.2 [A] and 2.6 [A], respectively. In previous studies, HC (Herbacetin) and CAPE (Caffeic acid phenethyl ester) have shown inhibitory activity against SAM-dependent viral MTase. To evaluate the inhibitory potential of HC and CAPE against KFDV NS5 MTase, we have performed isothermal titration calorimetry (ITC) and tryptophan fluorescence spectroscopy (TFS) to validate protein interaction with target compounds. MTase inhibition assay was performed using capillary electrophoresis (CE) assays. Additionally, fluorescence polarisation (FP) confirmed RNA binding inhibition by CAPE and HC. Together, these experiments suggest that HC and CAPE are promising inhibitors against KFDV NS5 MTase and could potentially act as lead compounds to design broad-spectrum anti-Orthoflavivirus drugs.

Mass gatherings are thought to create conditions for pathogen introduction and spread, yet whether such events measurably alter the viral community of a city has rarely been tested against an extensive historical baseline. We analyzed 5,137 hybrid-capture metagenomic wastewater samples collected from 55 sites across 17 Texas cities between June 2022 and July 2026, spanning the 2026 FIFA World Cup. We asked (1) whether host-city viral community composition during the tournament departed from its established summer-to-summer range, (2) whether targeted respiratory viruses rose above their characteristic summer trend, and (3) whether the load of rare and novel taxa was elevated in host cities relative to non-host cities. Despite the plausibility of these hypotheses, we detected no tournament-associated perturbations. Although based on a single event, these findings favor continuous wastewater surveillance over event-triggered deployment, both for detecting introductions that arrive unpredictably and for supplying the baseline against which any event should be judged.

Antimicrobial resistance (AMR) is a major threat to modern medicine and an emerging environmental pollutant. Wastewater-based monitoring offers a scalable alternative to traditional surveillance by enabling population-level monitoring across One Health compartments. However, it remains unclear how well bacterial variants detected in wastewater reflect those causing clinical infections. Here, we assessed the prevalence of carbapenem-resistant Escherichia coli (CREC) in wastewater from Uppsala, Sweden, and compared wastewater isolates with clinical isolates from the same region and period. We observed partial overlap between wastewater and clinical CREC populations, but several common clinical variants were not detected in wastewater and vice versa. CREC prevalence was an order of magnitude higher in wastewater receiving hospital effluent, yet this did not improve concordance with clinical isolates, suggesting that hospital wastewater may artificially inflate CREC prevalence estimates. These findings indicate that wastewater CREC prevalence does not necessarily reflect the regional clinical burden. Furthermore, metagenomic sequencing of wastewater-derived E. coli populations rarely detected CREC resistance genes, suggesting that CREC abundance is below the detection limit of this approach and that targeted enrichment is required for reliable monitoring. Together, our results show that while wastewater-based monitoring can complement AMR surveillance, it should be interpreted cautiously when used as an indicator of clinical risk.

Wastewater-based epidemiology uses both targeted and non-targeted bioanalytical approaches to detect and track disease-causing microbes in wastewater. Targeted approaches such as quantitative polymerase chain reaction (qPCR) remain widely used while sequencing-based approaches have slowly gained popularity. However, concordance between established targeted molecular assays and sequencing approaches remains incompletely characterized. To assess the validity of sequencing as an alternative to qPCR, we compared outcomes of both methods targeting five respiratory viruses in wastewater samples collected across 16 Texas cities over 4 years (May 2022-July 2026) of continuous sampling. We found positive correlations between qPCR and sequencing measurements, with both methods independently correlating with statewide clinical emergency department visits. qPCR positivity also increased with genomic sequencing coverage, and sequencing demonstrated greater potential than qPCR as an early respiratory virus outbreak detection method. Our findings support metagenomic sequencing as a sensitive and highly specific alternative to targeted qPCR for wastewater surveillance, and reinforce its utility towards long-term longitudinal monitoring of respiratory viruses.

