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.

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

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

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection.

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Genome-wide identification of Gα family in grass carp (Ctenopharyngodon idella) and reproductive regulation functional characteristics of Cignaq.

BACKGROUND: The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp. RESULTS: Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression. CONCLUSIONS: These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts.

Interactions between Balantidium ctenopharyngodoni and microbiota reveal its low pathogenicity in the hindgut of grass carp.

BACKGROUND: Hosts, parasites, and microbiota interact with each other, forming a complex ecosystem. Alterations to the microbial structure have been observed in various enteric parasitic infections (e.g. parasitic protists and helminths). Interestingly, some parasites are associated with healthy gut microbiota linked to the intestinal eubiosis state. So the changes in bacteria and metabolites induced by parasite infection may offer benefits to the host, including protection from other parasitesand promotion of intestinal health. The only ciliate known to inhabit the hindgut of grass carp, Balantidium ctenopharyngodoni, does not cause obvious damage to the intestinal mucosa. To date, its impact on intestinal microbiota composition remains unknown. In this study, we investigated the microbial composition in the hindgut of grass carp infected with B. ctenopharyngodoni, as well as the changes of metabolites in intestinal contents resulting from infection. RESULTS: Colonization by B. ctenopharyngodoni was associated with an increase in bacterial diversity, a higher relative abundance of Clostridium, and a lower abundance of Enterobacteriaceae. The family Aeromonadaceae and the genus Citrobacter had significantly lower relative abundance in infected fish. Additionally, grass carp infected with B. ctenopharyngodoni exhibited a significant increase in creatine content in the hindgut. This suggested that the presence of B. ctenopharyngodoni may improve intestinal health through changes in microbiota and metabolites. CONCLUSIONS: We found that grass carp infected with B. ctenopharyngodoni exhibit a healthy microbiota with an increased bacterial diversity. The results suggested that B. ctenopharyngodoni reshaped the composition of hindgut microbiota similarly to other protists with low pathogenicity. The shifts in the microbiota and metabolites during the colonization and proliferation of B. ctenopharyngodoni indicated that it may provide positive effects in the hindgut of grass carp.

Morphological examination of abdominal vertebral bodies from grass carp using high-resolution micro-CT scans.

The vertebral column, a defining trait of all vertebrates, is organized as a concatenated chain of vertebrae, and therefore its support to the body depends on individual vertebral morphology. Consequently, studying the morphology of the vertebral centrum is of anatomical and clinical importance. Grass carp (GC) is a member of the infraclass Teleostei (teleost fish), which accounts for the majority of all vertebrate species; thus, its vertebral anatomical structure can help us understand vertebrate development and vertebral morphology. In this study, we have investigated the morphology and symmetry of the grass carp vertebral centrum using high-resolution micro-CT scans. To this end, three abdominal vertebrae (V9, V10, & V11) from eight grass carp were micro-CT scanned and then segmented using Dragonfly (ORS Inc.). Grass carp vertebral centrum conformed to the basic teleost pattern and demonstrated an amphicoelous shape (biconcave hourglass). The centrum's cranial endplate was smaller, less circular, and shallower compared to the caudal endplate. While the vertebral centrum demonstrated bilateral symmetry along the sagittal plane (left/right), the centrum focus was shifted dorsally and cranially, breaking dorsoventral and craniocaudal symmetry. The sum of these findings implies that the caudal aspect of grass carp vertebral centrum is bigger and more robust. Currently, we have no information whether this is due to nature, for example, differences in gene expression, or nurture, for example, environmental effect. As the vertebral parapophyses and spinous processes are slanted caudally, the direction of muscle action during swimming may create a gradient of stresses from cranial to caudal, resulting in a more robust caudal aspect of the vertebral centrum. Expanding our study to include additional quadrupedal and bipedal (i.e., human) vertebrae, as well as testing if these morphological aspects of the vertebrae are indeed plastic and can be affected by environmental factors (i.e., temperature or other stressors) may help answer this question.

