Search Results (93)

Carpione rhabdovirus (CAPRV) is an emerging virus within the family Rhabdoviridae, posing potential threats to aquaculture species such as golden pompano (Trachinotus anak). However, since the 21st century, and for CAPRV strains isolated from marine fish, only a single CAPRV2023 sequence has previously been available in public databases, with no additional sequences reported. Because the virus undergoes genetic variation, relying on this single sequence likely introduced mismatches or off-target risks in earlier detection assay designs. Notably, the previously developed two-step N-targeting detection assay was designed based solely on that single CAPRV2023 sequence. Consequently, this study involved determining and analyzing the N gene sequences from CAPRV isolates gathered from 2023 to 2025, with the aim of pinpointing conserved regions for assay development, and sequence comparisons subsequently verified the existence of mismatches in the primer–probe binding sites of the previous assay. Since quantitative assays in aquatic virology often define copy numbers utilizing either plasmid DNA templates or RNA templates produced via in vitro transcription, which may lead to variations in amplification kinetics and sensitivity, this study compared both standards to ensure reliable quantification across different nucleic acid types. Based on these findings, a one-step TaqMan quantitative PCR (qPCR) assay was developed and validated using dual nucleic acid standards, namely plasmid DNA and in vitro–transcribed RNA. Compared with conventional two-step qPCR, the one-step format combines cDNA synthesis and subsequent DNA amplification in a single sealed tube, thereby effectively preventing cross-contamination, simplifying the workflow, and improving detection efficiency. The assay exhibited strong linearity (R² > 0.99) and consistent amplification efficiencies between 90% and 110%, demonstrating excellent quantitative performance. The detection limits were 2 copies per reaction for plasmid DNA and 20 copies for in vitro–transcribed RNA templates. No cross-reactivity was observed with other aquatic pathogens, and the assay showed strong repeatability and reproducibility (coefficients of variation below 2.0%), providing a sensitive and reliable tool for epidemiological surveillance and the analysis of CAPRV distribution in marine aquaculture systems of southern China. Full article

With emerging viruses and drug resistance on the rise, the discovery and development of innovative antiviral substances and agents are necessary for the effective treatment and control of viral outbreaks. Surrogate viruses are safer alternatives used in research to mimic dangerous or hard-to-culture viruses. They enable efficient, ethical, and cost-effective screening of antiviral compounds. In this study, we used a recombinant viral hemorrhagic septicemia virus (rVHSV) expressing enhanced green fluorescent protein as a surrogate for RNA viruses for the high-throughput screening of antiviral agents. An optimized mixture of viruses and EPC host cells was distributed in 96-well plates containing chemical compounds or plant extracts for screening. Using this system, 44,642 chemical compounds and 8104 plant and marine organism extracts were tested; 140 candidates were selected from primary screening, and 8 compounds and 5 plant extracts were further selected based on the selectivity index (SI), representing the ratio of the cytotoxic concentration (CC₅₀) to the inhibition concentration (IC₅₀). Among these, compound 3, which had the highest SI value of 1046, was further tested, considering in vitro activity against VHSV and another fish rhabdovirus, snakehead rhabdovirus (SHRV). Full article

Golden pompano (Trachinotus ovatus) is the largest-scale marine aquaculture fish species in China, with a significant economic and nutritional value as a high-quality seafood product. The recent outbreak of an epidemic caused by a novel Carpione rhabdovirus (CAPRV) occurred in cultured golden pompano. To address it, a CAPRV enzyme-mediated one-step sample processing–reverse transcription–enzyme-mediated duplex exponential amplification (EmOSP-RT-EmDEA) detection system was developed. This innovative molecular diagnostic tool integrates enzyme-mediated one-step sample processing (EmOSP) with enzyme-mediated duplex exponential amplification (EmDEA) technology. Unlike traditional RPA-Cas12a detection methods, this system directly incorporates fluorophores into RNA components, eliminating the need for exogenous fluorescent probes while maintaining high sensitivity. It enables rapid, sensitive, and specific detection of CAPRV2023 across various sample types, including clinical, invasive, minimally invasive, and environmental specimens. Performance evaluation of the CAPRV2023 EmOSP-RT-EmDEA detection system against conventional diagnostic methods, such as TaqMan qPCR and traditional PCR, demonstrated superior sensitivity, with a detection limit as low as 4 copies/μL, and exceptional specificity. The optimized EmOSP protocol for nucleic acid extraction from fecal, hepatic, and water samples provided robust and reproducible results. The EmOSP-RT-EmDEA system achieved a detection rate of 68.14% in fecal samples, matching the performance of the gold-standard TaqMan qPCR assay. Full article

