Grass Carp Reovirus Replication Research Articles

GCRV-encoded circRNA circ_20 forms a ternary complex with BIP and PERK to delay virus replication by inhibiting the PERK-eIF2α pathway

Viral circRNAs play important roles in host-virus interactions. Previous reports showed that grass carp reovirus (GCRV) encodes 32 circRNAs, and circ_20 from the negative strand of GCRV genome segment 7 has the potential to regulate GCRV replication. However, the regulatory mechanism of circ_20 on GCRV remains unknown. In this study, circ_20 was further validated, and circ_20 negatively regulated ERS, the PERK pathway, and ROS production in GCRV-infected cells. Furthermore, circ_20 inhibited the PERK pathway by forming a ternary complex with BIP and PERK, resulting in delaying GCRV replication. RNA pull-down results indicated that the 51–102 nt region of circ_20 interacts with BIP, while the 451–502 and 514–565 nt regions interact with PERK. After the deletion of these interaction regions, the ability of circ_20 to promote BIP-PERK interaction decreases, leading to a decrease in the ability to inhibit GCRV proliferation. These findings uncovered new insights into the complex interplay between viruses and host cells and provided a novel understanding of the significance of viral circRNAs in virus-host interactions.

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.

The dexd/H-box helicase DHX40 is a novel negative regulator during GCRV infection via targeting the host RLR helicases and viral nonstructural protein NS38

RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.

The role of MASP1 in the complement system and expression characteristics in response to GCRV infection in grass carp

The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30–91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.

GMPR2-MDA5 interaction bridged the cell junction and anti-GCRV immune

As a consequence of increased vascular endothelial permeability and vascular injury caused by GCRV (grass carp reovirus) infection, grass carp (Ctenopharyngodon idellus) could suffer from hemorrhagic disease and even death. MDA5 is a key RNA sensor that stimulates type I interferon as well as regulates vascular permeability. The guano monophosphate ductase 2 (GMPR2) plays a key role in regulating the permeability of vascular endothelial cells. This study examined the interaction between grass carp MDA5 and GMPR2 and their antiviral effects. GMPR2 interacted directly with MDA5 and promoted its protein level, whilst MDA5 conversely inhibited the GMPR2 protein expression. The expression of GCRV VP7 protein was inhibited by the increased levels of MDA5 and GMPR2. In addition, the cell junction molecule Claudin7b which is closely related to the change in vascular permeability, was inhibited by both MDA5 and GMPR2. Finally, in GMPR2 knockout zebrafish, the mRNA level of MDA5 was significantly down-regulated, which validated the positive functional relationship of GMPR2 on MDA5. Consistent changes of the Claudin 7b and VP7 protein levels were observed in the GMPR2 and MDA5 overexpression groups. In summary, MDA5-GMPR2 interaction affecting GCRV replication might through cell junction molecule Claudin7b. The results from this study provided systematic support for revealing the genetic basis of cell permeability change as well as the hemorrhagic diseases in grass carp.

Functional comparison of Rab3aa and Rab3ab in grass carp (Ctenopharyngodon idella) immune response and GCRV replication

Rab GTPases not only play a crucial role in the exocytosis and membrane transport of all eukaryotic cells, but are also gradually being found to be involved in innate immunity in mammals. However, the immunological function of Rab3a in fish remains unclear. In this study, two members of Rab3a, CiRab3aa and CiRab3ab, were cloned from grass carp (Ctenopharyngodon idella) for the first time. It was observed that CiRab3aa exhibited a prompt response to grass carp reovirus (GCRV) infection, and suppressing the expression of the CiRab3aa effectively inhibited GCRV replication, whereas the expression level of the CiRab3ab gene did not affect GCRV replication. Furthermore, overexpression of CiRab3aa significantly suppressed the mRNA levels of TLR9, TNF-α, IL-1β, IL-6, IL-10, ERK, and IFN-I following stimulation with CpG ODN 1670 A, suggesting that CiRab3aa is a negative regulator of TLR9 signaling pathway. In conclusion, this study not only revealed functional differences between CiRab3aa and CiRab3ab in grass carp immunity, providing new sights into understanding their function in teleost fish, but also provided a new target for the treatment strategy of grass carp hemorrhagic disease. These results will help address the harm and economic losses caused by GCRV infection in grass carp breeding industry.