Avian Influenza viruses (AIVs) infect a broad host range despite having a natural reservoir in wild aquatic birds. Whilst most strains stay within their host species, some break the species barrier through genetic adaptations. We are most concerned about zoonotic cases, where a human becomes infected. Despite these events being rare, they are associated with high mortality and introduce the risk of onward human-to-human transmission of AIV. As a novel pathogen within the human population, this could have pandemic potential. Using genetic composition features for 8 AIV proteins drawn from viral sequence data, we employ machine-learning algorithms to classify AIV cases as zoonotic or not. These genetic features encode host 'signatures' which can indicate zoonosis and include frequency measures such as dipeptide composition and amino acid physiochemical properties. We consistently find XGBoost to outperform all other algorithms. We optimise parameters for ten classification models: one for each of the 8 proteins and two combined models. Following this, we show that a multi-model approach gives the best performing prediction for AIV zoonosis. We have identified all 8 proteins as having a role in predicting zoonotic transmission. Of particular importance is the PB2 and HA proteins, with specific amino acid physiochemical properties such as charge, secondary structure and hydrophobicity amongst the most indicative features in our combined models. Our alignment-free computational study can identify AIV cases still within avian hosts which are genetically closest to zoonotic AIV cases, thereby identifying the cases most likely to cross the species barrier. In a resource limited environment, our model could be used to quickly identify high priority cases for further investigation.

The lack of effective anti-JEV therapy possesses significant challenge to control JEV. {beta}-catenin, a key mediator of Wnt signaling pathway regulates different viral replication and host immune responses. However, its role in JEV infection remains to be elucidated. Thus, the current study focused on evaluating iCRT-14, a specific {beta}-catenin inhibitor, against JEV. Treatment with iCRT-14 following JEV infection resulted efficient reduction in viral progeny release, viral RNA and protein levels in Huh7 and HEK293T cells. Further, active and total {beta}-catenin, Cyclin D-1 and GSK3-{beta}, the other key pathway players were also modulated in infected and inhibitor treated cells. Moreover, iCRT-14 showed an IC of 4.56 in Huh7 cell and maximal inhibition at the early stages of the JEV life cycle. Interestingly, the overexpression of {beta}-catenin in both the cells and siRNA-mediated {beta}-catenin knockdown (in Huh7 cells) significantly abrogated JEV replication, as evidenced by decreased viral titers, viral protein expression, and viral as well as total RNA levels. Moreover, the reduction in extracellular (84%) and intracellular (60%) viral titers following iCRT-14 treatment highlights its role in impairing JEV infection. Further, in silico molecular docking and co-immunoprecipitation studies demonstrated interactions between {beta}-catenin and the JEV NS5 and E proteins. Collectively, these findings suggest that optimum level of {beta}-catenin is required for efficient JEV infection, highlighting its potential as a target for designing host-directed control strategies to regulate viral infection.

Eukaryotic DNA viruses that replicate in the nucleus often exploit components of the host DNA replication machinery for genome replication. Geminiviruses, causal agents of devastating crop diseases worldwide, strictly depend on host factors to replicate their circular single-stranded (ss) DNA genomes, with only a single virus-encoded protein, Rep, required for this process. Rep recruits host replication proteins to the viral genome and catalyzes nicking and ligation at the initiation and termination sites of rolling-circle replication. Despite the reliance of geminiviral replication on plant proteins, the composition of the viral replisome remains largely unknown. Here, we use TurboID-based proximity labeling to identify plant proteins in the vicinity of the Rep proteins from the geminiviruses tomato yellow leaf curl virus (TYLCV) and abutilon mosaic virus (AbMV) during infection. Combining virus-induced gene silencing, infection assays, and chromatin immunoprecipitation, we identify host DNA replication-related factors required for viral genome replication and likely components of the viral replisome. Our results indicate that geminiviruses and related eukaryotic ssDNA viruses selectively repurpose components of the eukaryotic replication fork to support rolling-circle replication, and suggest that they follow a leading-strand replication mode while utilizing the lagging-strand DNA polymerase {delta}. These findings shed light on the molecular mechanism of geminiviral DNA replication and identify potential targets for engineering antiviral resistance in crops.