Evaluation of virulence of Aeromonas veronii strain GZ21-2 and development of a highly effective vaccine for grass carp with the potential for industrial application.

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C-34 °C, respectively. The median lethal dose (LD50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii, incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application.

The effects of starvation stress on intestinal morphology and flora of grass carp (Ctenopharyngodon idella).

Starvation stress can profoundly impact various physiological parameters in fish, including metabolism, behavior, meat quality, and reproduction. However, the repercussions of starvation on the intestinal microbiota of grass carp remain under-explored. This research aimed to elucidate the effects of a 28-day starvation period on the composition of the intestinal microbiota of grass carp. Tissue pathology assessments revealed significant alterations in the dimensions of intestinal villi in the foregut, midgut, and hindgut as compared to the controls. Specifically, dominant differences appeared in both the length and width of the villi. Moreover, a marked decline in the goblet cell population was observed across all the intestinal segments. 16S rDNA sequencing was used to investigate changes in the gut microbiota, which revealed distinct clustering patterns among the starved and control groups. While α diversity metrics remained consistent for the anterior intestine, significant deviations were recorded in the Shannon (midgut: ***P < 0.001; hindgut: *P < 0.05) and Simpson indices (midgut and hindgut: ***P < 0.001), demonstrating alterations in microbial richness and evenness. At the phylum level, Proteobacteria, Bacteroidetes, and Fusobacteria emerged as dominant groups post-starvation. Other bacterial taxa, such as Actinobacteria and Verrucomicrobia, decreased, whereas Bacteroidetes and Firmicutes showed a small increase. In summation, starvation induces considerable morphological and microbial shifts in the grass carp intestine, and thus, this study offers valuable insights into their cultivation strategies.

Implications on growth performance, glucose metabolism, PI3K/AKT pathway, intestinal flora induced by dietary taurine in a high-carbohydrate diet for grass carp (Ctenopharyngodon idella).

An 8-week experiment was performed to investigate the influence on growth performance, plasma biochemistry, glucose metabolism and the insulin pathway of supplementation of dietary taurine to a high-carbohydrate diet for grass carp. In this study, fish were fed diets at one of two carbohydrate levels, 31·49 % (positive control) or 38·61 % (T00). The high-carbohydrate basal diet (T00), without taurine, was supplemented with 0·05 % (T05), 0·10 % (T10), 0·15 % (T15) or 0·20 % (T20) taurine, resulting in six isonitrogenous (30·37 %) and isolipidic (2·37 %) experimental diets. The experimental results showed that optimal taurine level improved significantly weight gain, specific growth rate (SGR), feed utilisation, reduced plasma total cholesterol levels, TAG and promoted insulin-like growth factor level. Glucokinase, pyruvate kinase and phosphoenolpyruvate carboxykinase activities showed a quadratic function model with increasing dietary taurine level, while hexokinase, fatty acid synthetase activities exhibited a positive linear trend. Optimal taurine supplementation in high-carbohydrate diet upregulated insulin receptor (Ir), insulin receptor substrate (Irs1), phosphatidylinositol 3-kinase (pi3k), protein kinase B (akt1), glycogen synthase kinase 3 ß (gs3kß) mRNA level and downregulated insulin-like growth factor (igf-1), insulin-like growth factor 1 receptor (igf-1R) and Fork head transcription factor 1 (foxo1) mRNA level. The above results suggested that optimal taurine level could improve growth performance, hepatic capacity for glycolipid metabolism and insulin sensitivity, thus enhancing the utilisation of carbohydrates in grass carp. Based on SGR, dietary optimal tributyrin taurine supplementation in grass carp was estimated to be 0·08 %.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection.

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

Edwardsiella piscicida causes iron storage disorders by an autophagy pathway in fish monocytes/macrophages.