Identification of the C1qDC gene family in grass carp (Ctenopharyngodon idellus) and the response of C1qA, C1qB, and C1qC to GCRV infection in vivo and in vitro

Proteins from the C1q domain-containing (C1qDC) family recognize self-, non-self-, and altered-self ligands and serves as an initiator molecule for the classical complement pathway as well as recognizing immune complexes. In this study, C1qDC gene family members were identified and analyzed in grass carp (Ctenopharyngodon idellus). Members of the C1q subfamily were cloned, and their response to infection with the grass carp virus was investigated. In the grass carp genome, 54 C1qDC genes and 67 isoforms have been identified. Most were located on chromosome 3, with 52 shared zebrafish homologies. Seven substantially differentially expressed C1qDC family genes were identified in the transcriptomes of cytokine-induced killer (CIK) cells infected with grass carp reovirus (GCRV), all of which exhibited sustained upregulation. The opening reading frames of grass carp C1qA, C1qB, and C1qC, belonging to the C1q subfamily, were determined to be 738, 732, and 735 base pairs, encoding 245, 243, and 244 amino acids with molecular weights of 25.81 kDa, 25.63 kDa and 26.16 kDa, respectively. Three genes were detected in the nine collected tissues, and their expression patterns were similar, with the highest expression levels observed in the spleen. In vivo after GCRV infection showed expression trends of C1qA, C1qB, and C1qC in the liver, spleen, and kidney. An N-type pattern in the liver and kidney was characterized by an initial increase followed by a decrease, with the highest expression occurring during the recovering period, and a V-type pattern in the spleen with the lowest expression levels during the death period. In vitro, after GCRV infection showed expression trends of C1qA, C1qB, and C1qC, and this gradually increased within the first 24 h, with a notable increase observed at the 24 h time point. After CIK cells incubation with purified recombinant proteins, rC1qA, rC1qB, and rC1qC for 3 h, followed by GCRV inoculation, the GCRV replication indicated that rC1qC exerted a substantial inhibitory effect on viral replication in CIK cells after 24 h of GCRV inoculation. These findings offer valuable insights into the structure, evolution, and function of the C1qDC family genes and provide a foundational understanding of the immune function of C1q in grass carp.

Grass carp peroxiredoxin 5 and 6-mediated autophagy inhibit grass carp reovirus replication and mitigate oxidative stress

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H2O2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H2O2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress.

Grass carp superoxide dismutases exert antioxidant function and inhibit autophagy to promote grass carp reovirus (GCRV) replication

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two CuZn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H2O2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication.

First in vitro and in vivo evaluation of recombinant IL-1β protein as a potential immunomodulator against viral infection in fish

IL-1β is an important proinflammatory cytokine with multifaceted modulatory roles in immune responses. In fish, recombinant IL-1β has been employed in the control of bacterial diseases, while the antiviral mechanisms of IL-1β remain largely unknown, and the efficacy of recombinant IL-1β as an immunomodulator to prevent viral diseases is still not determined. This study evaluated the immunomodulatory effects of recombinant grass carp IL-1β against grass carp reovirus (GCRV) in vitro and in vivo. Firstly, the mature form (Ser111-Lys270) of grass carp IL-1β was identified, and its recombinant protein (designated as rgcIL-1β) was prepared through prokaryotic expression. Then, an in vitro evaluation model for rgcIL-1β activity was established in the CIK cells, with the appropriate concentration (600 ng/mL) and effect time (1 h). In vitro, rgcIL-1β could not only induce the production of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α but also a series of antiviral factors including IFN-1, IFN-2, IFN-γ, and ISG15. Mechanistically, transcriptome analysis and western blotting confirmed that rgcIL-1β activated multiple transcriptional factors, including NF-κB, IRF1, IRF3, and IRF8, and the signal pathways associated with inflammatory cytokines and antiviral factors expression. Expectedly, rgcIL-1β treatment significantly inhibited GCRV replication in vitro. In vivo administration of rgcIL-1β via intraperitoneal pre-injection significantly aroused an antiviral response to restrict GCRV replication and intense tissue inflammation in grass carp, demonstrating the immunomodulatory effects of rgcIL-1β. More importantly, rgcIL-1β administrated with 10 ng/g and 1 ng/g could improve the survival rate of grass carp during GCRV infection. This study represents the first time to comprehensively reveal the immunomodulatory and antiviral mechanisms of IL-1β in fish and may also pave the way for further developing recombinant IL-1β as an immunotherapy for the prevention and control of fish viral diseases.

