[Show abstract][Hide abstract] ABSTRACT: High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1, and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/ bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.
[Show abstract][Hide abstract] ABSTRACT: Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (TRIF) is an adapter in responding to activation of some toll-like receptors (TLRs), which provides early clearance of viral and bacterial pathogens. Here we identified and characterized a full-length genomic sequence of TRIF gene from grass carp Ctenopharyngodon idella (designated as CiTRIF). CiTRIF genomic sequence consists of 3534 base pairs (bp), containing 5' flank sequence (496 bp) and unique intron (815 bp). The full-length cDNA sequence is 2241 bp, including 5' untranslated region (UTR) of 352 bp, 3' UTR of 209 bp, and an open reading frame of 1680 bp encoding a polypeptide of 559 amino acids with an estimated molecular weight of 62.643 kDa and a predicted isoelectric point of 5.71. The deduced amino acid sequence just contains TIR domain, and is most similar to the zebrafish (Danio rerio) TRIF sequence with an identity of 64%. CiTRIF exhibits sequence divergence from its orthologs. Promoter region was predicted and promoter activity was verified. mRNA expression of CiTRIF gene is widespread in fifteen tissues investigated, highly in foregut and skin physiological immune barrier. The transcripts of CiTRIF were significantly and rapidly induced in spleen and head kidney tissues at early stage post grass carp reovirus (GCRV) challenge. The modulations are significant but mild in CIK (C. idella kidney) cells post GCRV infection or poly(I:C) stimulation. The over-expression vector was constructed and transfected into CIK cell line to get stably expressing recombinant proteins. In CiTRIF transfected cells, mRNA expressions of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I were up-regulated. After GCRV infection, the transcripts of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I fell a little bit after a rapidly and strongly rise. In CiTRIF over-expression cells, virus load and titer were significantly lower than those in controls post GCRV challenge, and virus replication was inhibited obviously. The results indicate that the novel TRIF gene from grass carp plays important roles in modulating antiviral innate immune responses, and serve the further functional studies on TRIF gene in teleosts and immune evolution.
[Show abstract][Hide abstract] ABSTRACT: TLR8 (toll-like receptor 8), a homolog of TLR3, TLR7 and TLR9 as prototypical intracellular members of TLR family, is generally associated with sensing single stranded RNA and plays a pivotal role in antiviral immune response. In this study, a TLR8 gene from grass carp Ctenopharyngodon idella (designated as CiTLR8) was obtained and characterized. The full-length cDNA of CiTLR8 was of 3766 bp. The open reading frame was of 3072 bp and encoded a polypeptide of 1023 amino acids, including seventeen LRR (leucine-rich repeat) motifs, one transmembrane domain and one TIR (toll/interleukin-1 receptor) domain. A single intron with the size of 839 bp was found on the neck of start codon (ATG). CiTLR8 mRNA was ubiquitously expressed in the 15 tested tissues and the expression level in gas bladder, spleen, brain, hindgut and trunk kidney tissues was high. Besides, the CiTLR8 expression in spleen and head kidney was significantly up-regulated and reached peak at 24 h post-injection of grass carp reovirus (GCRV). CiTLR8 transcription reached peak at 8 h and then declined below the normal level post-GCRV infection in the C. idella kidney (CIK) cell line; and it was rapidly and significantly down-regulated by the stimulation of the synthetic double-stranded RNA polyriboinosinic-polyribocytidylic acid sodium salt (poly I:C) in CIK in a dose and time-dependent manner. The inhibitor expression vectors were constructed and transfected into CIK cell line to obtain stably expressing shRNA targeting TLR8. In CiTLR8-knockdown cells, CiTLR7 transcript weakly increased, CiIFN-I mRNA was significantly down-regulated, and the expression of CiMyD88, CiIRF7 and CiMx1 scarcely changed. Post poly I:C stimulation, CiTLR8, CiTLR7 and CiMyD88 transcripts significantly increased, CiIRF7 was down-regulated after an initial phase of increase, and CiIFN-I and CiMx1 transcripts were up-regulated. After GCRV infection, the transcripts of CiTLR8, CiTLR7, CiMyD88 and CiIRF7 were up-regulated, but CiIFN-I and CiMx1 transcripts were inhibited. Nevertheless, cells transfected with pshTLR8 vectors had strong resistance against GCRV injection, suggesting CiTLR8 might play a negative role in antiviral immune response. These results collectively suggested that CiTLR8 was a novel member of TLR gene family, engaging in antiviral innate immune defense in C. idella, which laid a foundation for the further mechanism research of TLR8 in fishes.
