Mammalian reovirus µ1 protein attenuates RIG-I and MDA5-mediated signaling transduction by blocking IRF3 phosphorylation and nuclear translocation.

Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous µ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV µ1 protein expression by shRNA could impair MRV proliferation. Specifically, µ1 protein inhibited MRV or poly(I:C)-induced IFN-ß expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that µ1 protein significantly decreased IFN-ß mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that µ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein µ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection.

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.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Molecular cloning, functional characterization and expression analysis of P65 subunit in response to GCRV infection in rare minnow (Gobiocypris rarus).

P65, the all-important subunit of the transcription factor NF-κB, plays an important role in the regulation of immune response. In this study, the cDNA of P65 subunit of rare minnow Gobiocypris rarus (GrP65) was cloned, and its expression patterns and functional role in rare minnow were investigated. The GrP65cDNA encodes a polypeptide of 573 amino acids, containing a well-conserved Rel-homology domain (RHD). The amino acid sequence analysis showed that GrP65 shared 81% and 69% identity to the grass carp (Ctenopharyngodon idella) and human (Homo sapiens) orthologous, respectively. Phylogenetic analysis revealed that GrP65 clustered with homologues from other teleosts. Cellular distribution anallysis demonstrated that GrP65 was located in the cytoplasm and nucleus. Quantitative real-time PCR analysis showed that GrP65 was ubiquitously expressed in all examined tissues, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrP65 were significantly up-regulated in liver in response to GCRV infection, which suggested that GrP65 might play a crucial role in recognition and responses to GCRV infection in fish. In addition, GrP65 activated several interferon (IFN) promoters and induced the expression of downstream IFN-stimulated genes (ISGs). Furthermore, overexpression of P65 remarkably decreased the GCRV proliferation, while knockdown of P65 obtained opposite effects. In summary, we systematically characterized GrP65 and demonstrated its role in the innate immune response to GCRV infections.

Molecular identification and function characterization of four finTRIM genes from the immortal fish cell line, EPC.

In mammals, tripartite motif (TRIM)-containing proteins are involved in interferon (IFN)-mediated antiviral response as pivotal players endowed with antiviral effects and modulatory capacity. Teleost fish have a unique subfamily of TRIM, called finTRIM (fish novel TRIM, FTR) generated by genus- or species-specific duplication of TRIM genes. Herein, four TRIM genes are identified from Epithelioma papulosum cyprini (EPC) cells, and phylogenetically close to the members of finTRIM, thus named FTREPC1, FTREPC2, FTREPC3 and FTREPC4. Despite high similarity in nucleotide sequence, FTREPC1/2 genes encode two proteins with a typically consecutive tripartite motif followed by a C-terminal B30.2 domain, while FTREPC3/4-encoding proteins retain only a RING domain due to early termination of translation. They are induced by poly(I:C), GCRV and SVCV as IFN-stimulated genes (ISGs), and this induction is severely impaired by blockade of STAT1 pathway and is dependent on a typical ISRE motif within the 5' untranslated regions (5'UTRs) of FTREPC1/2/3/4 genes. Whereas overexpression of FTREPC1/2/3/4 alone does not activate fish IFN promoters, overexpression of FTREPC1 or FTREPC2, rather than FTREPC3 and FTREPC4, significantly impairs intracellular poly(I:C)-triggered activation of fish IFN promoters. Consistently, FTREPC1/2 promote virus replication through negatively regulating IFN response. Our results provide evidence for the involvement of EPC finTRIM proteins in IFN antiviral response and insights into genus- or species-specific regulation of fish innate immune pathways.

Protein Phosphatase PP1 Negatively Regulates IRF3 in Response to GCRV Infection in Grass Carp (Ctenopharyngodon idella).

Protein phosphatase-1 (PP1) has an important role in many cell functions, such as cell differentiation, development, immune response and tumorigenesis. However, the specific role of PP1 in the antiviral response in fish remains to be elucidated. In this study, the PPP1R3G homolog was identified in the grass carp (Ctenopharyngodon idella) and its role in defence against the GCRV infection was investigated. Phylogenetic analysis demonstrated that CiPPP1R3G clustered with homologues from other teleosts. Temporal expression analysis in vivo revealed that the expression level of CiPPP1R3G was significantly up-regulated in response to GCRV infection in grass carps, especially in the intestine and head-kidney. Cellular distribution analysis revealed that CiPPP1R3G was located in the nucleus and cytoplasm. Overexpression of CiPPP1R3G significantly negatively regulated the expression of CiIRF3, thus inhibiting its activation. In summary, we systematically analyzed the PPP1R3G gene in grass carp and illustrated its function as a negative regulator in the anti-GCRV immune responses.

