LGP2 Facilitates Bacterial Escape through Binding Peptidoglycan via EEK Motif and Suppressing NOD2-RIP2 Axis in Cyprinidae and Xenocyprididae Families.

RIG-I-like receptors and NOD-like receptors play pivotal roles in recognizing microbe-associated molecular patterns and initiating immune responses. The LGP2 and NOD2 proteins are important members of the RIG-I-like receptor and NOD-like receptor families, recognizing viral RNA and bacterial peptidoglycan (PGN), respectively. However, in some instances bacterial infections can induce LPG2 expression via a mechanism that remains largely unknown. In the current study, we found that LGP2 can compete with NOD2 for PGN binding and inhibit antibacterial immunity by suppressing the NOD2-RIP2 axis. Recombinant CiLGP2 (Ctenopharyngodon idella LGP2) produced using either prokaryotic or eukaryotic expression platform can bind PGN and bacteria in pull-down and ELISA assays. Comparative protein structure models and intermolecular interaction prediction calculations as well as pull-down and colocalization experiments indicated that CiLGP2 binds PGN via its EEK motif with species and structural specificity. EEK deletion abolished PGN binding of CiLGP2, but insertion of the CiLGP2 EEK motif into zebrafish and mouse LGP2 did not confer PGN binding activity. CiLGP2 also facilitates bacterial replication by interacting with CiNOD2 to suppress expression of NOD2-RIP2 pathway genes. Sequence analysis and experimental verification demonstrated that LGP2 having EEK motif that can negatively regulate antibacterial immune function is present in Cyprinidae and Xenocyprididae families. These results show that LGP2 containing EEK motif competes with NOD2 for PGN binding and suppresses antibacterial immunity by inhibiting the NOD2-RIP2 axis, indicating that LGP2 plays a crucial negative role in antibacterial response beyond its classical regulatory function in antiviral immunity.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Predicting hypovitaminosis C with LASSO algorithm in adult critically ill patients in surgical intensive care units: a bi-center prospective cohort study.

Vitamin C played pleiotropic roles in critical illness and vitamin C insufficiency was predictive of the development of multiple organ failure. Currently, the prevalence of vitamin C insufficiency in Chinese critically ill patients is rarely determined and there are no established bedside tools to predict hypovitaminosis C. To develop a nomogram to identify patients with high risk of hypovitaminosis C, we performed a bi-center prospective cohort study at two ICUs of the first and sixth medical center in PLA General Hospital, Beijing, China from May 6th to July 31st, 2021 We identified 322 eligible patients. 62.4% patients were hypovitaminosis C. 7 features, including source of infection, the level of serum albumin, age, male gender, sepsis, vascular disease, and wasting of vitamin C by the kidney, were selected using LASSO algorithm and therefore included in the nomogram. In the testing set, our model showed moderate discrimination ability with areas under the curve of 0.75 [0.64-0.84]. Variable importance evaluated by SHAP value highlighted two novel important predictors, i.e., abdominal infection and the level of serum albumin. In conclusion, we first reported a high burden of vitamin C insufficiency in Chinese adult patient in the ICU. We also constructed a prediction model to timely identify patients with high risk of hypovitaminosis C, which allows the clinicians to choose appropriate candidates for Vitamin C repletion in clinical practice or clinical trials.

IFN-γ enhances protective efficacy against Nocardia seriolae infection in largemouth bass (Micropterus salmoides).

