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.

Epidermal growth factor receptor promotes infectious spleen and kidney necrosis virus invasion via PI3K-Akt signaling pathway.

Infectious spleen and kidney necrosis virus disease (ISKNVD) caused significant economic losses to the fishery industry. Epidermal growth factor receptor (EGFR), phosphatidylinositide 3-kinase (PI3K) played an important role in ISKNV invasion. However, the molecular regulatory mechanisms among EGFR, PI3K-Akt, and ISKNV invasion are not clear. In this study, ISKNV infection rapidly induced EGFR activation. While, EGFR activation promoted virus entry, but EGFR inhibitors and specific RNA (siRNA) decreased virus invasion. The PI3K-Akt as downstream signalling of EGFR was activated upon ISKNV infection. Consistent with the trends of EGFR, Akt activation increased ISKNV entry into cells, Akt inhibition by specific inhibitor or siRNA decreased ISKNV invasion. Akt silencing combination with EGFR activation showed that EGFR activation regulation ISKNV invasion is required for activation of the Akt signalling pathway. Those data demonstrated that ISKNV-induced EGFR activation positively regulated virus invasion by PI3K-Akt pathway and provided a better understanding of the mechanism of EGFR-PI3K-Akt involved in ISKNV invasion.

Network meta-analysis on the effects of finerenone versus SGLT2 inhibitors and GLP-1 receptor agonists on cardiovascular and renal outcomes in patients with type 2 diabetes mellitus and chronic kidney disease.

OBJECTIVE: To evaluate the cardiovascular and renal benefits of finerenone, sodium-glucose cotransporter-2 inhibitors (SGLT2i) and glucagonlike peptide-1 receptor agonists (GLP-1 RA) in patients with Type 2 Diabetes Mellitus (T2DM) and chronic kidney disease (CKD) with network meta-analysis. METHODS: Systematic literature searches were conducted of PubMed, Cochrane Library, Web of Science, Medline and Embase covering January 1, 2000 to December 30, 2021. Randomized control trials (RCTs) comparing finerenone, SGLT-2i and GLP-1 RA in diabetics with CKD were selected. We performed a network meta-analysis to compare the two drugs and finerenone indirectly. Results were reported as risk ratio (RR) with corresponding 95% confidence interval (CI). RESULTS: 18 RCTs involving 51,496 patients were included. Finerenone reduced the risk of major adverse cardiovascular events (MACE), renal outcome and hospitalization for heart failure (HHF) (RR [95% CI]; 0.88 [0.80-0.97], 0.86 [0.79-0.93], 0.79 [0.67,0.92], respectively). SGLT-2i were associated with reduced risks of MACE (RR [95% CI]; 0.84 [0.78-0.90]), renal outcome (RR [95% CI]; 0.67 [0.60-0.74], HHF (RR [95% CI]; 0.60 [0.53-0.68]), all-cause death (ACD) (RR [95% CI]; 0.89 [0.81-0.91]) and cardiovascular death (CVD) (RR [95% CI]; 0.86 [0.77-0.96]) compared to placebo. GLP-1 RA were associated with a lower risk of MACE (RR [95% CI]; 0.86 [0.78-0.94]). SGLT2i had significant effect in comparison to finerenone (finerenone vs SGLT2i: RR [95% CI]; 1.29 [1.13-1.47], 1.31 [1.07-1.61], respectively) and GLP-1 RA (GLP-1 RA vs SGLT2i: RR [95% CI]; 1.36 [1.16-1.59], 1.49 [1.18-1.89], respectively) in renal outcome and HHF. CONCLUSIONS: In patients with T2DM and CKD, SGLT2i, GLP-1 RA and finerenone were comparable in MACE, ACD and CVD. SGLT2i significantly decreased the risk of renal events and HHF compared with finerenone and GLP-1 RA. Among GLP-1 RA, GLP-1 analogues showed significant effect in reducing cardiovascular events compared with exendin-4 analogues.