Oncolytic virotherapy has emerged as a promising and innovative approach to cancer treatment, leveraging viruses that selectively replicate in tumor cells and cause their destruction (oncolysis), while simultaneously stimulating anti-tumor immune responses. Vesicular stomatitis virus (VSV), a prototypic rhabdovirus, is among the most versatile oncolytic virus platforms due to its favorable biological characteristics, including rapid replication and cell lysis, lack of pre-existing immunity in humans, and amenability to genetic engineering. Over the past decade, significant progress has been made in VSV-based oncolytic virotherapy. This review presents a comprehensive update on developments since our last review, emphasizing improvements in VSV safety, oncoselectivity, tumor-specific replication, direct oncolysis, and induction of antitumor immunity. By integrating recent applied discoveries with foundational knowledge, this review aims to guide ongoing efforts to advance VSV-based oncolytic virotherapy toward broader clinical translation and improved cancer patient outcomes. Additionally, we provide an overview of three closely related rhabdoviruses (Maraba, Morreton, and Jurona viruses) as emerging oncolytic platforms currently under preclinical and clinical investigation. Full article

Infectious hematopoietic necrosis virus (IHNV), a salmonid rhabdovirus, causes severe mortality exceeding 90% in both wild and farmed salmon and trout. Frequent outbreaks of IHNV highlight the urgent need for rapid detection methods to support effective prevention and control. This study developed a double-antibody sandwich ELISA (DAS-ELISA) targeting the nucleocapsid (N) protein of IHNV. Two peptides derived from the N protein—selected for their strong antigenicity, high level of conservation, and surface accessibility—were used as immunogens to generate two specific monoclonal antibodies. Following optimization, the DAS-ELISA was established using monoclonal antibody N-15 as the capture antibody and horseradish peroxidase (HRP)-conjugated antibody N-106 as the detection antibody. The results of this study demonstrated that DAS-ELISA exhibited high specificity for multiple IHNV strains and showed no cross-reactivity with IPNV, SVCV, or VHSV. The detection sensitivity of DAS-ELISA for IHNV was determined to be 10³ TCID₅₀/mL. Parallel analysis of 293 clinical samples using DAS-ELISA and WOAH reference method demonstrated a concordance rate of 92.83% (κ = 0.856). These results confirm that the established DAS-ELISA exhibits high sensitivity, specificity, broad-spectrum applicability, and repeatability. In conclusion, this DAS-ELISA provides a reliable and efficient tool for high-throughput early detection of IHNV infection in clinical settings. Full article

Background/Objectives: Largemouth bass rhabdovirus (Micropterus salmoides rhabdovirus, MSRV) disease causes high mortality in largemouth bass farming. Therefore, vaccine development is critical for largemouth bass prevention against MSRV. Methods: An attenuated strain, denoted as MSRV-0509, was selected through intraperitoneal injection and immersion challenge assays, followed by plaque purification. The biological characteristics of MSRV-0509, including optimal inoculation dose, replication kinetics, thermostability, pH resistance, chloroform tolerance, and storage viability, were determined via viral titration. Spatiotemporal distribution patterns in largemouth bass post-intraperitoneal injection or immersion infection were quantified by qPCR. Immunoprotective efficacy was evaluated through intraperitoneal and immersion vaccination. Mechanistic insights were explored via relative qPCR and serum neutralization assays. Safety was assessed by single-dose overdose immunization and virulence reversion experiments. Results: An attenuated strain MSRV-0509 was screened through a challenge assay, exhibiting complete avirulence in largemouth bass compared to the virulent strain SCRV-T6. MSRV-0509 demonstrated optimal replication at low MOI (0.0001) in CPB cells, with peak titers (10^8.3 TCID₅₀/mL) at 96 h post-infection. The virus showed susceptibility to high temperatures, lipid solvents and acidic conditions, with prolonged stable storage viability at −80 °C. Tissue distribution revealed the spleen as the primary target after intraperitoneal injection, while immersion restricted infection to gills, with rapid clearance by 3–6 dpi. Vaccination trials identified 5 × 10² TCID₅₀/fish via intraperitoneal injection and 10^6.0 TCID₅₀/mL via immersion as effective immunizing doses, providing 100% relative survival post-challenge. Immune gene expression and serum neutralization showed Th1 and Th2 activation via intraperitoneal injection (elevated IL-12, IFN-γ, IL-10, IgM), whereas only the Th1 response was activated after vaccine immersion. No abnormality and mortality were observed in single overdose vaccination and virulence reversion experiments, confirming that MSRV-0509 was safe. Conclusions: These results proved that MSRV-0509 could be a promising vaccine candidate to protect largemouth bass from MSRV disease. Full article