Introduction: Nocardia seriolae adversely impacts a diverse range of fish species, exhibiting significant pathogenic characteristics that substantially impede the progress of aquaculture. N. seriolae infects in fish has a long incubation period, and clinical symptoms are not obvious in the early stages. There is presently no viable and eco-friendly approach to combat the spread of the disease. According to reports, N. seriolae primarily targets macrophages in tissues after infecting fish and can proliferate massively, leading to the death of fish. Interferon-gamma (IFN-γ) is a crucial molecule that regulates macrophage activation, but little is known about its role in the N. seriolae prevention. Methods: IFN-γ was first defined as largemouth bass (Micropterus salmoides, MsIFN-γ), which has a highly conserved IFN-γ characteristic sequence through homology analysis. The recombinant proteins (rMsIFN-γ) were obtained in Escherichia coli (E. coli) strain BL21 (DE3). The inflammatory response-inducing ability of rMsIFN-γ was assessed in vitro using monocytes/macrophages. Meanwhile, the protective effect of MsIFN-γ in vivo was evaluated by N. seriolae infection largemouth bass model. Results: In the inflammatory response of the monocytes/macrophages activated by rMsIFN-γ, various cytokines were significantly increased. Interestingly, interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-a) increased by 183- and 12-fold, respectively, after rMsIFN-γ stimulation. rMsIFN-γ improved survival by 42.1% compared with the control. The bacterial load in the liver, spleen and head kidney significantly decreased. rMsIFN-γ was also shown to better induce increased expression of IL-1ß, TNF-α, hepcidin-1(Hep-1), major histocompatibility complex I (MHCI), and MHC II in head kidney, spleen and liver. The histopathological examination demonstrated the transformation of granuloma status from an early necrotic foci to fibrosis in the infection period. Unexpectedly, the development of granulomas was successfully slowed in the rMsIFN-γ group. Discussion: This work paves the way for further research into IFN-γ of largemouth bass and identifies a potential therapeutic target for the prevention of N. seriolae.

Hepcidin contributes to largemouth bass (Micropterus salmoides) against bacterial infections.

The increasing emergence and dissemination of bacterial pathogens in largemouth bass culture accelerate the desire for new treatment measures. Antimicrobial peptides as the host's antimicrobial source dominate the preferred molecules for discovering antibacterial agents. Here, the potential of Hepcidin-1 from largemouth bass (Micropterus salmoides) (MsHep-1) against bacterial infection is demonstrated. MsHep-1 not only improved the survival rate in infection experiments involving Nocardia seriolae (12 %) and Aeromonas hydrophila (18 %) but also coped with iron overload conditions in vivo. Moreover, the antibacterial activity of MsHep-1 in vitro was identified against both gram-negative and gram-positive bacteria. Mechanistic studies show MsHep-1 leads to bacterial death by changing the bacterial membrane potential and disrupting the bacterial membrane structure. These findings demonstrate that MsHep-1 may play an important role in the host response to bacterial infection. It provides promising strategies in the application of immunosuppression prevention and control in fish. AMPs may be a promising and available reservoir for treating the current bacterial diseases.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

TLR5M and TLR5S Synergistically Sense Flagellin in Early Endosome in Lamprey Petromyzon marinus, Switched by the N-Glycosylation Site N239.

In mammals, TLR5 functions as a homodimer to recognize bacterial flagellin on the cytomembrane. The current investigations reveal the existence of two types of TLR5, a membrane-bound PmTLR5M, and a soluble variant PmTLR5S, in lamprey (Petromyzon marinus). Although both PmTLR5M and PmTLR5S can bind flagellin, only PmTLR5M is capable of eliciting a proinflammatory response, whereas PmTLR5S can detect the flagellin and facilitate the role of PmTLR5M in early endosomes. The trafficking chaperone UNC93B1 enhances the ligand-induced signaling via PmTLR5M or the combination of PmTLR5M and PmTLR5S. PmTLR5M recruits MyD88 as an adaptor. Furthermore, chimeric receptor studies demonstrate the indispensability of the intradomain of PmTLR5M in effective activation of the proinflammatory pathway upon flagellin stimulation, and the combination of PmTLR5S with a singular intradomain in both homodimer and heterodimer ectodomain arrangements can very significantly augment the immune response. Furthermore, the flagellin binding sites between PmTLR5M and PmTLR5S are conserved, which are essential for ligand binding and signal transduction. Moreover, investigations on N-linked glycosylation modifications reveal that the N239 site in PmTLR5M and PmTLR5S plays a switch role in both flagellin binding and immune responses. In addition, PmTLR5M exhibits the high-mannose-type and complex-type N-glycosylation modifications; however, PmTLR5S shows exclusive complex-type N-glycosylation modification. The key N239 site demonstrates complex-type N-glycosylation modification. The findings address the function and mechanism of TLR5 in ligand recognition, subcellular localization, and signaling pathway in lowest vertebrate and immune system transition species, highlight the regulatory role of N-glycosylation modification in TLRs, and augment immune evolutionary research on the TLR signaling pathway.

