Virology Journal

The Rosaceae family comprises thousands of species across over 100 genera, including Salmonberry (Rubus spectabilis), a Pacific Northwest native within the diverse Rubus genus. Its berries and leaves are used for food and medicinal purposes, and ecologically it functions as a pioneer species that supports biodiversity and limits erosion. Although many Rubus genomes were sequenced and analyzed, salmonberry remains undercharacterized: despite a recently sequenced genome, no publicly available annotation or gene expression analysis currently exists. Here, we used RNA sequencing to characterize the salmonberry leaf transcriptome and examine its phylogenetic relationship within Rubus. The assembled 63,285 unique transcripts included 1,389 high-confidence lncRNA transcripts expressed in salmonberry leaves, 218 of which are conserved across Rubus. Phylogenetic analysis indicates that salmonberry is closely related to Rubus arcticus. In addition, we detected a novel species of virus associated with salmonberry. These findings provide foundational genomic resources for R. spectabilis and offer new insights into its evolutionary relationships and endogenous viral integrations.

Introduction Recent respiratory illness, especially influenza, may trigger acute cardiac events via elevated inflammatory mediators. During the 2018 influenza season in Bangladesh, this study examined whether recent acute clinical respiratory illness (CRI) or laboratory-confirmed influenza was associated with elevated hs-CRP and IL-6, linked to acute cardiac events. Methods A total of 139 participants aged [&ge;]40 were recruited from a Dhaka cardiac hospital: 70 with acute myocardial infarction (AMI), 30 with other acute cardiac events, and 39 healthy individuals. CRI was defined as fever with cough and/or respiratory symptoms within seven days. Respiratory swabs were tested for influenza, and blood was analyzed for hs-CRP and IL-6. Results Median hs-CRP and IL-6 were higher in participants with CRI or influenza but not significantly. Cardiac patients had elevated hs-CRP (9.98 mg/L in other cardiac; 4.86 mg/L in AMI vs. 1.73 mg/L in healthy) and IL-6 (0.1 pg/mL in other cardiac; 0.145 pg/mL in AMI vs. 0.08 pg/mL in healthy) (p<0.001). CRI was not significantly associated with elevated hs-CRP or IL-6, though influenza in healthy participants was linked to higher IL-6. Cardiac patients had a higher risk of hs-CRP [&ge;]3 mg/L and elevated IL-6. Conclusion Cardiac patients showed significantly increased inflammatory markers, but CRI was not clearly linked to inflammation. Further research should assess biomarker utility for early cardiac risk.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

BackgroundHerpesviruses are a group of double-stranded DNA viruses known to infect a wide range of vertebrates and establish life-long latent infections. While bats serve as natural reservoir hosts for numerous viral families, relatively few bat herpesviruses have been successfully isolated. In this study, we report the isolation and characterization of two novel alphaherpesvirus strains obtained from Rousettus aegyptiacus bats in Coastal Kenya. MethodsThe samples of oral and rectal swabs were collected from three different species of bats from coastal Kenya between October 2024 and April 2025; the bat species collected include Hipposideros spp., Coleura afra, and Rousettus aegyptiacus. Virus isolation was performed by inoculation of samples in Vero E6 cells and subsequent monitoring for cytopathic effects (CPE). Total nucleic acids were extracted from CPE positive cultures and subjected to library preparation to enable unbiased detection of both RNA and DNA viruses. The libraries were sequenced using next-generation sequencing with Illumina MiSeq platform. Subsequently, bioinformatic analysis was carried out to identify the virus, generate consensus genomes as well as phylogenetic analysis to determine the placement of identified viruses. ResultsTwo samples from R. aegyptiacus (KIK_460_O and KIK_465_O) induced typical CPE within five days. Sequencing and assembly yielded partial consensus sequences of approximately 60 kb (KIK_460_O) and 70 kb (KIK_465_O), representing extended genomic data for a bat-associated alphaherpesvirus. This virus has a genome of about 140kb, indicating that our partial assemblies account for about 43-50% of the total genome. Both isolates were found to be closely related to Dzifa herpesvirus, an alphaherpesvirus previously identified in Kilifi, Kenya. Alphaherpesvirus was identified based on partial sequencing of UL19 (3,787bp) and UL30 (2,846bp) genes. The two isolates were found to be identical at the UL19 gene, showing that they belonged to the same virus strain. Phylogenetic analysis showed that the novel alphaherpesvirus belongs to primate alphaherpesviruses under the subfamily Alphaherpesvirinae. ConclusionThis study reports the isolation and genomic characterization of a novel fruit bat alphaherpesvirus from Kenyan Rousettus aegyptiacus bats. The partial genome assembly (60-70 kb) represent the first extended genomic data for this virus, covering approximately 43-50% of the estimated 140 kb complete genome. The phylogenetic placement of this alphaherpesvirus near primate viruses, especially Pteropodid alphaherpesvirus 1, suggests bat-association and needs further investigation into its zoonotic potential.