The establishment and partial characterization of three continuous cell lines from European freshwater fish species are provided. The three new cell lines, designated NPL-3, AF-1, and PF-1, were created from larvae of northern pike (Esox lucius) and fin tissues of asp (Leuciscus aspius) and European perch (Perca fluviatilis) fin tissues, respectively. All three cell lines have been subcultured more than 90 times since their establishment. Cells were optimally maintained at 25 °C in M199 medium supplemented with 10% fetal bovine serum. The NPL-3 and AF-1 cells are susceptible to spring viraemia of carp virus, pike fry rhabdovirus, ictalurid herpesvirus 2, and European catfish virus, while in the PF-1 cells, only the latter two viruses were successfully propagated. These newly established cell lines could serve as diagnostic tools for the aforementioned economically important viral diseases. They might be effective appliances for isolating novel viruses from northern pike, asp, European perch, and other closely related fish species. Full article

This study aimed to explore the RNA viruses affecting Passiflora species in Brazil. Our results enhance the understanding of the viruses that infect Passiflora plants by identifying and characterizing three previously unrecognized viruses: Passiflora cytorhabdovirus (PFCV), Passiflora nucleorhabdovirus 1 (PaNV1), and Passiflora nucleorhabdovirus 2 (PaNV2). These rhabdoviruses were identified through high-throughput sequencing and validated by reverse transcription-polymerase chain reaction (RT-PCR) in various Passiflora species. PFCV has a genome organization 3′-N-P-P3-P4-M-G-P7-L-5′ and was classified as a novel member of the Gammacytorhabdovirus genus. A particularly noteworthy feature of PFCV is its glycoprotein, as the genomes of other gammarhabdoviruses do not contain this gene. PFCV has a high incidence across multiple locations and was identified in plants from Northeastern, Central, and Southeastern Brazil. PaNV1 with genome structure 3′-N-P-P3-M-G-L-5′ and PaNV2 with genome organization 3′-N-X-P-Y-M-G-L-5′ are new members of the Alphanucleorhabdovirus genus and have a more restricted occurrence. Importantly, all three viruses were found in mixed infections alongside at least one other virus. In situ observations confirmed mixed infections, with PaNV2 particles co-located in tissues with a potyvirus and a carlavirus. Phylogenetic and glycoprotein sequence similarity network analysis provided insights into their evolutionary placement and potential vector associations. These findings expand the known diversity of rhabdoviruses in Passiflora and contribute to the understanding of their evolution and epidemiology. Full article

A novel ephemeral fever rhabdovirus and a CHeRI orbivirus of a previously unidentified genetic lineage were isolated in mosquito cell line C6/36 cells as co-infecting agents from the spleen tissue of a dead farmed white-tailed deer (WTD; Odocoileus virginianus) in Florida. We designated the ephemeral fever rhabdovirus as Hardee County ephemerovirus 1, strain CHeRI ephemerovirus 1. The genetic sequences of the CHeRI orbivirus isolated in this work differ significantly from those of three previously described CHeRI orbivirus lineages. We designated this new virus as CHeRI orbivirus 4, strain CHeRI orbivirus 4-1. Whereas it remains unknown whether one, both, or none of the viruses contributed to the pathology, gross observations revealed that the dead WTD had severely congested and hemorrhagic lungs, and that its heart, kidneys, and spleen were also congested. Full article

A primary cell culture derived from the gill tissues of largemouth bass (Micropterus salmoides) was successfully established and characterized, providing a physiologically relevant model for virological research. Gill tissues were enzymatically dissociated, and their cells were cultured in M199 supplemented with 20% fetal bovine serum at 25 °C, yielding optimal growth. Viral replication within these primary cells was confirmed by transmission electron microscopy, and further qRT-PCR demonstrated the upregulation of antiviral genes (IFN1, Mx1, ISG15, and Viperin). These primary gill cells of spindle-like morphology exhibited significantly higher susceptibility to Micropterus salmoides rhabdovirus (MSRV) compared to established cell lines, as evidenced by higher viral titers, thus establishing their suitability for studying host–virus interactions. Furthermore, these cells were amenable to genetic manipulation, with the successful transfection of an mCherry reporter gene using commercially available reagents. These findings highlight the utility of the largemouth bass gill-derived primary cell culture as an alternative in vitro system for investigating MSRV pathogenesis and host immune responses, which serves as a stepping stone for improved antiviral strategies in largemouth bass aquaculture. Full article