Vitamin D3 can effectively and rapidly clear largemouth bass ranavirus by immunoregulation.

Largemouth bass ranavirus (LMBV) is a highly destructive pathogen that causes significant mortality rates among largemouth bass populations. Unfortunately, there is a dearth of drug development efforts specifically aimed at treating LMBV. To address this, our study sought to investigate the potential effectiveness of incorporating varying doses of VD3 into the diet as a treatment for LMBV. Through qRT-PCR and semi-qPCR, we observed significant suppression and clearance of LMBV pathogens in largemouth bass fed with 15000 IU/Kg and 20000 IU/Kg of VD3 within 14 days. In addition, VD3 treatment significantly increased the expression levels of key immune-related genes such as IL-1ß, IFN-γ, Mx, and IgM. Encouragingly, we observed that VD3 significantly increased antioxidant and immune activities such as TSOD, TAOC and C3 in serum and maintained total protein levels. Additionally, tissue pathology sections highlighted a dose-dependent relationship between VD3 supplementation and tissue damage, with the 15000 IU and 20000 IU groups exhibiting minimal damage. In conclusion, a reasonable concentration of VD3 effectively reduced LMBV replication and tissue damages, while improved immune-related genes expression and serum biochemical indices. These findings declare the considerable therapeutic potential of VD3 supplementation for combating LMBV disease and provide an alternative treatment option for fish farming.

TLR7 neo-functionalizes to sense dsRNA and trigger antiviral and antibacterial immunity in non-tetrapod vertebrates.

TLR7 plays a crucial role in sensing viral ssRNA and initiating immune responses. Piscine TLR7 also responds to dsRNA challenge. dsRNA exists in almost all the viruses at specific stages. However, the mechanism on sensing dsRNA by TLR7 remains unknown. In the present study, we employed Ctenopharyngodon idella TLR7 (CiTLR7) to systematically explore the immune functions and mechanisms in teleost. CiTLR7 can directly bind not only ssRNA but also dsRNA at different patches in lysosome, recruit MyD88 as adaptor, and activate the downstream IFN pathway via SLC15A4/TASLa/TASLb/IRF5/IRF7 complex for antiviral and antibacterial infections and AP-1 pathway for pro-inflammatory cytokines. The key binding sites for dsRNA are L29 and L811 in CiTLR7. Further, we found that the function on recognizing dsRNA by TLR7 emerges in pisciformes and loses in tetrapods in evolution. This is the first report on sensing both ssRNA and dsRNA by a TLR member.

The Combination of ß-Glucan and Astragalus Polysaccharide Effectively Resists Nocardia seriolae Infection in Largemouth Bass (Micropterus salmoides).

Effectively treating and preventing outbreaks is crucial for improving the economic benefits of aquaculture. Therefore, utilizing immunostimulants, either alone or in combination, is regarded as a promising strategy. In this study, ß-glucan + APS (200 mg/kg + 200 mg/kg), ß-glucan (200 mg/kg), APS (200 mg/kg), enrofloxacin (15 mg/kg), and sulfadiazine (15 mg/kg) were added to feed to assess the effects against Nocardia seriolae infection in largemouth bass (Micropterus salmoides) within 14 days. The survival rates did not differ between the enrofloxacin group and the ß-glucan + APS group, but both were significantly higher than that of the control group. Additionally, the enrofloxacin group and the ß-glucan + APS group exhibited the lowest bacterial loads and tissue damage. Importantly, the ß-glucan + APS treatment significantly improved serum enzyme activities (total superoxide dismutase, lysozyme, total protein) and the expression of immune genes (IL-1ß, TNF-α, IFN-γ, IgM) compared to the other treatment groups. The enrofloxacin group showed similar efficacy to the ß-glucan + APS group in combating N. seriolae infection, but N. seriolae in the enrofloxacin group developed drug resistance. In summary, the combined use of ß-glucan and APS is a promising strategy for treating bacterial diseases, thereby contributing to the promotion of sustainable aquaculture development.