Influenza A virus (IAV) causes significant morbidity and mortality worldwide. Understanding how viral RNAs may regulate host genes through microRNA-like mechanisms can clarify pathogenesis and reveal therapeutic targets. In this study, we screened all eight IAV H3N2 RNA segments (PB2, PB1, PA, HA, NP, NA, M, and NS) using an ab initio computational pipeline; five segments (PB2, PB1, PA, HA, and M) met the VMir scoring threshold for further analysis, while NP, NA, and NS were excluded due to low pre-miRNA scores. Mature miRNAs were identified using MatureBayes, and target genes in the human genome were predicted with the miRDB server. From these targets, we selected two genes per qualifying segment (10 genes total) based on their functional relevance to influenza infection and supporting literature; all selected genes are unique to their respective segment. We identified 10 segment-specific target genes (IFNL1, DDX60, SAMHD1, MAVS, IRF4, BIRC2, AGO1, MAP3K1, NOD1, and TNFAIP1) and one common target across all five analyzed segments (CADM2). Gene Ontology and pathway analyses showed enrichment in interferon signaling, RIG-I-like receptor pathways, antiviral restriction, RNA interference, and inflammatory responses. Literature supports roles for these genes in pulmonary and antiviral innate immunity. Our findings provide a basis for experimental validation and may help the research community better understand influenza virus pathogenesis and identify novel therapeutic candidates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/725090v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2b14adorg.highwire.dtl.DTLVardef@5a9b2eorg.highwire.dtl.DTLVardef@81ffc1org.highwire.dtl.DTLVardef@be119b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

Tomato fruit blotch virus (ToFBV) is an emerging multipartite positive-sense RNA virus associated with blotchy symptoms on tomato fruits and classified within the genus Blunervirus (family Kitaviridae). Despite its increasing agricultural relevance, the study of ToFBV has been hindered by the lack of mechanical transmissibility and the difficulty in reproducing infections under controlled conditions. In this work, we report a preliminary step toward the development of the first infectious agroclone system for ToFBV, based on full-length cDNA copies of its four genomic RNAs. We demonstrate that the cloned viral genome is capable of initiating cell autonomous replication in Nicotiana benthamiana, as indicated by the accumulation of negative-sense RNA intermediates in infiltrated tissues. To further validate the system, RNA3 was engineered to express GFP, enabling visualization of infection foci and confirming active viral replication in both N. benthamiana and tomato. Functional assays of RNA4-encoded proteins demonstrated that it encodes a movement protein capable of complementing movement-deficient viral vectors and a putative suppressor of post-transcriptional gene silencing (PTGS). Together, these results establish a versatile reverse genetics platform for ToFBV, providing new insights into the replication and functional organization of blunerviruses and enabling future studies on virus-host interactions, pathogenicity, and control strategies.

Tomato brown rugose fruit virus (Tobamovirus fructirugosum) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus was reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of tomato fruits from local markets to investigate the variability of the virus in the country. We explored the different pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein, and the coat protein genes. Then, we used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with the ones from the world. We found that genetic diversity is low, which is consistent with the global situation. No signatures of diversifying selection were detected across the analyzed genes. However, the virus sequences from Morocco showed a clear geographic structure, suggesting that geographic factors probably combined with agricultural practices may contribute to shaping the population structure of ToBRFV in Morocco.