Tripartite Motif-Containing 44 (TRIM44) is responsible for cancers, neurodegenerative diseases, and viral infections. However, the role of Siniperca chuatsi TRIM44 (scTRIM44) during viral infection remains unclear. In the present study, we analyzed the molecular characteristics of scTRIM44 and its role in infectious spleen and kidney necrosis virus (ISKNV), largemouth bass virus (LMBV), and Siniperca chuatsi rhabdovirus (SCRV) infection. ScTRIM44 contained one B-box domain (B, 166–207 aa) and a coiled-coil domain (CC, 279–309 aa), but lacked the canonical RING domain of E3 ubiquitin ligases. The scTRIM44 mRNA was expressed relatively high in immune-related tissues. The mRNA expression of scTRIM44 significantly decreased in vivo and vitro post-ISKNV and -LMBV infection. However, the expression of scTRIM44 mRNA showed significant up-regulation post-SCRV infection. ScTRIM44 positively regulated SCRV infection in CPB cells, but copies of ISKNV and LMBV showed no significant alteration in over-expressed or knocked-down scTRIM44 cells. Moreover, scTRIM44 positively regulated RIG-I- and MDA5-mediated interferon molecule signaling. These data suggested that scTRIM44 promoted SCRV infection by positively regulating RIG-I- and MDA5-mediated interferon molecule signaling, but didn’t regulate ISKNV and LMBV infection. This research provided a comprehensive insight into the antiviral activity of scTRIM44. Full article

In this study, honeybee viruses were identified in naturally infected honeybee colonies (Apis mellifera carnica). From nine selected samples of clinically affected and ten samples of healthy honeybee colonies, different strains of honeybee viruses were first detected using quantitative real-time RT-PCR methods. Twenty-two nucleotide sequences of the complete genomes of honeybee viruses were identified using the Illumina Next-Generation Sequencing (NGS) method: acute bee paralysis virus (ABPV) (n = 4), black queen cell virus (BQCV) (n = 3), chronic bee paralysis virus (CBPV) (n = 2), deformed wing virus (DWV) (n = 5), Lake Sinai virus (LSV) (n = 4), sacbrood bee virus (SBV) (n = 1), Apis rhabdovirus-1 (ARV-1) (n = 1), bee macula-like virus (BeeMLV) (n = 1) and Hubei partiti-like virus 34 (HPLV34) (n = 1). The nucleotide sequences of ABPV, BQCV, DWV and SBV are the first complete genomes of these viruses identified in Slovenia and they represent an important contribution to our understanding of the genetic diversity of honeybee viruses. ARV-1, BeeMLV and HPLV34 were detected and sequenced for the first time in Slovenia. Full article

Rabies virus (RABV) is among the first recognized viruses of public health concern and has historically contributed to the development of viral vaccines. Despite these significances, the three-dimensional structure of the RABV virion remains unknown due to the challenges in isolating structurally homogenous virion samples in sufficient quantities needed for structural investigation. Here, by combining the capabilities of cryogenic electron tomography (cryoET) and microscopy (cryoEM), we determined the three-dimensional structure of the wild-type RABV virion. Tomograms of RABV virions reveal a high level of structural heterogeneity among the bullet-shaped virion particles encompassing the glycoprotein (G) trimer-decorated envelope and the nucleocapsid composed of RNA, nucleoprotein (N), and matrix protein (M). The structure of the trunk region of the virion was determined by cryoEM helical reconstruction, revealing a one-start N-RNA helix bound by a single layer of M proteins at an N:M ratio of 1. The N-M interaction differs from that in fellow rhabdovirus vesicular stomatitis virus (VSV), which features two layers of M stabilizing the N-RNA helix at an M:N ratio of 2. These differences in both M-N stoichiometry and binding allow RABV to flex its N-RNA helix more freely and point to different mechanisms of viral assembly between these two bullet-shaped rhabdoviruses. Full article

Leafhoppers are economically important pests and may serve as vectors for pathogenic viruses that cause substantial crop damage. In this study, using deep transcriptome sequencing, we identified three novel viruses within the order Mononegavirales, including two viruses belonging to the family Rhabdoviridae and one to the family Lispiviridae. The complete genome sequences were obtained via the rapid amplification of cDNA ends and tentatively named Recilia dorsalis rhabdovirus 1 (RdRV1, 14,251 nucleotides, nt), Nephotettix virescens rhabdovirus 1 (NvRV1, 13,726 nt), and Nephotettix virescens lispivirus 1 (NvLV1, 14,055 nt). The results of a phylogenetic analysis and sequence identity comparison suggest that RdRV1 and NvRV1 represent novel species within the family Rhabdoviridae, while NvLV1 is a new virus belonging to the family Lispiviridae. As negative-sense single-strand RNA viruses, RdRV1 and NvRV1 contain the conserved transcription termination signal and intergenic trinucleotides in the non-transcribed region. Intergenomic sequence and transcriptome profile analyses suggested that all these genes were co-transcriptionally expressed in these viral genomes, facilitated by specific intergenic trinucleotides and putative transcription initiation sequences. Full article

Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus. Cumulative mortality was 100% for salamanders injected with SVCV, 98–100% for tadpoles exposed to virus via immersion, and 0–100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events. Full article