The oral antigen-adjuvant fusion vaccine P-MCP-FlaC provides effective protective effect against largemouth bass ranavirus infection.

Largemouth bass ranavirus (LMBV) is highly contagious and lethal to largemouth bass, causing significant economic losses to the aquaculture industry. Oral vaccination is generally considered the most ideal strategy for protecting fish from viral infection. In this study, the fusion protein MCP-FlaC, consisting of the main capsid protein (MCP) as the antigen and flagellin C (FlaC) as the adjuvant, was intracellularly expressed in Pichia pastoris. Subsequently, the recombinant P. pastoris was freeze-dried to prepare the oral vaccine P-MCP-FlaC. Transmission electron microscopy and scanning electron microscopy analysis showed that the morphology and structure of the freeze-dried recombinant P. pastoris vaccine remained intact. The experiment fish (n = 100) was divided into five groups (P-MCP-FlaC, P-MCP, P-FlaC, P-pPIC3.5K, control) to evaluate the protective efficacy of the recombinant vaccine. Oral P-MCP-FlaC vaccine effectively up-regulated the serum enzymes activity (total superoxide dismutase, lysozyme, total antioxidant capacity, and complement component 3). The survival rate of P-MCP-FlaC group was significantly higher than that of the other groups. The mRNA expression of crucial immune genes (IL-1ß, TNF-α, MHC-II, IFN-γ, Mx, IgM, IgT) was also signally elevated in P-MCP-FlaC group. Vaccine P-MCP-FlaC markedly inhibited the replication of LMBV in the spleen, head kidney, and intestine, while reducing the degree of lesion in the spleen. These results suggest that the oral P-MCP-FlaC vaccine could effectively control LMBV infection, proving an effective strategy for viral diseases prevention in aquaculture.

Re-identification and characterization of grass carp Ctenopharyngodon idella TLR20.

Toll-like receptors (TLRs) play a crucial role in the recognition of microbial-associated molecular patterns in the innate immune system. Fish TLRs have undergone significant gene expansion to adapt to complex aquatic environments. Among them, TLR20 from the TLR11 family actively responds to viral and bacterial invasions. Previous studies have reported two TLR20s in grass carp (Ctenopharyngodon idella), and in this study, we revised this conclusion. Based on the latest grass carp genome, we identified a new TLR20 member. These three TLR20s are arranged in tandem on chromosome 9, indicating that they are generated by gene duplication events. They were renamed CiTLR20.1 to CiTLR20.3 based on their chromosomal positions. The CiTLR20s in C. idella exhibit higher similarities with those in Danio rerio, Cyprinus carpio, and Megalobrama amblycephala, and lower similarities with those in other distantly related fish species. Selective pressure analysis revealed low conservation and negative evolution of TLR20s during evolution. The 3D structures of the three TLR20s showed significant differences, reflecting functional variations and different downstream adaptor molecule recruitment. Transcriptome data revealed tissue distribution differences of TLR20s, with TLR20.1 showing relatively low expression levels in all the tissues, while TLR20.2 and TLR20.3 showed higher expression in the head kidney, spleen, and gill. Additionally, TLR20.2 and TLR20.3 actively responded to GCRV-II infection, with higher upregulation of TLR20.2 in response to Aeromonas hydrophila challenge. In conclusion, this study corrected the number of grass carp TLR20 members and analyzed TLR20 from an evolutionary and structural perspective, exploring its role in antiviral and antibacterial defense. This study provides reference for future research on fish TLR20.