Characterization and function of mandarin fish c-Myc during viral infection process.

c-Myc is a transcription factor and master regulator of cellular metabolism, and plays a critical role in virus replication by regulating glutamine metabolism. In this study, the open-reading frame (ORF) of c-Myc, designated as Sc-c-Myc, was cloned and sequenced. Multiple alignment of the amino acid sequence showed that the conserved domain of Sc-c-Myc, including the helix-loop-helix-zipper (bHLHzip) domain and Myc N-terminal region, shared high identities with other homologues from different species. Sc-c-Myc mRNA was widely expressed in the examined tissues of mandarin fish, and the higher mRNA levels was expressed in hind kidney. Moreover, mRNA and protein level of Sc-c-Myc was significantly increased in the Chinese perch brain (CPB) cells and spleen of mandarin fish post infection with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Sc-c-Myc overexpression promoted ISKNV and SCRV replication, on the contrary, knocking down Sc-c-Myc restrained ISKNV and SCRV replication. These results indicated that Sc-c-Myc involved in ISKNV and SCRV replication and proliferation, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

The dynamic immune responses of Mandarin fish (Siniperca chuatsi) to ISKNV in early infection based on full-length transcriptome analysis and weighted gene co-expression network analysis.

Mandarin fish (Siniperca chuatsi) been seriously harmed by infectious spleen and kidney necrosis virus (ISKNV) in recent years, but the early immune response mechanism of infection is still unknown. Here, we performed RNA sequencing on the spleens of mandarin fish infected with ISKNV at 0, 12, 24, 48, and 72 h post-infection (hpi) using short-read Illumina RNA sequencing and long-read Pacific Biosciences isoform sequencing to generate a full-length transcriptome. The immune responses of mandarin fish infected with ISKNV at the molecular level were characterized by RNA-seq analysis and weighted gene co-expression network analysis (WGCNA). A total of 26,528 full-length transcript sequences were obtained. There were 2,729 (1,680 up-regulated and 1,112 down-regulated), 1,874 (1,136 up-regulated and 738 down-regulated), 2,032 (1,158 up-regulated and 847 down-regulated), and 4,176 (2,233 up-regulated and 1,943 down-regulated) differentially expressed genes (DEGs) in mandarin fish at 12, 24, 48, and 72 hpi, compared with uninfected fish, respectively. A total of four modules of co-expressed DEGs identified by WGCNA were significantly positively correlated to the four time points after infection, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the immune-related DEGs in all these modules were mainly enriched in Phagosome, Endocytosis, Herpes simplex infection, and Cytokine-cytokine receptor interaction pathways. Further analysis showed that oher signaling pathways, including CAMs, NOD-like receptor and ER protein processing, Intestinal immune network for IgA production, TLR pathway, and Apoptosis significantly enriched in four modules corresponding to 12, 24, 48, and 72 hpi respectively, had specifically participated in the immune response. Hub genes identified based on the high-degree nodes in the WGCN, including CAM3, IL-8, CCL21, STING, SNX1, PFR and TBK1, and some DEGs such as MHCI, MHCII, TfR, STING, TNF α, TBK1, IRF1, and NF-kB, BCR, IgA and Bcl-XL had involved in dynamic molecular response of mandarin fish to ISKNV infection. In sum, this study provides a set of full-length transcriptome of the spleen tissue of mandarin fish for the first time and revealed a group of immune genes and pathways involved in different temporal responses to ISKNV infection, which has implications for resource conservation and aiding the development of strategies to prevent virus early infection for mandarin fish.

The composition and antiviral activity of scTRIM59 in Mandarin fish.

The tripartite motif (TRIM) proteins play critical roles in viral infection by modulating innate immunity. However, the molecular and antiviral activity of TRIM59 in mandrain fish is not fully understood. In present study, we cloned and sequenced the TRIM59 core sequence and explored its characteristics in Mandarin fish. The Siniperca chuatsi TRIM59 (scTRIM59) showed relatively high expression in immune-related organs. scTRIM59 expression was significantly down-regulated post ISKNV infection in vivo and vitro, but up-regulated at the early stages of SCRV infection in CPB cells. The overexpression of scTRIM59 inhibited ISKNV and SCRV infection, but decreased the expression of IRF3/IRF7-mediated signal genes. However, knockdown of scTRIM59 promoted the ISKNV and SCRV infection, but increased the expression of IRF3/IRF7-mediated signal genes. Those results indicated that scTRIM59 negatively regulated ISKNV, SCRV infection and IRF3/IRF7-mediated signal genes. This study provided new ideas about the function of scTRIM59.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

Characterization of mandarin fish (Siniperca chuatsi) IL-6 and IL-6 signal transducer and the association between their SNPs and resistance to ISKNV disease.