Tomato yellow leaf curl virus (TYLCV) is a devastating viral pathogen threatening agricultural crops globally. In this study, we identified a novel TYLCV isolate (TYLCV-YN6244), which caused viral epidemic in resistant tomato cultivars at Yuanmo county, Yunnan Province of China. We determined the complete genome of TYLCV-YN6244 and found it encoded six viral proteins characteristic of Geminivirus. We identified its V2 protein as a potent viral suppressor of RNA silencing (VSR), and generated infectious clone of wildtype TYLCV-YN6244, or V2-defective TYLCV-YN6244 (TYLCV-YN6244-{Delta}V2) in which V2 was deleted. Both of infectious clones were capable of systemically infecting tobacco and tomato. However, TYLCV-YN6244 but not TYLCV-YN6244-{Delta}V2 could cause disease symptoms in wildtype tobacco or tomato plants, and viral accumulation was drastically reduced in plants infected with TYLCV-YN6244-{Delta}V2 compared to TYLCV-YN6244 while the efficiency of virus-derived small interfering RNAs (vsiRNAs) biogenesis was conversely increased in plants infected with TYLCV-YN6244-{Delta}V2. Surprisingly, small RNA profiling indicated that 21nt and 22nt rather than 24nt vsiRNAs were predominantly produced in tomato plants infected with either TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2. Furthermore, transcriptome analyses revealed that TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2 infection differentially modulated metabolism and defense-related pathways in tomato, probably underlying distinct viral pathogenicity and disease symptoms induced in plants. Overall, our research not only identified a novel pathogenic TYLCV isolate but also characterized molecular biology and host response in tomato with infectious clones firstly developed, with implications in untangling virus-host interaction for developing novel resistance in crop tomato.

We prepared siRNA libraries against the H5N2 virus NP gene, and the PA, PB1 and PB2 genes that express the proteins that form the virus polymerase complex. The antiviral activity of the siRNA libraries in H5N2 virus infected cells was initially assessed by using qPCR to measure the corresponding mRNA levels in the siRNA-treated cells. In this way siRNA molecules within each library were identified that exhibited to a greater than 70% reduction in levels of each target mRNA. A selection of these siRNA molecules was further evaluated for their antiviral activity in a multi-cycle H5N2 MDCK cell model. The siRNA molecules identified were successful in blocking virus transmission and lead to a reduction in influenza virus progeny virus production. This antiviral activity correlated with both the inhibition of nuclear export of the newly formed RNP complexs that arise from the transcriptional activity of the input virus, and the inhibition of the polymerase activity of the newly formed virus polymerase complexes. This study highlights the potential use of siRNA as a strategy to block virus transmission by targeting the avian influenza virus polymerase complex.

Hydrangea ringspot virus (HdRSV) is an emerging plant virus infecting ornamental hydrangea species worldwide, yet its genomic diversity and host associations remain poorly understood. To expand the available genomic resources and assess HdRSV variability, we screened 210 publicly available Hydrangea spp. transcriptomes from diverse tissues, complemented with four newly generated H. macrophylla transcriptomes from Colombia. Viral genomes were assembled from infected samples and analyzed to infer phylogenetic relationships, lineage distribution, and relative viral RNA abundance. Two well-supported phylogenetic lineages (HdRSV-L1 and HdRSV-L2) were recovered from both full-genome and replicase coding sequence (CDS) analyses. HdRSV was detected across all host tissues examined, with the highest median viral loads in roots, followed by stems and leaves. H. macrophylla harbored both viral lineages, while H. serrata was exclusively infected by HdRSV-L1. Cultivar-level analysis revealed marked differences in viral abundance, with lineages showing distinct tissue preferences but no co-infection patterns, except in the Bailer cultivar. Comparative analysis of the replicase CDS identified a single lineage-defining nonsynonymous mutation (C1578T; Thr[-&gt;]Ile), fixed in 90% of HdRSV-L2 genomes, corresponding to a polar-to-nonpolar amino acid change potentially associated with structural adaptation. Together, these findings provide the most comprehensive overview to date of HdRSV genomic diversity, host and tissue distribution, and molecular variation, offering new insights into the evolution and epidemiology of this understudied plant virus.