Nanopeptide CI20 remarkably enhances growth performance and disease resistances by improving the mucosal structure, antioxidant capacity, and immunity in mandarin fish (Siniperca chuatsi).

Soybean meal, excessively used in place of fish meal (FM) in aquaculture, has a detrimental impact on fish. In this study, the nanopeptide CI20, which was created by conjugating antimicrobial peptide gcIFN-20H and CMCS, were evaluated the feeding effect in mandarin fish (Siniperca chuatsi). Compared with the control group, 150 mg/kg C-I20-fed fish showed the second highest growth performance with no significant changes in body composition. C-I20-fed fish showed more goblet cells and thicker mucin after feeding. The 150 mg/kg CI20 diet boosted the antioxidant capacity, immunity, and digestive enzymes. After Aeromonas hydrophila and infection spleen and kidney necrosis virus infection, the survival rates in the 150 mg/kg CI20 group were highest. Meanwhile, many tissues in the 150 mg/kg CI20 group had significantly lower pathogen loads than the other groups. Treatment with 150 mg/kg CI20 was effective in increasing antioxidant capacity and immunity. The minimum tissue lesions were observed in the 150 mg/kg CI20 group. The goblet cell number and mucin thickness were significantly increased by CI20 treatment after infection. The study results herein showed that a reasonable dietary concentration of CI20 feed promoted growth performance and disease resistances in fish, suggesting a prospective nano antimicrobial peptide for the aquaculture.

VP4/VP56/VP35 Virus-like Particles Effectively Protect Grass Carp (Ctenopharyngodon idella) against GCRV-II Infection.

Grass carp reovirus (GCRV) seriously threatens the grass carp (Ctenopharyngodon idella) industry. Prophylactic GCRV vaccines prepared by virus-like particle (VLP) assembly biotechnology can improve effectiveness and safety. The highly immunogenic candidate antigens of GCRV vaccines that have been generally considered are the outer capsid proteins VP4, VP56, and VP35. In this study, VP4, VP56, and VP35 were expressed in an Escherichia coli expression system and a Pichia pastoris expression system. The successful assembly of uniform, stable, and non-toxic VP4/VP56/VP35 VLPs was confirmed through various assays. After vaccination and GCRV infection, the survival rate in the VLPs + adjuvant Astragalus polysaccharide (APS) group was the highest (62%), 40% higher than that in control group (22%). Through the antibody levels, tissue viral load, and antioxidant immunity assays, the P. pastoris VLP vaccine effectively improved IgM levels, alleviated tissue virus load, and regulated antioxidant immune-related indicators. The treatment with P. pastoris VLPs enhanced the mRNA expression of important immune-related genes in the head kidney, as measured by qRT-PCR assay. Upon hematoxylin-eosin staining examination, relatively reduced tissue pathological damage was observed in the VLPs + APS group. The novel vaccine using P. pastoris VLPs as an effective green biological agent provides a prospective strategy for the control of fish viral diseases.

Nanopeptide C-I20 as a novel feed additive effectively alleviates detrimental impacts of soybean meal on mandarin fish by improving the intestinal mucosal barrier.