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p < 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p < 0.01) and improved markedly at 168 h in the spleen (p < 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.

Identification of intron in ORF003 gene and its application for inactivation test of ISKNV.

The virus inactivation test is a critical skill in inactivated vaccine production. Active viruses produced viral mRNA in susceptible cells or the host can be used to infer whether a DNA virus is replicating by RT-PCR. But it is generally difficult to avoid genomic DNA contamination in the samples. However, the use of primers spanning an intron is an effective alternative for virus inactivation test. Therein, a nested RT-PCR was developed to detect active ISKNV in the inactivated vaccine. At first, the transcriptome analysis of CPB cell infected with ISKNV revealed several gaps in some viral transcripts compared to ISKNV genome. One intron in ORF003L with 80 bp (designated IN-3) was confirmed by PCR and sequencing analysis. Then, two primer sets (primer A and primer B) spanning the IN-3 intron were designed to detect ISKNV transcription. The nested RT-PCR conditions were optimized with 0.4 µM primer A and 0.2 µM primer B, and 68 °C and 55 °C for annealing temperature, respectively. The sensitivity results indicated that the nested RT-PCR could detect one copy of live ISKNV propagating in CPB cells for seven days. The nested RT-PCR method was more sensitive and accurate than the method of blind passages in cells and fish challenge experiments. Together, above results indicate that this assay is a time-saving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.

Molecular characterization and function of EGFR during viral infectionprocess in Mandarin fishSiniperca chuatsi.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase protein and plays a critical role in virus infection by modulating innate immunity. In this study, we cloned and sequenced the EGFR coding sequence of mandarin fish, designed as scEGFR, and explored its characteristics. scEGFR mRNA was widely expressed in the tested tissues of mandarin fish, and the higher mRNA levels were expressed in kidney and spleen. scEGFR expression was up-regulated in spleen and CPB cells at early stage of ISKNV and SCRV infection. Gefitinib (EGFR inhibitor) inhibited ISKNV and SCRV replication, and increased the expression of the interferon-stimulated genes (ISG). However the EGF (EGFR activator) promoted ISKNV and SCRV replication, and decreased the interferon-stimulated genes. Those results indicated that scEGFR and its signaling involved in ISKNV and SCRV infection, and EGFR activation negatively regulated the interferon response, providing a potential target for the development of new therapic strategy against ISKNV and SCRV.

Administration of dietary recombinant hepcidin on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection under cage aquaculture conditions.

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.

Transcriptomic profiles reveal that inactivated iridovirus and rhabdovirus bivalent vaccine elicits robust adaptive immune responses against lethal challenge in marbled sleepy goby.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Siniperca chuatsi rhabdovirus (SCRV) induces autophagy via PI3K/Akt-mTOR pathway in CPB cells.

Autophagy is an important mechanism for organisms to eliminate viruses and other intracellular pathogens. Siniperca chuatsi rhabdovirus (SCRV) is an agent that has caused devastating losses in Chinese perch (Siniperca chuatsi) industry. But the role of autophagy in Siniperca chuatsi rhabdovirus (SCRV) infection is not clearly understood. In this study, we identified that SCRV infection triggered autophagy in CPB cells, which was demonstrated by the appearance of the membrane vesicles, GFP-LC3 punctuate pattern, conversion of LC3-I to LC3-II, and the co-localization of autophagosomes and lysosomes. The changes of autophagy flux in SCRV infection indicated that autophagy was inhibited at the early stage of SCRV infection, but was promoted at the late stage. UV-inactivated SCRV can induce autophagy, suggesting that SCRV replication is not essential for the induction of autophagy. Furthermore, we found inducing autophagy with Rapa inhibited SCRV proliferation, but inhibiting autophagy with 3-MA or CQ increased SCRV production in CPB cells. Then we assessed the effects of PI3K/Akt-mTOR signaling pathway on SCRV induced autophagy. We found that SCRV infection activated PI3K/AKT signaling pathway at 4 hpi, but inhibited it at 8 hpi. SCRV-N mRNA and protein level were decreased by inhibiting PI3K with LY294002, but increased by activating PI3K with 740Y-P. Those results indicated that SCRV infection induced autophagy via the PI3K/Akt-mTOR signal pathway, which will provide new insights into SCRV pathogenesis and antiviral treatment strategies.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of an immersion DNA vaccine against infectious spleen and kidney necrosis virus in mandarin fish.