Accurate quantitation of therapeutic bacteriophages (phages) remains a challenge for clinical development. Plaque-based enumeration is the current standard but is laborious, host-dependent, and variable, particularly when distinguishing individual phages in cocktails. Targeted mass spectrometry of virion structural proteins offers an orthogonal, structure-based approach amenable to reproducible and scalable phage quantitation. Here, we describe a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was performed on an LTQ Orbitrap XL to assess sequence coverage and select surrogate peptide candidates based on specificity and sensitivity. High-resolution peptide mapping identified multiple structural proteins of LUZ19 and provided 55% sequence coverage for the major head protein (YP_001671977.1). Fifteen peptides were detected and evaluated, from which the tryptic peptide EVAELDGQELAR was selected based on abundance, stability, and chromatographic performance. Quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring transitions for targeted peptide detection. Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%, intra-day precision ranging from 0.5 to 9.8%, and inter-day precision ranging from 6.3 to 9.7%. Peptide concentrations from digested lysate samples were related to phage concentrations determined by double layer agar assay, yielding an estimated three copies of the major head protein per virion. ImportanceBacteriophages are the most abundant biological entities on the planet and represent a promising therapeutic class for combating drug-resistant bacterial infections. Realizing the clinical potential of bacteriophage therapy requires analytical methods capable of meeting the standards of modern drug development. Targeted mass spectrometry offers unmatched specificity and resolution for precise quantitation of individual bacteriophages within complex biological samples, a capability that conventional enumeration methods cannot match. Only one prior study has applied mass spectrometry to bacteriophage quantitation, using a well-characterized model bacteriophage at a single concentration without calibration or a validated analytical range. Using Pseudomonas aeruginosa podovirus LUZ19, we present the first targeted mass spectrometry-based bacteriophage quantitation assay developed and validated following FDA bioanalytical guidance. This work establishes a rigorous analytical foundation that moves bacteriophage therapy closer to the standards required for informed dose selection, candidate evaluation, and clinical development.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

Purpose: Human Parvovirus B19 (B19V) infection is associated with a broad spectrum of clinical manifestations, including erythema infectiosum, arthropathy, transient red cell aplasia, hepatitis, and adverse fetal outcomes. Due to nonspecific presentations and limited routine testing, B19V infection often remains undiagnosed. Despite recent reports of increased B19V activity worldwide, contemporary data on its presence in Brazil remain scarce. We investigated the circulation of underdiagnosed pathogens in patients with suspected infectious diseases, prioritizing severe and fatal cases. Methods: In this descriptive study, hybrid capture-based whole-genome sequencing was used for a broad-range viral detection and characterization. We analyzed 472 clinical specimens selected for diagnostic investigation between 2024 and 2025, according to surveillance criteria for respiratory and arboviral infections, referred to the Public Health Laboratory of the state of Rio de Janeiro, Brazil. Results: B19V was the second most frequently detected viral pathogens, being identified in 190/472 specimens. Of these, just one sample had been previously tested for this pathogen. Thirty-one cases had higher B19V genomic coverage and were therefore selected for further analysis. Notably, B19V was the only virus detected with substantial genomic coverage in nine individuals, including elderly patients, and meningitis cases with B19V-positive cerebrospinal fluid. Phylogenetic analysis showed that recovered genomes clustered within genotype 1A2. Conclusions: B19V was frequently detected in patients undergoing investigation for infectious diseases and its circulation may represent an important and underrecognized pathogen. These findings support the incorporation of B19V testing into diagnostic algorithms for unexplained infectious diseases, especially in patients presenting atypical symptoms.