Oral pcDNA3.1-VP4/VP56-FlaC DNA vaccine encapsulated by chitosan/sodium alginate nanoparticles confers remarkable protection against GCRV infection in grass carp

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) results in huge losses to the grass carp industry. In previous studies, DNA vaccination provided an efficient strategy for combating viral infection, but biodegradation and adverse environment hindered its application. In this study, Chitosan/sodium alginate (CS/SA) nanoparticles (NPs) loading system was applied to develop the oral DNA nano vaccine CS/SA/VP4C NPs and the oral DNA nano vaccine CS/SA/VP56C NPs to enhance its application effect. The recombinant plasmids pcDNA3.1-VP4C and pcDNA3.1-VP56C were constructed to express GCRV major outer capsid protein VP4 fusing Aeromonas hydrophila flagellin C (VP4C) and GCRV fiber protein VP56 fusing A. hydrophila flagellin C (VP56C), respectively. The oral DNA nano vaccines were about 100 nm in size with a regular spherical structure observed by transmission electron microscope. In addition, the fusion proteins VP4C and VP56C could be efficiently expressed in both fathead minnow cells and grass carp intestine epithelial cells by indirect immunofluorescence and western blotting analysis. The CS/SA/VP4C NPs and CS/SA/VP56C NPs were orally vaccinated with grass carp to evaluate the immune protective effect. The results showed that CS/SA/VP4C + VP56C NPs group had the highest protective effect that the survival rate increased by 42% compared with the control group. In addition, CS/SA/VP4C + VP56C NPs treatment could rapidly up-regulate serum innate immune indexes (total superoxide dismutase, lysozyme and complement C3), and significantly increase the mRNA expression of IFN1, MHC-IIα, IL-1β, TNFα, IgM, and IgT. Meanwhile, CS/SA/VP4C + VP56C NPs treatment greatly inhibited the replication of GCRV in the hindgut, spleen and head kidney. These results suggested that oral administration of CS/SA/VP4C + VP56C NPs could effectively prevent GCRV infection by improving adaptive immunity and innate immunity. Therefore, our study provides a new strategy for the fish viral disease prevention, which also establishes a theoretical basis for developing multiple DNA vaccines in the aquaculture industry.

Piscine Vitamin D Receptors Vdra/Vdrb in the Absence of Vitamin D Are Utilized by Grass Carp Reovirus for Promoting Viral Replication.