Study of the activation of the PI3K/Akt pathway by the motif of σA and σNS proteins of avian reovirus.

The present study was conducted to determine whether avian reovirus (ARV) activates the phosphatidylinositol 3-kinase-dependent Akt (PI3K/Akt) pathway according to the PXXP or YXXXM motifs of σA and σNS proteins. Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motifs of the σA and σNS genes. Plasmid constructs that contain mutant σA and σNS genes were generated and transfected into Vero cells, and the expression levels of the corresponding genes were quantified according to immunofluorescence and Western blot analyses. The Akt phosphorylation (P-Akt) profile of the transfected Vero cells was examined by flow cytometry and Western blot. The results showed that the σA and σNS genes were expressed in the Vero cells, and P-Akt expression in the σA mutant groups (amino acids 110-114 and 114-117) was markedly decreased. The results indicated that the σA protein of ARV activates the PI3K/Akt pathway via the PXXP motif. The results of this study reveal the mechanisms by which ARV manipulates the cellular signal transduction pathways, which may provide new ideas for novel drug targets.

Characterization and expression of galectin-3 in grass carp (Ctenopharyngodon idella).

Galectins are members of evolutionary conserved lectin family and play important roles in the innate and adaptive immunity of both vertebrates and invertebrates. Galectin-3 is the only chimera galectin with one C-terminal carbohydrate recognition domain (CRD) connected to the N-terminal end. Here, a galectin-3 (named CiGal3) from grass carp was identified and characterized, which encodes polypeptides 362 amino acids with a predicted molecular mass of 36.45 kDa and theoretical isoelectric point of 4.91. The sugar binding motifs involved in carbohydrate binding activity (H-N-R, V-N and W--E-R) were detected in CRD. In comparison to other species, CiGal3 showed the highest similarity and identity to Cyprinus carpio (95.3% sequence similarity and 92.5% sequence identity). The subcellular localization of CiGal3 was distributed in the cytoplasm and nucleus of transfected cells. The CiGal3 transcripts were ubiquitously expressed in all checked tissues and highly expressed in immune tissues. In addition, the expression of CiGal3 in liver and spleen was induced post grass carp reovirus (GCRV), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C) challenge. These results suggest that CiGal3 plays a vital role in the immune system.

Sequence and expression analysis of the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 from the barbel chub Squaliobarbus curriculus.

MDA5 is a cytoplasmic viral double-stranded RNA recognition receptor that plays a pivotal role in the aquatic animal innate immune system. To decipher the role of MDA5 of Squaliobarbus curriculus (ScMDA5) in the immune response, full-length cDNA of ScMDA5 was cloned using the RACE technology, mRNA and protein expression levels of ScMDA5 signalling pathway members in response to stimulation were detected and effects of overexpression of ScMDA5 on the immune response were investigated. ScMDA5 comprises 3597 bp and is composed of an open reading frame (2958 nucleotides long) that translates into a putative peptide of 985 amino acid residues. ScMDA5 possesses two N-terminal caspase-recruiting domains, DEAD-like helicases superfamily, helicase superfamily C-terminal and RIG-I_C-RD domains, and differences in these domains among species were mainly observed with respect to their length and location. ScMDA5 was closely clustered with those of Carassius auratus, Ctenopharyngodon idellus and Mylopharyngodon piceus. ScMDA5 transcripts were most abundant in the spleen and the lowest in the liver. Expression levels of ScMDA5 in healthy tissues were significantly correlated with those of ScIRF3, ScIRF7 and ScIFN. Besides, mRNA expression levels of ScIRF3 were significantly correlated with those of ScIRF7 (0.956, P < 0.01). Expression level changes, including downregulation, upregulation and initial upregulation followed by downregulation, were found in ScMDA5 signalling pathway molecules in tissues after grass carp reovirus infection. Protein levels of ScMDA5 were the highest in the liver and the lowest in the spleen in detected healthy tissues. Overexpression of ScMDA5 led to significantly enhanced CiIRF7 and CiMx transcription in grass carp ovary cells (P < 0.05). The results of this study helped to clarify the role of ScMDA5 in the immune reaction against grass carp reovirus and provided fundamental information for fish breeding to achieve strong resistance to infection.