Most adenovirus (Ad) vectors are based on the genome of human Ad type 5 (Ad5), which targets their entry to cells that express the Coxsackie and Adenovirus Receptor (CAR or CXADR). However, certain human Ads do not use CAR, for example Ad35 interacts with cell-surface CD46 and Ad3 uses desmoglein 2 (DSG2) for cell entry. In this study, a comparison of different Ad receptors using transcriptomic and proteomic databases showed that CD46 is widely expressed across human cells and tissues whereas CAR and DSG2 are more restricted to epithelial cells. We have used a hybrid virus, Ad5F35, that comprises an Ad5 genome in which the Ad5 fibre was replaced with that of Ad35, thus retargeting the virus from CAR- to CD46-expressing cells and enabling transduction of primary human NK and T cells. However, lymphocytes required approximately 10 to 20-fold more Ad5F35 particles per cell (ppc) compared to A549 epithelial cells to achieve a similar level of transduction. Consistent with this, quantitation of the cell-surface density of CD46 molecules revealed approximately 100 CD46 molecules per {micro}m2 in primary NK cells compared with approximately 2000 CD46 per {micro}m2 in HeLa cells, a 20-fold difference. Cell-surface CD46 density was reduced by approximately 95% by RNA interference in HeLa cells to levels that approximate those found on NK cells. Lower CD46 density reduced transduction by Ad5F35 but this could be compensated for with increased MOI. Our results identify the density of cell surface CD46 as a critical determinant of Ad5F35 transduction and demonstrate that Ad5F35 is an efficient vector for gene delivery in primary human NK cells.

Citrus yellow vein clearing virus (CYVCV) is the causal agent of an emerging disease representing a potentially high-impact threat for citrus production. Despite remaining outside Europe for decades, CYVCV has now expanded towards two important European citrus producers, Italy and, more recently, Spain. The presence of this virus in the EPPO region represents a current threat with unpredictable and potentially devastating consequences for European citriculture. Therefore, urgent protective measures need to be taken to prevent CYVCV spread and minimize its impact. Diagnostics is a key measure in the management of viral diseases, highlighting the need for harmonized methods suitable for reliable routine detection of the currently known CYVCV diversity. In this study, an inclusive, efficient and highly sensitive real-time RT-qPCR for the detection of CYVCV in plant material and transmission vectors has been developed and validated according to EPPO standards. Moreover, the validated method has been successfully adapted to both PCR digital platforms, that allow high-sensitive absolute quantitative detection, essential in the diagnostics at low viral concentrations; and PCR portable tools, that can be applied in a real diagnostic context for on-site detection. This versatility combines standard validated performance, absolute sensitive quantitation and real on-site detection. The study has also addressed sampling strategies to support reliable molecular diagnostic performance. Our results represent an improvement in the detection of CYVCV to be applied in epidemiological studies and different real diagnostic contexts for the containment of this important citrus pathogen.

Virophages are double-stranded DNA viruses that hyperparasitize giant viruses infecting unicellular eukaryotes. Parasitization by virophages often reduces the replication of giant viruses, thereby modulating microbial communities in the environment. However, the molecular mechanisms underlying the tripartite relationship are largely unknown due to methodological limitations. In the present study, we developed a reverse-genetics system for a Sputnik virophage that parasitizes the amoeba-infecting giant virus, mimivirus. We demonstrated that transfection of genomic DNA could recover infectious virophage particles. Transfection of genomic DNA synthesized by circular polymerase extension reaction (CPER) also resulted in the recovery of infectious viruses. As a proof of concept, we successfully modified two Sputnik genes by transfecting CPER-assembled mutant genomic DNA. Collectively, our reverse-genetics system provides a framework for assessing the functional importance of Sputnik genes and should facilitate future genetic studies of virophages. Significance statementVirophages are viruses that hyperparasitize giant viruses, which infect unicellular eukaryotes and have extremely large particles and genomes. Giant viruses modulate microbial communities not only by killing their hosts but also by altering host cellular functions. Virophages modulate the replication of giant viruses, thereby driving ecosystem dynamics. Previous studies have demonstrated their widespread distribution through isolation and metagenomic analyses. However, the functions of most virophage genes remain unknown. Due to the lack of genetic tools, the molecular mechanisms underlying the interactions between virophages and giant viruses remain largely elusive. Here, we established a virophage reverse-genetics system based on circular polymerase extension reaction. Our results demonstrate that the system can dissect virophage gene functions and will accelerate virophage genetics.