Edwardsiella piscicida (E. piscicida) is a gram-negative pathogen that survives in intracellular environment. Currently, the interplay between E. piscicida and host cells has not been completely explored. In this study, we found that E. piscicida disturbed iron homeostasis in grass carp monocytes/macrophages to maintain its own growth. Further investigation revealed the bacteria induced an increase of intracellular iron, which was subjected to the degradation of ferritin. Moreover, the autophagy inhibitor impeded the degradation of ferritin and increase of intracellular iron in E. piscicida-infected monocytes/macrophages, implying possible involvement of autophagy response in the process of E. piscicida-broken iron homeostasis. Along this line, confocal microscopy observed that E. piscicida elicited the colocalization of ferritin with LC3-positive autophagosome in the monocytes/macrophages, indicating that E. piscicida mediated the degradation of ferritin possibly through the autophagic pathway. These results deepened our understanding of the interaction between E. piscicida and fish cells, hinting that the disruption of iron homeostasis was an important factor for pathogenicity of E. piscicida. They also indicated that autophagy was a possible mechanism governing intracellular iron metabolism in response to E. piscicida infection and might offer a new avenue for anti-E. piscicida strategies in the future.

miR-193b-5p promotes GCRV replication by inhibiting autophagy via targeting deptor in grass carp (Ctenopharyngodon idellus).

miRNAs are increasingly recognized for their crucial role in autophagy processes. Recent research has highlighted the significant function of autophagy in modulating immune responses. Within this context, specific miRNAs have been identified as indirect mediators of immune functions through their modulation of autophagy. In this study, we verified that miR-193b-5p simultaneously targeted the grass carp autophagy-related gene deptor, thereby reducing autophagy levels in CIK cells. Moreover, we found the expression levels of miR-193b-5p and deptor responding to pathogen infections in the GCRV-infected CIK cells. Notably, the overexpression of miR-193b-5p was found to induce the GCRV replication and reduce the irf3, irf7 and IFN1 expression. These findings also demonstrated that grass carp miR-193b-5p impacted the proliferation, migration, and antiapoptotic abilities of CIK cells. All the above results indicated that miR-193b-5p was linked to grass carp autophagy and played a vital role in antiviral immunity by targeting deptor. Our study may provide important insights into autophagy-related miRNAs and their roles in defense and immune mechanisms against pathogens in teleost.

A molecular mechanism underlies grass carp (Ctenopharyngodon idella) TARBP2 regulating PKR-mediated cell apoptosis.

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation.

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

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.

Oral immunization with recombinant L. lactis expressing GCRV-II VP4 produces protection against grass carp reovirus infection.

The hemorrhagic disease causing by grass carp reovirus (GCRV) infection, is associated with major economic losses and significant impact on aquaculture worldwide. VP4 of GCRV is one of the major outer capsid proteins which can induce an immune response in the host. In this study, pNZ8148-VP4/L. lactis was constructed to express recombinant VP4 protein of GCRV, which was confirmed by the Western-Blot and enzyme-linked immunosorbent assay. Then we performed the oral immunization for rare minnow model and the challenge with GCRV-II. After oral administration, pNZ8148-VP4/L. lactis can continuously reside in the intestinal tract to achieve antigen presentation. The intestinal and spleen samples were collected at different time intervals after immunization, and the expression of immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that VP4 recombinant L. lactis could induce complete cellular and humoral immune responses in the intestinal mucosal system, and effectively regulate the immunological effect of the spleen. The immunogenicity and the protective efficacy of the oral vaccine was evaluated by determining IgM levels and viral challenge to vaccinated fish, a significant level (P < 0.01) of antigen-specific IgM with GCRV-II neutralizing activity was able to be detected, which provided a effective protection in the challenge experiment. These results indicated that an oral probiotic vaccine with VP4 expression can provide effective protection for grass carp against GCRV-II challenge, suggesting a promising vaccine strategy for fish.

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.

Effects of Aeromonas infection on the immune system, physical barriers and microflora structure in the intestine of juvenile grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1ß and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and ß-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.

Evaluation of four novel non-grain protein sources completely replacing soybean meal on growth performance, serum biochemistry, amino acid transport and intestinal health of grass carp (Ctenopharyngodon idella) at different water temperatures.