The vitamin D receptor (VDR) plays a pivotal role in the biological actions of vitamin D (VitD). However, little is known about the functions of VDR in the production of viral inclusion bodies (VIBs). Using a representative strain of grass carp reovirus (GCRV) genotype I, GCRV-873, we show that GCRV-873 recruits grass carp Vdrs for promoting the production of VIBs in the absence of VitD. Inhibition of cholesterol synthesis by lovastatin impairs the production of VIBs and blocks the effects of grass carp Vdrs in promoting the production of VIBs in the absence of VitD. Furthermore, grass carp Vdrs are found to form the Vdra-Vdrb heterodimer, which is vital for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgcr)-dependent cholesterol synthesis and GCRV replication. Intriguingly in the presence of VitD, grass carp Vdra but not Vdrb forms the heterodimer with the retinoid X receptor beta b (Rxrbb), which induces the transcription of those genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway for inhibiting GCRV infection. Furthermore, the VitD-activated Vdra-Vdrb heterodimer attenuates the transcription of the RLR antiviral signaling pathway induced by VitD. In the presence of VitD, a balance between the Vdra-Rxrbb heterodimers as coactivators and Vdra-Vdrb heterodimers as corepressors in affecting the transcriptional regulation of the RLR antiviral signaling pathway may eventually determine the outcome of GCRV infection. Transfection with VitD can abolish the effect of grass carp Vdrs in promoting GCRV replication in a dose-dependent manner. Taken together, these findings demonstrate that GCRV utilizes host Vdrs to increase hmgcr-dependent cholesterol synthesis for promoting its replication, which can be prevented by VitD treatment. IMPORTANCE Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhagic disease, which seriously harms freshwater fish. Although many positive or negative regulators of GCRV infection have been identified in teleosts, little is known about the molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection. Here, we show that in the absence of VitD, the GCRV-873 strain utilizes host vitamin D receptors Vdra/Vdrb to increase hmgcr-dependent cholesterol synthesis for promoting the production of VIBs, which are important functional sites for aquareovirus replication and assembly. The negative regulation of Vdrs during viral infection can be prevented by VitD treatment. Thus, this present work broadens understanding of the pivotal roles of Vdrs in the interaction between the host and GCRV in the absence or presence of VitD, which might provide a rational basis for developing novel anti-GCRV strategies.

Structure, function and immune evasion strategies of aquareoviruses, with focus on grass carp reovirus

AbstractAquareoviruses (AqRVs) can infect various wild or cultured bony fishes with serious muscle and viscera haemorrhage syndrome and have caused severe economic losses worldwide. Accumulating evidences have revealed the functions of viral proteins of AqRVs in RNA transcription, virion assembly, cell attachment and/or viral replication. In addition, structural information and transcription mechanism of AqRVs have been in‐depth studied through cryo‐electron microscopy combined with three‐dimensional image reconstruction. Although far from enough, structural and functional studies of AqRVs provide substantial insights into how viral proteins coordinate the replication of AqRVs and escape the host immune response. Grass carp reovirus (GCRV) is the most pathogenic AqRV isolated from aquatic animals. The completion of GCRV lifecycle requires the participation of host proteins. So far, many host factors involved in the entry and replication of GCRV have been identified. Based on the function of viral proteins and the viral lifecycle, many vaccines and antiviral agents have been developed to prevent the replication and infection of GCRV. This review summarises the structural characteristics and the composition of core proteins of AqRVs, as well as synergistic mechanisms of structural proteins on genome transcription and virus entry of AqRVs. In addition, the review highlights our current knowledge for the infection and replication, functions of non‐structural proteins, immune escape mechanisms, as well as the vaccines and antiviral agents of AqRVs, especially for GCRV. A better understanding of structure, function and immune evasion strategies of AqRVs will offer new targets for prevention and control of AqRVs in aquaculture.

ITRAQ proteome analysis of insight into TLR20.2 functions through IFN1 signaling exerts regulatory effects on GCRV replication

Maternal immunization is an efficacious way to protect offspring from pathogen infections. To clarify the immune-enhancing mechanism of maternal immunization in grass carp, the iTRAQ assay was applied to screen differentially expressed proteins (DEPs) among eggs produced from maternal-immunized and non-immunized female grass carp. A total of 138 DEPs were obtained and TLR20.2 was the sole identified consensus DEP among the maternal-immunized and non-immunized groups. In general, the gene and protein expressions of TLR20.2 in the egg cell and early developmental stage embryos could be enhanced by maternal-immunized treatment. The mRNA levels of tlr20.2 were up-regulated in the liver, head kidney, and spleen in response to grass carp reovirus (GCRV) infection. Mechanistically, TLR20.2 functions through the IFN1 signaling and displayed inhibitory functions on GCRV replication by remarkably down-regulation of vp2 and vp7 expressions. The HSP90 was identified to be one of the proteins that interacted with TLR20.2, which coexpressed with TLR20.2 and mainly concentrated around the central veins. HSP90 promotes GCRV replication by inhibiting the irf3, irf7, and ifn1 expressions, which resulted in remarkably increased vp2 and vp7 gene expressions. The HMGB1a and HMGB1b could bind to the TLR20–2 promoter, and showed positive effects on tlr20.2 expression by significantly promoting its promoter activity (P < 0.01). Moreover, HMGB1a and HMGB1b exerted negative effects on HSP90 expression and thus the GCRV replication. Overall, this study provided molecular evidence that maternal immunization treatment protect against GCRV infection through the enhancement of TLR20.2 expression, and the HMGB1-TLR20.2-HSP90-IFN axis confers regulatory effects on GCRV replication. The results are novel findings to help elucidate the resistance-enhancing mechanism in fish species.