Antibacterial peptide has been widely developed in cultivation industry as feed additives. However, its functions in reducing the detrimental impacts of soybean meal (SM) remain unknown. In this study, we prepared nano antibacterial peptide CMCS-gcIFN-20H (C-I20) with excellent sustained-release and anti-enzymolysis, and fed mandarin fish (Siniperca chuatsi) with a SM diet supplemented with different levels of C-I20 (320, 160, 80, 40, 0 mg/Kg) for 10 weeks. 160 mg/Kg C-I20 treatment significantly improved the final body weight, weight gain rate and crude protein content of mandarin fish and reduced feed conversion ratio. 160 mg/Kg C-I20-fed fish maintained appropriate goblet cells number and mucin thickness, as well as improved villus length, intestinal cross-sectional area. Based on these advantageous physiological changes, 160 mg/Kg C-I20 treatment effectively reduced multi-type tissue (liver, trunk kidney, head kidney and spleen) injury. The addition of C-I20 did not change the muscle composition and muscle amino acids composition. Interestingly, dietary 160 mg/Kg C-I20 supplementation prevented the reduction in myofiber diameter and change in muscle texture, and effectively increased polyunsaturated fatty acids (especially DHA + EPA) in muscle. In conclusion, dietary C-I20 in a reasonable concentration supplementation effectively alleviates the negative effects of SM by improving the intestinal mucosal barrier. The application of nanopeptide C-I20 is a prospectively novel strategy for promoting aquaculture development.

Type II Grass Carp Reovirus Rapidly Invades Grass Carp (Ctenopharyngodon idella) via Nostril-Olfactory System-Brain Axis, Gill, and Skin on Head.

Type II grass carp reovirus (GCRV-II) with high pathogenicity and infectivity causes severe hemorrhagic disease, which leads to extensive death in the grass carp and black carp aquaculture. However, the early invasion portal remains unclear. In this study, we explored the invasion portal, time, and pathway of GCRV-II by immersion infection in grass carp. Through the detection of the infected grass carp external body surface tissues, most of them could be detected to carry GCRV-II within 45 min except for the skin covered by scales. Further shortening the duration of infection, we proved that GCRV-II rapidly invades through the nostril (especially), gill, and skin on head at only 5 min post-immersion, rather than merely by adhesion. Subsequently, visual localization investigations of GCRV-II were conducted on the nostril, olfactory system (olfactory bulb and olfactory tract), and brain via immunofluorescence microscopy and transmission electron microscopy. We found that few viruses were located in the nostril at 5 min post-immersion infection, while a significantly increased quantity of viruses were distributed in all of the examined tissues at 45 min. Furthermore, the semi-qRT-PCR and Western blotting results of different infection times confirmed that GCRV-II invades grass carp via the nostril-olfactory system-brain axis and then viral replication unfolds. These results revealed the infection mechanism of GCRV-II in terms of the invasion portal, time, and pathway in grass carp. This study aims to understand the invasion mode of GCRV-II in grass carp, thus providing theoretical support for the prevention and control strategies of hemorrhagic disease.

Temperature-regulated type II grass carp reovirus establishes latent infection in Ctenopharyngodon idella brain.

Grass carp reovirus (GCRV) causes extensive infection and death in grass carp and black carp fingerlings, with a highly seasonal prevalence. Previous studies suggested that GCRV can become latent after primary infection. In this study, we investigated type II GCRV (GCRV-II) latency in asymptomatic grass carp with GCRV infection or exposure history. We found that during latent infection, GCRV-II was detectable only in the brain of grass carp, unlike the multi-tissue distribution observed in natural infection. GCRV-II only caused damage to the brain during latent infection, while in natural infection, brain, heart, and eye tissues had relatively higher viral loads. We also discovered viral inclusion bodies in infected fish brains. Additionally, GCRV-II distribution in grass carp was notably affected by ambient temperature, with the virus targeting the brain only during low temperatures and multi-tissue distribution during high temperatures. This study provides insights into the mechanisms of GCRV-II latent infection and reactivation and contributes to the prevention and control of GCRV pandemics.

Chitooligosaccharide boosts the immunity of immunosuppressed blunt snout bream against bacterial infections.