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture.

First report of megalocytivirus (iridoviridae) in cultured bluegill sunfish, Lepomis macrochirus, in China.

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.

Development of strand-specific real-time RT-PCR for the analysis of SCRV transcription and replication dynamics.

To distinguish between three types of Siniperca chuatsi rhabdovirus (SCRV) viral RNA (vRNA, cRNA, and mRNA) and investigate SCRV transcription and replication dynamics in Chinese perch brain CPB cells, a novel, strand-specific, reverse transcriptase quantitative real-time PCR (RT-qPCR) assay was established. The method is based on strand-specific reverse transcription, using tagged primers to add a 'tag' sequence at the 5' end. We used the 'tag' sequence as the forward primer and a strand-specific reverse primer to quantify the three types of RNA. Three types of synthetic viral RNA were used as reference standards for validation and quantification. These assays were optimized to produce a standard curve from 102 to 107 copies/µL, with an efficiency of 91-101% and an R2 value of 0.9949-0.9999. The coefficients of variation for repeatability and reproducibility were less than 2.85% and 5.52%, respectively. Using this method, specific target RNA was detected at a 3500-70,000 fold higher level than other types of RNA. This method was also used to evaluate the dynamics of vRNA, cRNA and mRNA synthesis in CPB cells infected with SCRV. The results indicate that the intracellular dynamics of vRNA, cRNA and mRNA are different. In the earliest phase of SCRV infection, all three types of viral RNA increased very slowly. The copy number of vRNA and mRNA increased exponentially from 4 h post infection, while cRNA increased from 6 h post infection. The amount of cRNA was lower than vRNA and mRNA throughout the infection. The novel, strand-specific RT-qPCR method developed in this study provides critical data to aid the understanding of transcription and replication during SCRV infection.

MiR-214 inhibits snakehead vesiculovirus (SHVV) replication by targeting host GS.

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.

MicroRNAs profiles of Chinese Perch Brain (CPB) cells infected with Siniperca chuatsi rhabdovirus (SCRV).

MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection.

Chitosan and anisodamine improve the immune efficacy of inactivated infectious spleen and kidney necrosis virus vaccine in Siniperca chuatsi.

Siniperca chuatsi is an economically important fish in China, but infectious spleen and kidney necrosis virus (ISKNV) causes high mortality and significant economic losses. Currently, vaccination is the most promising strategy to prevent infectious diseases, while adjuvant can effectively enhance immune responses. In this study, inactivated ISKNV vaccine was prepared, then poly (I:C), chitosan, anisodamine and ims1312 were used as adjuvants to evaluate the effect on the immune responses and ISKNV replication. Chitosan could strongly boost the protection of liver and spleen tissues by pathological sections. In serum, poly (I:C) and chitosan group had protective effect on catalase, acid phosphatase, blood urea nitrogen. mRNA expressions showed these adjuvants induced the cytokines of early immune responses (TNF-α, Viperin) in both spleen and mesonephron by real time quantitative RT-PCR assays. Meanwhile, poly (I:C), chitosan and anisodamine were significantly improved the antiviral function and inhibited ISKNV replication. Chitosan and anisodamine played a significantly protective role in the immune protective rate test. The results indicated that all the four adjuvants are valid in the inactivated ISKNV vaccine, and chitosan is recommended preferentially. The present study provides reference for other animal vaccine adjuvants.

Immersion vaccination of Mandarin fish Siniperca chuatsi against infectious spleen and kidney necrosis virus with a SWCNTs-based subunit vaccine.

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.