Breast Tumor-Associated Metalloproteases Restrict Reovirus Oncolysis by Cleaving the σ1 Cell Attachment Protein and Can Be Overcome by Mutation of σ1.

Reovirus is undergoing clinical testing as an oncolytic therapy for breast cancer. Given that reovirus naturally evolved to thrive in enteric environments, we sought to better understand how breast tumor microenvironments impinge on reovirus infection. Reovirus was treated with extracellular extracts generated from polyomavirus middle T-antigen-derived mouse breast tumors. Unexpectedly, these breast tumor extracellular extracts inactivated reovirus, reducing infectivity of reovirus particles by 100-fold. Mechanistically, inactivation was attributed to proteolytic cleavage of the viral cell attachment protein σ1, which diminished virus binding to sialic acid (SA)-low tumor cells. Among various specific protease class inhibitors and metal ions, EDTA and ZnCl2 effectively modulated σ1 cleavage, indicating that breast tumor-associated zinc-dependent metalloproteases are responsible for reovirus inactivation. Moreover, media from MCF7, MB468, MD-MB-231, and HS578T breast cancer cell lines recapitulated σ1 cleavage and reovirus inactivation, suggesting that inactivation of reovirus is shared among mouse and human breast cancers and that breast cancer cells by themselves can be a source of reovirus-inactivating proteases. Binding assays and quantification of SA levels on a panel of cancer cells showed that truncated σ1 reduced virus binding to cells with low surface SA. To overcome this restriction, we generated a reovirus mutant with a mutation (T249I) in σ1 that prevents σ1 cleavage and inactivation by breast tumor-associated proteases. The mutant reovirus showed similar replication kinetics in tumorigenic cells, toxicity equivalent to that of wild-type reovirus in a severely compromised mouse model, and increased tumor titers. Overall, the data show that tumor microenvironments have the potential to reduce infectivity of reovirus.IMPORTANCE We demonstrate that metalloproteases in breast tumor microenvironments can inactivate reovirus. Our findings expose that tumor microenvironment proteases could have a negative impact on proteinaceous cancer therapies, such as reovirus, and that modification of such therapies to circumvent inactivation by tumor metalloproteases merits consideration.

Enteric viruses exploit the microbiota to promote infection.

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission.

Black carp IRF5 interacts with TBK1 to trigger cell death following viral infection.

Interferon regulated factor 5 (IRF5) is a key regulator of inflammatory responses in human and mammals; however, its role in teleost remains largely unknown. In this study, IRF5 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized, which possesses conservation in structure and sequence to its mammalian counterparts. Black carp IRF5 (bcIRF5) was characterized as a predominantly cytosolic protein by immunofluorescent staining and showed little IFN promoter-inducing ability in reporter assay. The direct association between bcIRF5 and black carp TBK1 (bcTBK1) were identified through co-immunoprecipitation assay, and co-expressed bcIRF5 in EPC cells suppressed bcTBK1-mediated IFN promoter transcription in reporter assay. Surprisingly, the titer of grass carp reovirus (GCRV) in the media of EPC cells co-expressing bcIRF5 and bcTBK1 was obviously lower than that of EPC cells expressing bcTBK1 alone. It was interesting that expression of bcIRF5 and/or bcTBK1 in EPC cells showed little effect on cell growth; however, the survival ratio of EPC cells co-expressing bcTBK1 and bcIRF5 post GCRV infection was much lower than that of EPC cells expressing bcIRF5 or bcTBK1 alone. These results indicate that bcIRF5 negatively regulates bcTBK1-mediated IFN signaling in healthy cells; however, it correlates with bcTBK1 and triggers cell death to inhibit the virus replication during the innate immune activation.

Identification and molecular characterization of peroxiredoxin 2 in grass carp (Ctenopharyngodon idella).

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.

Transcriptome analysis of rare minnow (Gobiocypris rarus) infected by the grass carp reovirus.