Fuselloviridae infecting thermoacidophilic Sulfolobales are among the best-studied archaeal viruses, with the Saccharolobus Spindle Shaped Virus 1 (SSV1)-Saccharolobus solfataricus interaction serving as a model system. After infection, SSV1 establishes a chronic infection with new virions continuously budding from the infected cell. UV irradiation triggers virus replication, increased virion production and a tightly orchestrated temporal transcription cycle. However, gene regulatory mechanisms post-UV-induction remain elusive. In this study, we investigated the SSV1 transcriptomic landscape post-UV-induction using high-resolution, long-read ONT-cappable-RNA-sequencing. Through end-to-end sequencing of primary transcripts, we generated a comprehensive transcript map of SSV1, identifying 21 transcription start sites (TSSs), including 12 newly described sites, and 19 termination sites (TTSs), of which 9 are novel. This enabled refinement of transcription units and operon structures across the genome. Five new antisense RNAs were discovered, including one encoded antisense to the open reading frame of a putative viral toxin gene a291, which we hypothesize to be a cis-encoded regulatory RNA. In addition, spontaneous recombination between two direct repeats in the SSV1 structural genes vp1 and vp3 was confirmed. To our knowledge, this is the first implementation of cappable-seq for an archaeal virus, and the first base-resolution analysis of the SSV1 transcriptome. It reveals a previously unappreciated level of transcriptional complexity, illustrating how high-resolution transcriptomics can deepen understanding of viral transcriptome architecture and regulatory networks. ImportanceArchaeal viruses remain among the least understood viruses in the virosphere, despite their ecological and evolutionary significance. Saccharolobus Spindle Shaped Virus 1 (SSV1) is a key model for the study of virus-host interactions in archaea. Yet, its gene regulatory mechanisms have remained poorly resolved. Here, we provide the first base-pair resolution transcriptome of an archaeal virus, revealing a complex and multi-layered transcriptional architecture. We uncover alternative transcription start and termination sites, as well as antisense transcription, pointing to regulatory mechanisms beyond a simple temporal gene expression program. These findings suggest that archaeal viruses use sophisticated transcriptional and post-transcriptional mechanisms similar to those in bacterial and eukaryotic viruses. By establishing a high-resolution map of the SSV1 transcriptome, this work advances our understanding of archaeal virus biology.

African swine fever virus (ASFV), the etiologic agent of African Swine Fever (ASF), is a high-consequence pathogen requiring experiments to be conducted in containment in non-endemic countries, thereby restricting diagnostic development, the creation of reference standards, and proficiency testing (PT). Safe and reliable inactivation methods are essential to expand diagnostic capacity while preserving nucleic acid integrity for molecular assays in unaffected countries. This study employed gamma irradiation to achieve complete inactivation of ASFV without compromising downstream molecular detection, as gamma irradiation offers deep penetration and uniform dose delivery. ASFV-cell culture supernatants were subjected to gamma irradiation doses ranging from 2 to 50 kGy. Viral replication was evaluated using TCID{square}{square} and serial passages, revealing a consistent dose{square}dependent reduction in infectivity across increasing irradiation dose levels and a complete loss of ASFV infectivity at 30 and 50 kGy. Molecular detection remained unaffected at all of the tested doses as confirmed by qPCR Ct values and sequence identity of the p72 gene. Whole genome sequencing demonstrated >99% genome coverage and consistent read depth profiles across irradiated and non-irradiated samples, indicating preservation of genomic integrity at all tested doses. These findings demonstrate that gamma irradiation at 50 kGy fully inactivates ASFV-cell supernatants while maintaining nucleic acid quality suitable for molecular diagnostics. The resulting inactivated material meets quality assurance requirements for molecular reference standards and PT panels and can be safely distributed to laboratories outside high containment facilities, supporting broader diagnostic readiness and harmonization of ASFV testing.