Determination of the minimum infective dose of viral hemorrhagic septicemia virus (VHSV) in juvenile olive flounder, Paralichthys olivaceus using an immersion challenge model.

Viral hemorrhagic septicemia virus (VHSV) affects over 80 fish species, leading to viral hemorrhagic septicemia (VHS). Horizontal VHSV transmission is widely studied, with researchers utilizing various doses to establish infection models. Infected hosts shed the virus into the environment, elevating the risk of transmission to naïve fish within the same system. This study aimed to ascertain the minimum infective dose of VHSV in olive flounder (Paralichthys olivaceus). In olive flounder, the detection of VHSV within the kidney exhibited the highest infection rate on the third day among days 1, 3 and 5. Doses of 103.0 to 104.7 TCID50/ml were administered to juvenile olive flounder across three farms. Results showed resistance to infection below 103.4 TCID50/ml at 15 °C. While infection frequency varied by concentration, higher concentrations correlated with more infections. Nonetheless, viral copy numbers did not differ significantly among infected fish at varying concentrations. This study underscores the need for early VHSV management and contributes essential data for pathogenicity assessment and foundational knowledge.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection.

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFß. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

NLRC3 attenuates antiviral immunity and activates inflammasome responses in primary grouper brain cells following nervous necrosis virus infection.

NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.

Beta glucan induced immune priming protects against nervous necrosis virus infection in sevenband grouper.

In the present study, we studied the effect of ß-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of ß-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1ß production were reduced in ß-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the ß-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with ß-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that ß-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Altered expression of immune factors in sevenband grouper, Hyporthodus septemfasciatus following nervous necrosis virus challenge at optimal and suboptimal temperatures.

The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1ß and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 104 TCID50/100µL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.

Proteasome subunit beta type-8 from sevenband grouper negatively regulates cytokine responses by interfering NF-κB signaling upon nervous necrosis viral infection.

During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome ß-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1ß, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.