Identification and analysis of long non-coding RNAs that are involved in response to GCRV infection in grass carp (Ctenopharyngodon idella)

Long noncoding RNAs (lncRNAs) play important roles in many biological processes including the immune response against virus infection. However, their roles in grass carp reovirus (GCRV) pathogenicity are largely unknown. In this study, the next-generation sequencing (NGS) technology was used to analyze the profiles of lncRNAs in GCRV-infected and mock-infected grass carp kidney (CIK) cells. Our results showed that 37 lncRNAs and 1039 mRNA transcripts exhibited differential expression in CIK cells after GCRV infection compared with the mock infection. Functional analysis through the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases (KEGG) indicated that target genes of the differentially expressed lncRNAs were mainly enriched in the biological processes - biological regulation, cellular process, metabolic process and regulation of the biological process, such as MAPK signaling pathway and Notch signaling. Furthermore, we observed that the lncRNA3076 (ON693852) was markedly upregulated after the GCRV infection. In addition, silencing lncRNA3076 decreased the GCRV replication, which indicates that it might play an important role in the replication of GCRV.

Oral Lactobacillus casei expressing VP56310–500 and adjuvant flagellin C delivered by alginate-chitosan microcapsules remarkably enhances the immune protection against GCRV infection in grass carp

Due to grass carp haemorrhagic disease caused by grass carp reovirus (GCRV), aquaculture industry suffers huge economic loss. Oral Lactobacillus casei vaccine, with its excellent safety and effectiveness, is a promising strategy to prevent infectious diseases. While, the oral vaccine encapsulated with alginate–chitosan (SA/CS) can further improve its immune function. In this study, recombinant L. casei (rLc) expressing multiple fragments of VP56 was constructed, which was a key outer capsid protein of GCRV. By mixing rLcs with fodder and fed grass carp, the corresponding anti-VP56 fragment sera was produced, and VP56310–500 was screened as optimal fragment of GCRV-II by adopting enzyme linked immunosorbent assay (ELISA). Furthermore, SA/CS was used as the encapsulating material of L. casei live vaccine expressing VP56310–500, thereby preparing oral microcapsule live vaccine SCL-56310-500. With good resistance to bile (0–0.4%), NaCl (0–0.8%), simulated intestinal fluid, and simulated gastric fluid (pH = 1.5–4.5). Also, it had the ability to maintain high viability after colonizing intestine of grass carp in 7 days. In addition, Flagellin C (FlaC) was adopted as an immune adjuvant, and rLc was constructed to express VP56310–500-FlaC (56310–500C), thereby enhancing immune effect of oral live vaccine. After oral administration of encapsulated L. casei vaccine, its protective effect on GCRV-II infection was evaluated. As indicated by the results, SCL-56310-500C effectively up-regulated the activity of serum biochemical enzymes (including TSOD, lysozyme, and complement C3), and induced mRNA expression of important immune genes (including IL-1β, IFN1, MHC-IIα, IgM, and IgT in immune tissues). Simultaneously, SCL-56310-500C greatly suppressed GCRV replication in head kidney, spleen, and intestine. Correspondingly, grass carp vaccinated orally with SCL-56310-500C have distinctly higher survival rate (58% versus 24% in control) against GCRV infection. These results illustrated that an oral probiotic vaccine SCL-56310-500C effective in GCRV infection prevention was developed. Thus, this study offered an inspiring strategy for the prevention of fish viral diseases.

TBK1 Isoform Inhibits Grass Carp Reovirus Infection by Targeting the Degradation of Viral Nonstructural Proteins NS80 and NS38.

TANK-binding kinase 1 (TBK1) undergoes alternative splicing, and the previously reported TBK1 isoforms are negative regulators of RIG-I-like receptor-mediated type I IFN production. Although a study has suggested that grass carp TBK1 has an opposite effect at high- and low-titer of grass carp reovirus (GCRV) infection, the functions of grass carp TBK1 isoforms in GCRV infection remain unclear. In this study, we show that a TBK1 isoform from grass carp (Ctenopharyngodon idellus) named as gcTBK1_tv3, which has a 1-aa difference with zebrafish TBK1_tv3, inhibits the replication and infection of GCRV both at high and low titers of infection in C. idellus kidney cells. gcTBK1_tv3 can colocalize and interact with the NS80 and NS38 proteins of GCRV. Furthermore, gcTBK1_tv3 specifically degrades the NS80 and NS38 proteins of GCRV through the ubiquitin-proteasome pathway. Mechanistically, gcTBK1_tv3 promotes the degradation of NS80 or NS38 for K48-linked ubiquitination by targeting the Lys503 residue of NS80 or Lys328 residue of NS38, respectively, which ultimately impairs the production of cytoplasmic viral inclusion bodies and limits GCRV replication and infection. Taken together, our findings provide insight into the function of TBK1 isoform in the antiviral immune response and demonstrate that TBK1 isoform can target the nonstructural proteins of GCRV for impairing the formation of viral inclusion bodies.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Grass Carp Prx 3 Elevates Host Antioxidant Activity and Induces Autophagy to Inhibit Grass Carp Reovirus (GCRV) Replication.

Peroxiredoxins are a family of antioxidant proteins that protect cells from oxidative damage caused by reactive oxygen species (ROS). Herein, the peroxiredoxin 3 gene from grass carp (Ctenopharyngodon idellus), named CiPrx3, was cloned and analyzed. The full-length cDNA of CiPrx3 is 1068 bp long, with a 753 bp open reading frame (ORF) that contains a thioredoxin-2 domain, two peroxiredoxin signature motifs, and two highly conserved cysteine residues. CiPrx3 was ubiquitously expressed in all the tested tissues, while its expression level was altered significantly after exposure to grass carp reovirus (GCRV) and pathogen-associated molecular patterns (PAMPs). CiPrx3 was localized in the mitochondria of transfected cells and concentrated in the nucleus after poly (I:C) treatment. Transformation of CiPrx3 into Escherichia coli enhanced host resistance to H2O2 and heavy metals. Purified recombinant CiPrx3 proteins could protect DNA against oxidative damage. Overexpression of CiPrx3 in fish cells reduced intracellular ROS, increased cell viability, and decreased cell apoptosis caused by H2O2 stimulation and GCRV infection. Further study indicated that CiPrx3 induced autophagy to inhibit GCRV replication in fish cells. Collectively, these results imply that grass carp Prx3 elevates host antioxidant activity and induces autophagy to inhibit GCRV replication.

MiR-2188-5p promotes GCRV replication by the targeted degradation of klf2a in Ctenopharyngodon idellus

Studies on host immunity evasion by aquatic viruses have largely focused on coding genes. There is accumulating evidence for the important biological functions of non-coding miRNAs in virus–host interactions. The regulatory functions of non-coding miRNAs in fish reovirus–host interactions remain unknown. Here, miR-2188-5p in grass carp (Ctenopharyngodon idellus), a miRNA specific to teleosts, was predicted to target the 3′ UTR of the transcription factor klf2a. A correlation analysis and dual-luciferase reporter assay revealed that miR-2188-5p could induce the degradation of klf2a. The expression of miR-2188-5p induced the degradation of klf2a in a dose-dependent manner, suppressing the type I interferon response and promoting grass carp reovirus (GCRV) replication. As determined by a co-expression analysis, klf2a inhibited viral infection when miR-2188-5p was overexpressed. The targeted degradation of klf2a by miR-2188-5p could inhibit the type I interferon response and promote the replication of GCRV; however, this targeted degradation ability was insufficient to fully inhibit GCRV infection. These results provide novel insights into the regulatory effects and biological functions of non-coding miRNAs in fish–virus interactions.