Grass carp shares the largest portion of the aquaculture production in China, but hemorrhagic disease caused by grass carp reovirus (GCRV) often results in tremendous loss of fingerlings and yearlings, causing significant economic damages. However, it is difficult to study antiviral mechanisms in grass carp in vivo due to its large size and long reproductive cycle. Therefore, a small cyprinid species named rare minnow with high sensitivity to GCRV, is regarded as a useful model to study the mechanisms of this disease. In this study, rare minnows were infected with the type-IIGCRV (GCRV-HZ08), and pathogenesis was investigated by BGISEQ-500 transcriptome sequencing of four cDNA libraries, hepatopancreas, gills, head-kidney and spleen, and real time quantitative PCR (qRT-PCR). We obtained 51.22 Gb bases in total, and de novo assembled 107,756 unigenes with an average length of 1,441 bp. GO analysis revealed that the differentially expressed genes (DEGs) involved in the defense mechanisms were the most enriched GO terms in all four tissues. KEGG analysis revealed that the most enriched pathways were "Influenza A", "Herpes simplex infection", "Transcriptional misregulation in cancer" and "Metabolic" pathways. We also performed a comparative transcriptomic study between GCRV-infected rare minnow and grass carp data. This revealed that "IL-17 signaling pathway", "NF-kappa B signaling pathway" and "Influenza A" pathways are conserved (important) in the regulation of anti-GCRV infection in both species, and need to be further investigated. Furthermore, a total of four immune-related DEGs were selected for qRT-PCR validation, and the results confirmed the RNA-seq data. These results enhance our understanding of the antiviral responses of cyprinid fish infected by GCRV.

Characterization of cathepsin C from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response.

SIKE of black carp is a substrate of TBK1 and suppresses TBK1-mediated antiviral signaling.

RIG-I like receptor (RLR) signaling functions importantly in host innate immune response against RNA virus, which is tightly regulated by a number of mechanisms to prevent aberrant interferon production. The suppressor of IKKε (SIKE) has been identified as a suppressor of IKKε and TBK1, which are key components of RLR signaling. In this study, SIKE homologue (bcSIKE) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcSIKE varied in host cells in response to the stimulation of LPS, poly (I:C) and viruses. bcSIKE migrated around 27 KDa in immunoblot assay and distributed in both cytoplasm and nucleus of host cell in immunofluorescent (IF) staining test. bcSIKE showed no IFN-inducing ability in reporter assay and EPC cells expressing bcSIKE showed no enhanced antiviral ability against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV). However, bcSIKE obviously dampened the IFN-inducing ability of RLR signaling members in reporter assay when bcSIKE was co-expressed with these molecules in EPC cells. The association between bcSIKE and bcTBK1 has been identified through IF and co-immunoprecipitation (co-IP) assay. The plaque assay demonstrated clearly that bcTBK1-mediated antiviral activity in EPC cells against both GCRV and SVCV was down regulated by bcSIKE. All the data generated in this paper support the conclusion that bcSIKE interacts with bcTBK1 and inhibits bcTBK1-mediated antiviral signaling during host innate immune activation, which is reported in teleost for the first time.

Identification, characterisation and preliminary functional analysis of IRAK-M in grass carp (Ctenopharyngodon idella).

Interleukin-1 receptor-associated kinase (IRAK) family members play important roles in myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) signaling, the crucial innate immune pathway in vertebrates. In the present study, the IRAK family gene IRAK-M (also called IRAK3) from grass carp (Ctenopharyngodon idella) was cloned and characterised. IRAK-M was mainly enriched in the spleen, and the significantly altered expression was observed after grass carp reovirus (GCRV) infection. Subcellular localisation showed that IRAK-M protein distributed uniformly in the entire cell and co-localised with MyD88 in the cytoplasm of transfected cells. Additionally, the interaction between IRAK-M and MyD88 was confirmed by bimolecular fluorescence complementation (BiFC) system. Moreover, deficient of IRAK-M in C. idella kidney cell line (CIK) with small interference RNA (siRNA) upregulated polyinosinic:polycytidylic acid (poly(I:C))-induced inflammatory cytokines production, including interleukin 8 (IL-8), IL-6, and tumour necrosis factor α (TNF-α), which reveals that IRAK-M functions as a negative regulator of inflammatory cytokines. Taken together, our results demonstrate that IRAK-M gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the IRAK-M in teleosts.

Molecular cloning and expression analysis of coagulation factor VIII and plasminogen involved in immune response to GCRV, and immunity activity comparison of grass carp Ctenopharyngodon idella with different viral resistance.

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella.

Molecular characterization and expression of TLR7 and TLR8 in barbel chub (Squaliobarbus curriculus): Responses to stimulation of grass carp reovirus and lipopolysaccharide.

The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus, designated as ScTLR7 and ScTLR8, were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8, MyD88, and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P: 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P: 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways.