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.

Parabacteroides distasonis regulates the infectivity and pathogenicity of SVCV at different water temperatures.

BACKGROUND: Spring viremia of carp virus (SVCV) infects a wide range of fish species and causes high mortality rates in aquaculture. This viral infection is characterized by seasonal outbreaks that are temperature-dependent. However, the specific mechanism behind temperature-dependent SVCV infectivity and pathogenicity remains unclear. Given the high sensitivity of the composition of intestinal microbiota to temperature changes, it would be interesting to investigate if the intestinal microbiota of fish could play a role in modulating the infectivity of SVCV at different temperatures. RESULTS: Our study found that significantly higher infectivity and pathogenicity of SVCV infection in zebrafish occurred at relatively lower temperature. Comparative analysis of the intestinal microbiota in zebrafish exposed to high- and low-temperature conditions revealed that temperature influenced the abundance and diversity of the intestinal microbiota in zebrafish. A significantly higher abundance of Parabacteroides distasonis and its metabolite secondary bile acid (deoxycholic acid, DCA) was detected in the intestine of zebrafish exposed to high temperature. Both colonization of Parabacteroides distasonis and feeding of DCA to zebrafish at low temperature significantly reduced the mortality caused by SVCV. An in vitro assay demonstrated that DCA could inhibit the assembly and release of SVCV. Notably, DCA also showed an inhibitory effect on the infectious hematopoietic necrosis virus, another Rhabdoviridae member known to be more infectious at low temperature. CONCLUSIONS: This study provides evidence that temperature can be an important factor to influence the composition of intestinal microbiota in zebrafish, consequently impacting the infectivity and pathogenicity of SVCV. The findings highlight the enrichment of Parabacteroides distasonis and its derivative, DCA, in the intestines of zebrafish raised at high temperature, and they possess an important role in preventing the infection of SVCV and other Rhabdoviridae members in host fish. Video Abstract.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.).

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Drug delivery system based on metal-organic framework improved 5-Fluorouracil against spring viremia of carp virus.

Spring viremia of carp virus (SVCV), as a high pathogenicity pathogen, has seriously restricts the healthy and sustainable development of cyprinid farming industry. In this study, we selected 5-Fluorouracil (5-Fu) as the drug model based on zeolitic imidazolate framework-8 (ZIF-8) to construct a drug delivery system (5-Fu@ZIF-8), and the anti-SVCV activity was detected in vitro and in vivo. The results showed 5-Fu@ZIF-8 was uniform cubic particle with truncated angle and smooth surface, and the particle size was 90 nm. The anti-SVCV activity in vitro results showed that the highest inhibition rate of 5-Fu was 77.93% at 40 mg/L and the inhibitory concentration at half-maximal activity (IC50) was 20.86 mg/L. For 5-Fu@ZIF-8, the highest inhibition rate was 91.36% at 16 mg/L, and the IC50 value was 5.85 mg/L. In addition, the cell viability was increased by 18.1% after 5-Fu treatment. Similarly, after 5-Fu@ZIF-8 treatment, the cell viability increased by 27.3%. Correspondingly, in vivo experimental results showed the viral loads reduced by 18.1% on the days 7 and the survival rate increased to 19.4% at 80 mg/L after 5-Fu treatment. For 5-Fu@ZIF-8, the viral loads reduced by 41.2% and the survival rate increased to 54.8%. Mechanistically, 5-Fu inhibits viral replication by regulating p53 expression and promoting early apoptosis in infected cells. All results indicated that 5-Fu@ZIF-8 improved the anti-SVCV activity; it may be a potential strategy to construct a drug-loaded system with ZIF-8 as a carrier for the prevention and treatment of aquatic diseases.

Effects on viral suppression and the early-immune expression of ribavirin against spring viremia of carp virus in vitro.

Spring viremia of carp virus (SVCV) is a globally distributed virus that causes severe clinical symptoms and high mortality in fish belonging to the families Cyprinidae and Siluridae. To protect the host against viral infection, understanding the relatedness between viral susceptibility and antiviral mechanisms must be crucial. Thus, we evaluated the viral suppression efficacy of ribavirin by measuring the transcription levels of viral and immune genes in vitro. The results showed that following ribavirin treatment after SVCV infection (MOI 0.1), ribavirin inhibited SVCV replication in epithelioma papulosum cyprini (EPC) cells and completely inhibited viral gene (G and N) expression at concentrations above 10 µg/mL at 48 h post-infection. Ribavirin does not directly damage SVCV particles but inhibits early viral replication. In the absence of SVCV infection, the immunological dynamics triggered by ribavirin resulted in upregulated pattern recognition receptors and proinflammatory cytokine-related genes (i.e., PI3K, MYD88, IRAK1, RIG-І, MAVS, Mx1, TNF-α, and NF-κB). Furthermore, EPC cells treated with ribavirin following SVCV infection showed upregulation of PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB genes within 24 h post-SVCV infection, suggesting that ribavirin positively inhibits the SVCV infection in vitro.

Spring Viremia of Carp Virus Infection Induces Carp IL-10 Expression, Both In Vitro and In Vivo.

Interleukin-10 (IL-10) is a pleiotropic cytokine with both immune enhancement and immunosuppression activities, but the main role is immunosuppression and anti-inflammatory ability. In order to use the immunosuppressive function of IL-10, many viruses, such as SARS-CoV-2, hepatitis B virus and EB virus, can evade the host's immune surveillance and clearance by increasing the expression of host IL-10. However, it has not been reported whether the aquatic animal infection virus can upregulate the expression of host IL-10 and the mechanisms are still unknown. Spring viremia of carp (SVC) is a fatal viral disease for many fish species and is caused by spring viremia of carp virus (SVCV). This disease has caused significant economic losses in the aquaculture industry worldwide. In this study, the expression of carp IL-10 with or without infection of SVCV in epithelioma papulosum cyprinid (EPC) cells, carp head kidney (cHK) primary cells and common carp tissues were analyzed using RT-PCR and ELISA. The results show that SVCV infection induced carp IL-10 mRNA and protein expression, both in vitro and in vivo. However, the upregulation of carp IL-10 by SVCV was hindered by specific inhibitors of the JAK inhibitor (CP-690550), STAT3 inhibitor (STA-21), NF-κB inhibitor (BAY11-7082) and p38 MAPK (mitogen-activated protein kinase) inhibitor (SB202190), but not JNK inhibitor (SP600125). Furthermore, the results demonstrated that JAK1, JAK2, JAK3, TYK2 and STAT5 played important roles in carp IL-10 production induced by SVCV infection. Taken together, SVCV infection significantly induced carp IL-10 expression and the upregulation trigged in JAK-STAT, NF-κB and p38MAPK pathways. To our knowledge, this is the first time that a fish infection virus upregulated the host IL-10 expression through the JAK-STAT, NF-κB and p38MAPK pathways. Altogether, fish viruses may have a similar mechanism as human or other mammalian viruses to escape host immune surveillance and clearance.

Characterization of STING from common carp (Cyprinus carpio L.) involved in spring viremia of carp virus infection.

Stimulator of interferon genes (STING) serve as an endoplasmic reticulum (ER) protein and modulates innate immune responses to viral contagion. Most investigations involving teleost STING antiviral immunity have examined DNA viruses. Therefore, fish STING signaling events against RNA viruses require additional exploration. Here, common carp STING (named CcSTING) was cloned and characterized. The bioinformatics analyses of CcSTING showed evolutionary conservations and were most closely related to other cyprinid STINGs. Immunofluorescence staining discovered that the CcSTING was chiefly placed in the cytoplasm, specifically within the ER. CcSTING was ubiquitously generated in all analyzed organs, with especially strong expression in the gills and head kidney. Spring viremia of carp virus (SVCV) stimulation and poly(I:C) infection induced the generation of CcSTING in immune-associated organs, as well as in peripheral blood leukocytes. Additional investigations revealed that CcSTING overexpression strongly suppressed SVCV replication in EPC cells. Mechanistically, CcSTING enhanced IFN-1 and ISGs expression following SVCV infection. CcSTING also substantially increased both IFN and NF-κB promoter luciferase activity via a dosage-dependent fashion. Lastly, CcSTING significantly up-regulated both TBK1 and p65 phosphorylation. Collectively, these findings demonstrated the critical role and underlying mechanism of fish STING in response to RNA virus.

Application of rhein as an immunostimulant controls spring viremia of carp virus infection.

In recent years, the exploration of natural compounds possessing both immunostimulatory and antiviral activities has attracted growing attention in aquaculture research. Consequently, the pursuit of identifying natural products exhibiting anti-SVCV potential as immunostimulants holds significant promise, offering a pathway to mitigate the economic ramifications inflicted by SVCV outbreaks in aquaculture settings. Among them, rhein emerges as a particularly compelling contender. Boasting a widespread distribution, well-established extraction methods, and multiple biological activities, it has exhibited the capacity to enhance the antiviral activity of host cells in vitro by blocking the viral internalization process, with a peak inhibition rate of 44.0%. Based on this intervention, rhein inhibited apoptosis and mitochondrial damage triggered by SVCV infection, ultimately producing a significant antiviral effect. Moving beyond the laboratory setting, rhein's efficacy translates effectively into in vivo scenarios. It has demonstrated substantial antiviral potency by increasing the expression of antiviral-related genes, most notably, retinoic acid-inducible gene I (RIG-I), interferon-φ (IFN-φ) and IFN-stimulated gene product 15 (ISG15). In concert with this genetic modulation, rhein efficiently reduces the viral load, precipitating a consequential enhancement in the survival rate of SVCV-infected fish, elevating it to an encouraging 16%. In conclusion, the outcomes of our investigation offer a compelling testament to rhein's potential as a valuable immunomodulator in the battle against SVCV infections in aquaculture, and the remarkable attributes exhibited by rhein underscore its viability for future commercial deployment.

The effect of stabilized culture of Lactobacillus rhamnosus GCC-3 on gut and liver health, and anti-viral immunity of zebrafish.

Probiotics are promising antibiotics alternatives to improve growth and disease resistance of cultured fish. Our study aimed to investigate the effect of dietary stabilized culture of Lactobacillus rhamnosus GCC-3 on growth performance, gut and liver health and anti-viral ability of zebrafish (Danio rerio). Zebrafish (0.161 ± 0.001 g) were fed control and the experimental diet containing 1% GCC-3 culture (1 × 107 CFU/g diet) for four weeks. Growth performance and gut and liver health parameters were monitored after four weeks feeding. The gut microbiota was analyzed by 16S rRNA gene sequencing. In another experiment, zebrafish (0.212 ± 0.001 g) were fed with basal or GCC-3 diets and challenged by spring viremia of carp virus (SVCV) at the end of feeding. The antiviral immune response was evaluated at 2nd and 4th days post SVCV infection and survival rate was calculated 14 days after challenge. The results showed that adding 1% GCC-3 significantly improved growth performance of zebrafish (P < 0.05). The intestinal expression of hypoxia-inducible factor Hif-1α, tight junction protein ZO-1α and ZO-1ß was significantly up-regulated in 1% GCC-3 group compared with control (P < 0.05). Besides, 1% GCC-3 decreased the content of MDA and increased total antioxidant capacity in the intestine, and the relative expression of SOD, GST and Gpxa was improved. The abundance of Proteobacteria was reduced while Firmicutes was enriched in the intestinal microbiota of 1% GCC-3 group compared with control (P < 0.05). Zebrafish fed 1% GCC-3 showed higher survival rate after SVCV challenge. Accordingly, the expression of antiviral genes in the spleen was increased at 2nd and 4th days post infection. In conclusion, our results indicate that dietary 1% GCC-3 supplementation can improve gut and liver health as well as antiviral immunity of zebrafish.

Infection of epithelioma papulosum cyprini (EPC) cells with spring viremia of carp virus (SVCV) induces autophagy and apoptosis through endoplasmic reticulum stress.

Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.

Prohibitin mediates the cellular invasion of spring viremia of the carp virus.

Spring viremia of carp virus (SVCV) is strongly contagious and pathogenic to common carp and cyprinoid species. However, knowledge of how SVCV enters host cells is still inadequate. In this study, mass spectrometry (MS) was incorporated with tandem affinity purification (TAP) to identify host proteins that interact with SVCV glycoprotein, the main attachment protein of SVCV. Specifically, prohibitin (PHB) received the utmost attention from all the candidate proteins, and its interaction with the SVCV-G protein was confirmed by immunoprecipitation and immunofluorescence assays. Treatment with PHB-specific inhibitors or knockdown of the expression of PHB by siRNAs resulted in a marked reduction in binding and entry of SVCV on host cells, while overexpression of PHB increased SVCV attachment and invasion. Furthermore, binding of SVCV to ZF4 and FHM cells was inhibited by pre-incubating the virus with recombinant PHB protein (rPHB) or blocking the cell surface PHB with its polyclonal antibodies. In addition, overexpression of PHB on SVCV-nonpermissive Grouper spleen cells (GSs) conferred susceptibility to SVCV infection. In vivo, treatment of rPHB could significantly inhibit SVCV propagation within zebrafish and benefit the survival rate of SVCV-infected zebrafish. These results demonstrate that PHB plays a crucial role in both the attachment and entry stages of SVCV infection.

GCRV NS38 counteracts SVCV proliferation by intracellular antagonization during co-infection.

Viral co-infection has been found in animals; however, the mechanisms of co-infection are unclear. The abundance and diversity of viruses in water make fish highly susceptible to co-infection. Here, we reported a co-infection in fish, which resulted in reduced host lethality and illustrated the intracellular molecular mechanism of viral co-infection. The spring viremia of carp virus (SVCV) is a highly lethal virus that infects Cyprinidae, such as zebrafish. The mortality of SVCV infection was significantly reduced when co-infected with the grass carp reovirus (GCRV). The severity of tissue damage and viral proliferation of SVCV was also reduced in co-infection with GCRV. The transcriptome bioinformatics analysis demonstrated that the effect on the host transcripts in response to SVCV infection was significantly reduced in co-infection. After excluding the extracellular interactions of these two viruses, the intracellular mechanisms were studied. We found that the GCRV NS38 remarkably decreased SVCV infection and viral proliferation. The interaction between GCRV NS38 and SVCV nucleoprotein (N) and phosphoprotein (P) proteins was identified, and NS38 downregulated both N and P proteins. Further analysis demonstrated that the N protein was degraded by NS38 indispensable of the autophagy receptor, sequestosome 1 (p62). Meanwhile, K63-linked ubiquitination of the P protein was reduced by NS38, leading to ubiquitinated degradation of the P protein. These results reveal that the intracellular viral protein interactions are a crucial mechanism of co-infection and influence the host pathology and expand our understanding in intracellular viral interactions co-infection.

Transcriptome analysis identifies LGP2 as an MDA5-mediated signaling activator following spring viremia of carp virus infection in common carp (Cyprinus carpio L.).

The common carp (Cyprinus carpio L.) is an important farmed species worldwide. Mucosal-associated lymphoid tissues play an essential role in the fight against pathogen infection. Spring viremia of carp virus (SVCV) poses a serious threat to the common carp aquaculture industry. Understanding the molecular mechanisms driving mucosal immune responses to SVCV infection is critical. In this study, the mucosal tissues (gills, foregut and hindgut) were collected from normal and infected fishes for transcriptome analysis. A total of 932,378,600 clean reads were obtained, of which approximately 80% were successfully mapped to the common carp genome. 577, 1,054 and 1,014 differential expressed genes (DEGs) were identified in the gills, foregut and hindgut, respectively. A quantitative polymerase chain reaction assay indicated that the DEGs expression in the foregut following SVCV infection was consistent with the transcriptome results. Among them, two key genes of the retinoic acid-inducible gene I (RIG-I)-like receptor family, melanoma-differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) (i.e., CcMDA5 and CcLGP2), underwent further analysis. Overexpression of CcMDA5 or CcLGP2 increased phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3 and the expression of interferon-1 (ifn-1), myxovirus resistance (mx), viperin and interferon-stimulated gene 15 (isg15), and inhibited SVCV replication in epithelioma papulosum cyprini cells. Furthermore, CcLGP2 significantly upregulated the CcMDA5-induced ifn-1 mRNA expression and the activation of the ifn-1 promoter. Finally, confocal microscopy and coimmunoprecipitation experiments revealed that CcLGP2 colocalizes and interacts with CcMDA5 via the C-terminal regulatory domain. This study provides essential gene resources for understanding the fish immune response to SVCV infection and sheds light on the potential role of fish LGP2 in the MDA5 regulation.

Antiviral effects of natural small molecules on aquatic rhabdovirus by interfering with early viral replication.

Spring viremia of carp virus (SVCV) is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range. To develop effective agents to control SVCV infection, we selected 16 naturally active small molecules to assess their anti-SVCV activity. Notably, dihydroartemisinin (DHA) (100 µmol/L) and (S, S)-(+)-tetrandrine (TET) (16 µmol/L) exhibited high antiviral effects in epithelioma papulosum cyprinid (EPC) cells, with inhibitory rates of 70.11% and 73.54%, respectively. The possible antiviral mechanisms were determined as follows: 1. Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells, suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted; 2. Although neither had an effect on viral adhesion, TET (but not DHA) interfered with SVCV entry into host cells (>80%), suggesting that TET may have an antiviral function in early viral replication. For in vivo study, both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%, significantly decreased viral load, and modulated the expression of antiviral-related genes, indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection. Overall, our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication, thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.

Dual-Targeting Polymer Nanoparticles Efficiently Deliver DNA Vaccine and Induce Robust Prophylactic Immunity against Spring Viremia of Carp Virus Infection.

Spring viremia of carp virus (SVCV) is highly contagious and lethal to most cyprinid fish, causing serious economic losses to the carp aquaculture industry. Although DNA vaccines can generate long-term humoral and cellular immune responses, which provide protective immunity against SVCV, the major drawback of DNA vaccines is their low immunogenicity in clinical tests. Here, we construct a dual-targeted polymer DNA vaccine delivery platform (MCS-PCHG) by using mannosylated chitosan to encapsulate the poly(d,l-lactide-co-glycolide)-loaded DNA vaccine containing the heavy-chain CH3 region (CH3) of common carp IgM and the antigenic domain (G131c). The developed nanovaccine delivery platform showed good biocompatibility in vivo and in vitro. With the modification of the mannose moiety and the modification of CH3, the constructed MCS-PCHG could efficiently activate the maturation of antigen-presenting cells. Moreover, we observe significantly high level of immune-related genes expression, serum antigen-specific IgM, SVCV-neutralizing antibody titers in fish vaccinated with MCS-PCHG. Next, the protective efficacy of MCS-PCHG was further evaluated by challenge test. The highest survival rate (ca. 84%) was observed in fish vaccinated with MCS-PCHG after challenging with SVCV. This study presents a novel design for smart, dual-targeted polymer nanoparticles, which are inherently biocompatible, promising for targeted vaccine delivery. IMPORTANCE Spring viremia of carp virus (SVCV) affects global cyprinid fish farming industry, with no available commercial vaccine. Herein, we developed a dual-targeting polymer nanovaccine (MCS-PCHG) by using mannose and common carp IgM heavy chain CH3 region (CH3) as antigen presenting cell (APCs) recognition moiety, attaining the effective delivery of antigen. This dual-targeting polymer vaccine can efficiently activate the APCs, and further induce robust and durable adaptive immune response with good protection against SVCV infection. Our study provides valuable theoretical basis for developing efficient vaccine against infectious diseases in aquaculture.

Dietary supplementation of exopolysaccharides from Lactobacillus rhamnosus GCC-3 improved the resistance of zebrafish against spring viremia of carp virus infection.

Spring viremia of carp virus (SVCV) can cause high mortality of fish. The aim of this study was to investigate the effects of Lactobacillus rhamnosus GCC-3 exopolysaccharides (GCC-3 EPS) on zebrafish (Danio rerio) infected with SVCV and elucidate the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.5% and 1% of GCC-3 EPS for 2 weeks. The results showed that supplementation of GCC-3 EPS significantly improved the survival rate of zebrafish compared with the control group. In addition, dietary 0.5% and 1% GCC-3 EPS significantly up-regulated the expression of genes related to type I interferon (IFN) antiviral immunity. Consistent with in vivo results, GCC-3 EPS significantly inhibited SVCV replication in zebrafish embryonic fibroblast (ZF4) cells while significantly increased the expression of type I IFN signaling pathway related genes. Furthermore, knocking down TANK-binding kinase 1 significantly blocked the antiviral effect of GCC-3 EPS. Dietary GCC-3 EPS improved gut microbiota, and the culture supernatant of GCC-3 EPS-associated microbiota significantly inhibited SVCV replication in ZF4 cells compared with the control-microbiota counterpart. In conclusion, our results indicate that dietary GCC-3 EPS can improve the resistance of zebrafish against SVCV infection, and the mechanism may involve enhanced type I interferon signaling.

Protective immunity against spring viremia of carp virus by mannose modified chitosan loaded DNA vaccine.

Spring viremia of carp virus (SVCV) usually be considered as one of the serious in viral diseases of aquaculture, and DNA vaccine with novel delivery mechanism or adjuvant has proven to be a promising and effective strategy to control aquatic animal diseases. In this study, the mannose-modified chitosan, a carrier system for vaccine delivery, were used to developed a chitosan-encapsulated DNA vaccine (CS-M-G) against SVCV, then investigated immune response induced by the vaccine. Our results showed that CS-M-G was confirmed the spherical or elliptical with even distribution and ranging from approximately 50 to 150 nm in size, the expression of the antigen gene could still be detected after 21 d post vaccination. The CS-M-G induces the highest antibody levels in the 20 µg dose group which is about 3 times than naked plasmid group at 21 d post vaccination, and still hold a higher level than control group at 28 d post vaccination. On the side, strongest protection with relative percent survival of 62.1% in the 20 µg CS-M-G group, which could produce significantly higher enzyme activities and up-regulated expression of immune-associated genes than control group. Thus, our results indicate that DNA vaccine loaded with mannose-modified chitosan induces strong immune response and provided an effective protection against SVCV infection, may be helpful and extended for developing more aquatic animal vaccines in the future.

Dynamic Interaction Between Mucosal Immunity and Microbiota Drives Nose and Pharynx Homeostasis of Common Carp (Cyprinus carpio) After SVCV Infection.

The crosstalk between the immune system and microbiota drives an amazingly complex mutualistic symbiosis. In mammals, the upper respiratory tract acts as a gateway for pathogen invasion, and the dynamic interaction between microbiota and mucosal immunity on its surface can effectively prevent disease development. However, the relationship between virus-mediated mucosal immune responses and microbes in lower vertebrates remains uncharacterized. In this study, we successfully constructed an infection model by intraperitoneally injecting common carp (Cyprinus carpio) with spring viremia of carp virus (SVCV). In addition to the detection of the SVCV in the nose and pharynx of common carp, we also identified obvious histopathological changes following viral infection. Moreover, numerous immune-related genes were significantly upregulated in the nose and pharynx at the peak of SVCV infection, after which the expression levels decreased to levels similar to those of the control group. Transcriptome sequencing results revealed that pathways associated with bacterial infection in the Toll-like receptor pathway and the Nod-like receptor pathway were activated in addition to the virus-related Rig-I-like receptor pathway after SVCV infection, suggesting that viral infection may be followed by opportunistic bacterial infection in these mucosal tissues. Using 16S rRNA gene sequencing, we further identified an upward trend in pathogenic bacteria on the mucosal surface of the nose and pharynx 4 days after SVCV infection, after which these tissues eventually reached new homeostasis. Taken together, our results suggest that the dynamic interaction between mucosal immunity and microbiota promotes the host to a new ecological state.

Bayesian latent class model estimates of diagnostic accuracy for three test methods designed to detect spring viremia of carp virus.

Spring viremia of carp virus (SVCV) causes a systemic hemorrhagic disease that poses a significant risk to wild and cultured fish and is listed as notifiable by the World Organization for Animal Health. Validated molecular diagnostic tools for SVCV are required to accurately describe and analyze the ecology of the virus. Here, the diagnostic specificity (DSp) and sensitivity (DSe) (i.e. accuracy) of three SVCV diagnostic tests - 2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays Q1G and Q2N and virus isolation by cell culture (VI) - were evaluated using 2-class latent class models run in maximum likelihood (ML) and Bayesian frameworks. Virus-free or experimentally-infected koi were sorted into three populations with low, moderate or high prevalence levels of SVCV (n = 269 fish in total). Koi kidney tissues were tested using Q2N and Q1G and for the VI assay, pools of kidney, spleen and gill tissues were used. All samples were blinded and analyzed in one laboratory. The ML and Bayesian approaches successfully estimated the diagnostic accuracy of the 3 tests with the exception of 1 ML model. The estimates were consistent across the two frameworks. The DSe estimates were higher for Q1G (>98 %) and Q2N (>96 %) compared to VI (>60 %). The DSp of all three tests varied by 12-15 % (79-91 % for Q1G, 79-94 % for Q2N and 81-97 % for VI) across same-fish samples revealing the potential range in test performance for one sample. The 3 fish populations had distinct SVCV prevalence levels estimated at 0-3 % (low), 70-73 % (moderate) and 95-96 % (high). The Bayesian covariance models revealed minor DSe dependence between Q1G and Q2N. The results suggested that SVCV diagnostic tests Q2N and Q1G are suitable for use as diagnostic assays and are fit for presumptive diagnosis, surveillance, and certification of populations or individuals as SVCV free.