The immunosuppression hazard of fish brought by intensive aquaculture needs to be addressed urgently, while chitooligosaccharide (COS) shows the potential application in the prevention the immunosuppression of fish due to its superior biological properties. In this study, COS reversed the cortisol-induced immunosuppression of macrophages and improved the immune activity of macrophages in vitro, promoting the expression of inflammatory genes (TNF-α, IL-1ß, iNOS) and NO production, and increasing the phagocytic activity of macrophages. In vivo, the oral COS was absorbed directly through the intestine, significantly ameliorating the innate immunity of cortisol-induced immunosuppression of blunt snout bream (Megalobrama amblycephala). Such as facilitated the gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and pattern recognition receptors (TLR4, MR) and potentiated bacterial clearance, resulting in an effective improvement in survival and tissue damage. Altogether, this study demonstrates that COS offers potential strategies in the application of immunosuppression prevention and control in fish.

IFN1 Enhances Thrombocyte Phagocytosis through IFN Receptor Complex-JAK/STAT-Complement C3.3-CR1 Pathway and Facilitates Antibacterial Immune Regulation in Teleost.

Type I IFNs with strong positive charges exhibit robust bactericidal activity and a protective effect against bacterial infections. However, the antibacterial mechanism in vivo remains unknown. In this study, Ab blockade of IFN1, a member of type I IFNs in grass carp (Ctenopharyngodon idella), resulted in high mortality, tissue bacterial loads, and low expression of immune factors after bacterial challenge, which indicates that the antibacterial activity of IFN1 has physiological significance. Meanwhile, we injected grass carp with the recombinant and purified intact IFN1 protein after bacterial injection, and the result demonstrated a remarkable therapeutic effect. Furthermore, we found that IFN1 expression was remarkably induced in blood cells after bacterial challenge, and prophagocytosis via IFN1 mostly increased in thrombocytes. Then, we isolated peripheral blood thrombocytes by polyclonal Ab of CD41 and stimulated thrombocytes with recombinant IFN1, and the results indicated that immune factors and complement components (especially C3.3) were induced. Unexpectedly, complements demonstrated not only bacteriolysis but also bacterial aggregation. Furthermore, Ab blockades of the three subunits (CRFB1/CRFB2/CRFB5) of the IFN1 receptor or inhibition of STAT1 almost abolished the prophagocytosis via IFN1 and reduced C3.3 and immune factor expression in thrombocytes. Meanwhile, Ab blockade of the complement receptor CR1 greatly attenuated the prophagocytosis of IFN1. In contrast, mouse IFN-ß did not show the promotion of antibacterial activity. These results clarify the prophagocytosis and immune regulation pathways of IFN1 in antibacterial immunity in teleosts. This study reveals the antibacterial mechanisms of type I IFNs in vivo and inspires functional studies of IFN in bacterial infections.

Interleukin-2 family cytokines: An overview of genes, expression, signaling and functional roles in teleost.

The interleukin-2 (IL-2) family cytokines include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which share γ chain (γc) subunit in receptors. The IL-2 family cytokines have unique biological effects that regulate differentiation, survival and activation of multiple lymphocyte lineages. Deficiency of IL-2 family signaling pathway in mammals prevents CD4+ T cells from developing effector functions and CD8+ T cells from developing immunological memory. In the present review, we addressed available information from teleost IL-2 family cytokines and discussed implications in teleost immunity. Also, we described and discussed their expression profiles, receptors, signaling transductions and functions. In teleost, IL-2 family has 5 members (IL-2, IL-4/13, IL-7, IL-15, IL-21) without IL-9, and their receptors share a common γc subunit and include other 6 subunits (IL-2Rß1/2, IL-4Rα1/2, IL-13Rα1/2, IL-7Rα, IL-15Rα, and IL-21Rα1/2). Some paralogues have changes in domain structure and show differential expression, modulation, functions. IL-2 family cytokines constitutively express in many immune associated tissues and are largely induced after pathogenic microbial stimulation. In general, there are relatively conserved functions in the IL-2 family throughout vertebrates, and many of the key IL-2 family members are important in lymphocyte proliferation and differentiation, development, inflammation from fishes to mammals. This review will give an update on the effective information of teleost IL-2 family cytokines. Thus, it will provide a source of reference for other researchers/readers and inspire further interest.