Singapore grouper iridovirus VP12 evades the host antiviral immune response by targeting the cGAS-STING signalling pathway.

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus. Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

Pathogens from salmon aquaculture in relation to conservation of wild Pacific salmon in Canada.

The spread of pathogens from farmed salmon is a conservation concern for wild Pacific salmon in British Columbia (BC), Canada. Three pathogens are prevalent in farmed Atlantic salmon in BC, spill over to wild Pacific salmon, and are linked to negative impacts on wild salmon: Piscine orthoreovirus, Tenacibaculum spp., and sea lice (Lepeophtheirus salmonis). Molecular screening of infectious agents in farmed and wild salmon and environmental DNA highlights a further 4 agents that are likely elevated near salmon farms and 37 that co-occur in wild and farmed salmon. Pathogens likely affect wild salmon indirectly by mediating migration, competition, and predation. Current net-pen aquaculture practices pose these risks to numerous populations of all species of wild salmon in BC, most of which are not covered in Government of Canada science and advisory reports. Climate change, pathogen evolution, and changes to disease management and aquaculture regulations will influence future risks.

Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line.

Cells are equipped with intracellular RIG-like Receptors (RLRs) detecting double stranded (ds)RNA, a molecule with Pathogen-Associated Molecular Pattern (PAMPs) generated during the life cycle of many viruses. Melanoma Differentiation-Associated protein 5 (MDA5), a helicase enzyme member of the RLRs encoded by the ifih1 gene, binds to long dsRNA molecules during a viral infection and initiates production of type I interferon (IFN1) which orchestrates the antiviral response. In order to understand the contribution of MDA5 to viral resistance in fish cells, we have isolated a clonal Chinook salmon Oncorhynchus tshawytscha epithelial-like cell line invalidated for the ifih1 gene by CRISPR/Cas9 genome editing. We demonstrated that IFN1 induction is impaired in this cell line after infection with the Snakehead Rhabdovirus (SHRV), the Salmon Alphavirus (SAV) or Nervous Necrosis Virus (NNV). The cell line, however, did not show any increase in cytopathic effect when infected with SHRV or SAV. Similarly, no cytopathic effect was observed in the ifih1-/- cell line when infected with Infectious Pancreatic Necrosis Virus (IPNV), Infectious Haemorrhagic Necrotic Virus (IHNV). These results indicate the redundancy of the antiviral innate defence system in CHSE-derived cells, which helps with circumventing viral evasion strategies.

Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Expression and regulation of the CXCL9-11 chemokines and CXCR3 receptor in Atlantic salmon (Salmo salar).

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN- γ) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon.

Genome-Wide Association Study of Resistance to Largemouth Bass Ranavirus (LMBV) in Micropterus salmoides.

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass (Micropterus salmoides). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes (MHC II, p38 MAPK, AMPK, SGK1, FOXO3, FOXO6, S1PR1, IL7R, RBL2, and GADD45) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

Immune response in diseased and healthy common carp exposed to carp edema virus.

The common carp is one of the most economically valuable freshwater fish worldwide and its aquaculture can be severely affected by the koi sleepy disease (KSD)/carp edema virus disease (CEVD). This study explores a natural outbreak of CEVD in a pond containing both clinically healthy and diseased fish of various origins exposed to the virus. We investigated mRNA expression of genes associated with known antiviral immune mechanisms, such as type I interferon signalling and cell-mediated cytotoxicity, and performed a comprehensive protein expression analysis to highlight differences between the two groups in various organs. Significant differences in expression profiles of common carp with and without clinical signs were found to be strongly dependent on the organ from which the sample originated. Components of the complement cascade, including various C3 proteins, exhibited upregulation only in less affected organs, specifically the head kidney and spleen. Other complement proteins such as B/C2 and C9 showed upregulation in the kidney, spleen, and gills but not in the skin. Conversely, lysozymes C and G, were observed to be upregulated in the most affected organs of the skin and gills. This study submits the first description of the immune system related proteome using a mass spectrometry on the samples isolated from fish infected with CEV. It also offers a unique comparison of immune reaction of CEV infected and healthy fish under an infectious pressure.

Genome-wide identification, characterization, and expression analysis of the transient receptor potential gene family in mandarin fish Siniperca chuatsi.

BACKGROUND: Temperature is a crucial environmental determinant for the vitality and development of teleost fish, yet the underlying mechanisms by which they sense temperature fluctuations remain largely unexplored. Transient receptor potential (TRP) proteins, renowned for their involvement in temperature sensing, have not been characterized in teleost fish, especially regarding their temperature-sensing capabilities. RESULTS: In this study, a genome-wide analysis was conducted, identifying a total of 28 TRP genes in the mandarin fish Siniperca chuatsi. These genes were categorized into the families of TRPA, TRPC, TRPP, TRPM, TRPML, and TRPV. Despite notable variations in conserved motifs across different subfamilies, TRP family members shared common structural features, including ankyrin repeats and the TRP domain. Tissue expression analysis showed that each of these TRP genes exhibited a unique expression pattern. Furthermore, examination of the tissue expression patterns of ten selected TRP genes following exposure to both high and low temperature stress indicated the expression of TRP genes were responsive to temperatures changes. Moreover, the expression profiles of TRP genes in response to mandarin fish virus infections showed significant upregulation for most genes after Siniperca chuatsi rhabdovirus, mandarin fish iridovirus and infectious spleen and kidney necrosis virus infection. CONCLUSIONS: This study characterized the TRP family genes in mandarin fish genome-wide, and explored their expression patterns in response to temperature stress and virus infections. Our work will enhance the overall understanding of fish TRP channels and their possible functions.

A naturaly attenuated largemouth bass ranavirus strain provided protection for Micropterus salmoides by immersion immunization.

Largemouth bass ranavirus (LMBV) causes disease outbreaks and high mortality at all stages of largemouth bass farming. Therefore, live vaccine development is critical for largemouth bass prevention against LMBV by immersion immunization. Herein, an attenuated LMBV strain with good immunogenicity, designated as LMBV-2007136, was screened from the natural LMBV strains bank through challenge assay and immersion immunization experiment. After determing the safe concentration range of LMBV-2007136, the minimum immunizing dose of immersion immunization was verified. When largemouth bass were vaccinated by immersion at the lowest concentration of 102.0 TCID50/mL, all of fish were survival post virulent LMBV challenge, and the relative percent survival (RPS) was 100 %. And the immune gene expression levels of IL-10, IL-12, IFN-γ, and IgM in the spleen and kidney post-vaccination were significantly up-regulated compared to the control group, but TNF-α expression showed no significant changes. The safety and efficacy of LMBV-2007136 at passages P8, P13, and P18 were futher assessed, and no death of largemouth bass was observed within 21 days post-immunization and RPS of three vaccination groups was 100 %, suggesting that the safety and efficacy of the attenuated strain at different passages was stable. Furthermore, in the virulence reversion test, the attenuated strain was propagated through 5 times in largemouth bass by intraperitoneal injection and no abnormality and mortality were observed, further proving the attenuated vaccine candidate LMBV-2007136 was safe. These results proved that LMBV-2007136 could be a promising candidate for a live vaccine to protect largemouth bass from LMBV disease.

Host Jump of an Exotic Fish Rhabdovirus into a New Class of Animals Poses a Disease Threat to Amphibians.

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.

Nonvirulent Infectious Salmon Anemia Virus (ISAV-HPR0) Not Detectable in Eggs or Progeny of Infected Captive Atlantic Salmon Brood.

The potential for infectious salmon anemia virus (ISAV)-an internationally regulated pathogen of salmon-to transmit vertically from parent to offspring is currently unclear. While the highly virulent ISAV phenotype known as ISAV-HPRΔ has been observed intra-ova, evidence for vertical transmission of the avirulent ISAV phenotype known as ISAV-HPR0 is lacking. In this study, we identified ISAV-HPR0-infected Atlantic salmon broodstock during spawning within a government research recirculating aquaculture facility using qPCR. Eggs and milt from infected brood were used to initiate 16 unique family dam-sire crosses from which 29-60 fertilized eggs per cross were screened for ISAV using qPCR (limit of detection ~100 virus genome copies/egg). A portion of eggs (~300) from one family cross was hatched and further reared in biosecure containment and periodically screened for ISAV by gill clipping over a 2-year period. ISAV was not detected in any of the 781 eggs screened from 16 family crosses generated by infected brood, nor in 870 gill clips periodically sampled from the single-family cohort raised for 2 years in biocontainment. Based on these findings, we conclude that ISAV-HPR0 has a limited likelihood for vertical parent-to-offspring transmission in cultured Atlantic salmon.

Transcriptomic response of lumpfish (Cyclopterus lumpus) head kidney to viral mimic, with a focus on the interferon regulatory factor family.

The economic importance of lumpfish (Cyclopterus lumpus) is increasing, but several aspects of its immune responses are not well understood. To discover genes and mechanisms involved in the lumpfish antiviral response, fish were intraperitoneally injected with either the viral mimic polyinosinic:polycytidylic acid [poly(I:C)] or phosphate-buffered saline (PBS; vehicle control), and head kidneys were sampled 24 hours post-injection (hpi) for transcriptomic analyses. RNA sequencing (RNA-Seq) (adjusted p-value <0.05) identified 4,499 upregulated and 3,952 downregulated transcripts in the poly(I:C)-injected fish compared to the PBS-injected fish. Eighteen genes identified as differentially expressed by RNA-Seq were included in a qPCR study that confirmed the upregulation of genes encoding proteins with antiviral immune response functions (e.g., rsad2) and the downregulation of genes (e.g., jarid2b) with potential cellular process functions. In addition, transcript expression levels of 12 members of the interferon regulatory factor (IRF) family [seven of which were identified as poly(I:C)-responsive in this RNA-Seq study] were analyzed using qPCR. Levels of irf1a, irf1b, irf2, irf3, irf4b, irf7, irf8, irf9, and irf10 were significantly higher and levels of irf4a and irf5 were significantly lower in the poly(I:C)-injected fish compared to the PBS-injected fish. This research and associated new genomic resources enhance our understanding of the genes and molecular mechanisms underlying the lumpfish response to viral mimic stimulation and help identify possible therapeutic targets and biomarkers for viral infections in this species.

Development and validation of reverse-transcription cross-priming amplification-based lateral flow assay for the detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×105 copies/µL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period.

Zebrafish phd1 enhances mavs-mediated antiviral responses in a hydroxylation-independent manner.

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara).

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

Liquid-liquid phase separation is essential for reovirus viroplasm formation and immune evasion.

Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, belonging to the family Spinareoviridae. Members of the Spinareoviridae family are known to replicate and assemble in cytoplasmic inclusion bodies termed viroplasms; however, the detailed mechanism underlying GCRV viroplasm formation and its specific roles in virus infection remains largely unknown. Here, we demonstrate that GCRV viroplasms form through liquid-liquid phase separation (LLPS) of the nonstructural protein NS80 and elucidate the specific role of LLPS during reovirus infection and immune evasion. We observe that viroplasms coalesce within the cytoplasm of GCRV-infected cells. Immunofluorescence and transmission electron microscopy indicate that GCRV viroplasms are membraneless structures. Live-cell imaging and fluorescence recovery after photobleaching assay reveal that GCRV viroplasms exhibit liquid-like properties and are highly dynamic structures undergoing fusion and fission. Furthermore, by using a reagent to inhibit the LLPS process and constructing an NS80 mutant defective in LLPS, we confirm that the liquid-like properties of viroplasms are essential for recruiting viral dsRNA, viral RdRp, and viral proteins to participate in viral genome replication and virion assembly, as well as for sequestering host antiviral factors for immune evasion. Collectively, our findings provide detailed insights into reovirus viroplasm formation and reveal the specific functions of LLPS during virus infection and immune evasion, identifying potential targets for the prevention and control of this virus. IMPORTANCE: Grass carp reovirus (GCRV) poses a significant threat to the aquaculture industry, particularly in China, where grass carp is a vital commercial fish species. However, detailed information regarding how GCRV viroplasms form and their specific roles in GCRV infection remains largely unknown. We discovered that GCRV viroplasms exhibit liquid-like properties and are formed through a physico-chemical biological phenomenon known as liquid-liquid phase separation (LLPS), primarily driven by the nonstructural protein NS80. Furthermore, we confirmed that the liquid-like properties of viroplasms are essential for virus replication, assembly, and immune evasion. Our study not only contributes to a deeper understanding of GCRV infection but also sheds light on broader aspects of viroplasm biology. Given that viroplasms are a universal feature of reovirus infection, inhibiting LLPS and then blocking viroplasms formation may serve as a potential pan-reovirus inhibition strategy.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation.

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

Molecular Mechanism of pH-Induced Protrusion Configuration Switching in Piscine Betanodavirus Implies a Novel Antiviral Strategy.

Many viruses contain surface spikes or protrusions that are essential for virus entry. These surface structures can thereby be targeted by antiviral drugs to treat viral infections. Nervous necrosis virus (NNV), a simple nonenveloped virus in the genus of betanodavirus, infects fish and damages aquaculture worldwide. NNV has 60 conspicuous surface protrusions, each comprising three protrusion domains (P-domain) of its capsid protein. NNV uses protrusions to bind to common receptors of sialic acids on the host cell surface to initiate its entry via the endocytic pathway. However, structural alterations of NNV in response to acidic conditions encountered during this pathway remain unknown, while detailed interactions of protrusions with receptors are unclear. Here, we used cryo-EM to discover that Grouper NNV protrusions undergo low-pH-induced compaction and resting. NMR and molecular dynamics (MD) simulations were employed to probe the atomic details. A solution structure of the P-domain at pH 7.0 revealed a long flexible loop (amino acids 311-330) and a pocket outlined by this loop. Molecular docking analysis showed that the N-terminal moiety of sialic acid inserted into this pocket to interact with conserved residues inside. MD simulations demonstrated that part of this loop converted to a ß-strand under acidic conditions, allowing for P-domain trimerization and compaction. Additionally, a low-pH-favored conformation is attained for the linker connecting the P-domain to the NNV shell, conferring resting protrusions. Our findings uncover novel pH-dependent conformational switching mechanisms underlying NNV protrusion dynamics potentially utilized for facilitating NNV entry, providing new structural insights into complex NNV-host interactions with the identification of putative druggable hotspots on the protrusion.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7.

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.