To investigate the effects of non-grain protein source and water temperature on growth and feed utilization differences of grass carp, the effects of different protein sources on the growth performance, serum biochemistry, digestive enzymes, amino acid transport and intestinal health of grass carp were studied at 24 °C, 28 °C and 32 °C. In this study, a total of 1350 grass carp (Ctenopharyngodon idella) (initial weight 5.00 ± 0.02 g) were selected, and Clostridium autoethanogenum protein (CAP), Tenebrio molitor meal (TMM), cottonseed protein concentrate (CPC) and Chlorella powder (CHP) were used as a single protein source to completely replace soybean meal for 56 days. The results showed that the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of grass carp increased significantly with the increasing temperature (P < 0.001). The CHP and SBM groups showed no significant differences in FBW, WGR, SGR and PER (P > 0.05), which were higher than the CAP, TMM and CPC groups (P < 0.001). The alanine transaminase (ALT), aspartate aminotransferase (AST), total protein (TP) and triglyceride (TG) concentrations of grass carp at 32 °C were significantly lower than those at 24 °C and 28 °C (P < 0.001). The acid phosphatase (ACP) activity decreased significantly with the increase of temperature (P = 0.001). The amylase (AMS) activity of the TMM, CPC and CHP groups was significantly lower than that of the SBM and CAP groups (P < 0.001), and the ACP and lipase (LPS) activities in the TMM group were significantly lower than those in the SBM group (P < 0.001). In addition, the interaction between temperatures and protein sources significantly affected the gene expression levels of amino acid transport including solute carrier family 1 member 3 (SLC1A3), solute carrier family 7 member 1 (SLC7A1), solute carrier family 7 member 5 (SLC7A5), solute carrier family 15 member 1b (SLC15A1b), solute carrier family 7 member 7 (SLC7A7), target of rapamycin (TOR), 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1), intestinal inflammatory including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-8 (IL-8), interleukin-10 (IL-10) and tight junction proteins (occludin, claudin1, claudin3, claudin7 and claudin11) (P ≤ 0.001). Collectively, our results indicated that CHP could be a potential protein source in the case of complete replacement of soybean meal in grass carp.

The effect of selenium on the intestinal health of juvenile grass carp based on the ERS-autophagy pathway.

Selenium (Se), a trace element, is vital for the maintenance of cellular redox balance, thyroid hormone metabolism, inflammation, and immunity. Aeromonas hydrophila (A. hydrophila) is a common Gram-negative conditional pathogenic bacterium in fish culture, posing a serious threat to intensive aquaculture. Our study investigated the influence of dietary Se on the intestinal immune function of grass carp (Ctenopharyngodon idella) and the related regulatory mechanisms. The 2160 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 6 test groups of 6 replicates each, and fed graded selenomethionine (0.05, 0.20, 0.40, 0.61, 0.77, 0.98 mg Se/kg diet) for 70 days and then injected with A. hydrophila for a 6-day attack test. The results indicated that appropriate Se levels (0.40 mg/kg diet) alleviated intestinal damage caused by A. hydrophila and increased intestinal immune substances C3 and C4 levels as well as the activity of acid phosphatase (ACP) and lysozyme (LZ) (P > 0.05). Appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) decreased intestinal pro-inflammatory cytokines (IFN-γ2, IL-6, IL-12p35, IL-17 A F and IL-17D) mRNA levels (P > 0.05) and increased intestinal anti-inflammatory factors (TGF-ß1, IL-4/13A, IL-4/13B, IL-10 and IL-22) mRNA levels (P > 0.05) in juvenile grass carp. Further studies revealed that Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal endoplasmic reticulum stress (ERS)-related signaling pathway. Furthermore, we found that appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal autophagy in juvenile grass carp, which may be related to ULK1, Beclin 1, ATG5, ATG12, LC3, and P62. In conclusion, appropriate levels of Se can alleviate intestinal inflammation and inhibit ERS and autophagy in juvenile grass carp. A quadratic regression analysis of intestinal ACP and LZ also indicated that the Se requirements of juvenile grass carp were 0.59 and 0.51 mg/kg, respectively.