[Show abstract][Hide abstract] ABSTRACT: Trunk kidney is a vital organ for excretion in teleosts. There have been sporadic reports of processing pathogens for the immune function in trunk kidney. However, molecular processes of pathogen recognition receptors (PRRs) responding to virus and viral/bacterial pathogen-associated molecular patterns (PAMPs) are poorly elucidated in trunk kidney. In the present study, we investigated transcriptional profiles of twelve representative immune-related genes (TLRs (TLR3, TLR7 and TLR22); RLRs (RIG-I, MDA5 and LGP2); NLRs (NOD1 and NOD2); adaptor molecules (MyD88 and IPS-1); effector molecule type I interferon (IFN-I) and immunoglobulin M (IgM)) in trunk kidney tissue of grass carp (Ctenopharyngodon idella) (designated as Ci) injection of grass carp reovirus (GCRV) utilizing quantitative real-time RT-PCR (qRT-PCR). Furthermore, mRNA expression patterns of these genes (IgM excepted) were examined post GCRV infection and polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) or peptidoglycan (PGN) stimulation in primary trunk kidney cells of grass carp. The relative values of CiTLR3, CiTLR22 and CiMyD88 were increased post GCRV challenge and viral/bacterial PAMPs stimulation. The mRNA transcriptions of CiTLR7 were obviously activated with GCRV challenge. Remarkably, the mRNA expressions of CiRIG-I, CiMDA5, CiLGP2 and CiIPS-1 were largely up-regulated with GCRV challenge and viral/bacterial PAMPs stimulation. Interestingly, the expression tendencies of CiNOD1 and CiNOD2 were differential not only in GCRV challenge and poly(I:C) stimulation, but also in LPS and PGN stimulation. It was demonstrated that CiIFN-I induced powerful anti-viral and anti-bacterial effects in trunk kidney. In addition, the expression of CiIgM was induced at 72 h post GCRV injection in vivo. Collectively, these results suggest that trunk kidney of grass carp serves as an important immune organ, and plays crucial roles in triggering anti-viral and anti-bacterial immune responses both in vivo and in vitro.
[Show abstract][Hide abstract] ABSTRACT: ADAR (adenosine deaminase acting on RNA) is an RNA editing enzyme that targets both coding and noncoding dsRNAs (double stranded RNAs) and converts adenosine to inosine, which is read by translation machinery and by polymerases during RNA-dependent RNA replication as if it is guanosine. This editing is a widespread post-transcriptional modification event in animals. In this study, we identified the full-length cDNA sequence of Ctenopharyngodon idella ADAR1 (designated as CiADAR1) and detected the mRNA expression patterns in response to dsRNA (polyinosinic-polycytidylic acid sodium salt, poly(I:C)) and grass carp reovirus (GCRV). CiADAR1 is a large gene in size, consisting of 4833 nucleotides encoding a protein of 1392 amino acids. The deduced amino acid sequence contains seven putative domains: one proline-rich region (Pro-R), two Z-DNA-binding domains (Zalpha), three dsRNA binding motifs (DSRM) and one tRNA-specific and dsRNA adenosine deaminase domain (ADEAMc). It is most homologous to Danio rerio ADAR (E-value = 0.0, identities = 80% (1110/1395)), also close homology to Homo sapiens ADAR1 (E-value = 0.0, identities = (47%) 530/1122). CiADAR1 mRNA was investigated in fifteen tissues, and was low expressed in muscle and head kidney tissues, high in blood and spleen tissues by quantitative real-time RT-PCR (qRT-PCR). mRNA expressions of CiADAR1 were significantly up-regulated and reached peak at 24 h post GCRV challenge in vivo and in vitro (P < 0.05). After poly(I:C) stimulation at different concentrations, CiADAR1 transcripts were rapidly and significantly up-regulated and recovered in dose-dependent and time-dependent manners (P < 0.05). The results indicate CiADAR1 was implicated in the antiviral immune response and laid the foundation for further studies on functions and mechanisms of RNA editing in fishes.
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptor 22 (TLR22) plays a crucial role in response to virus infection by recognizing double stranded RNA (dsRNA) in aquatic animals. In the present study, a TLR22 homologue gene was identified and characterized from grass carp (Ctenopharyngodon idella) (CiTLR22). CiTLR22 genomic sequence comprises 4754 base pairs (bp), containing one intron. The cDNA sequence consists of 3831bp, encoding a protein of 954 amino acid residues. CiTLR22 was constitutively expressed in all 15 investigated tissues, highly in gill and lowly in liver and spleen. The expression profile of CiTLR22 in spleen was rapidly and significantly up-regulated at 6h (456.13-fold, P<0.05), then rapidly recovered to normal level at 12h (P>0.05) post-injection of grass carp reovirus (GCRV). The expression levels of CiTLR22 were rapidly elevated post-poly(I:C) stimulation in dose- and time-dependent manners in CIK (C. idella kidney) cell line. After GCRV infection, CiTLR22 transcripts were inhibited at the early stage, then were up-regulated and reached a peak at 24h post-infection, latterly down-regulated in CIK cell culture. In the whole genomic sequence, six single nucleotide polymorphisms (SNPs) were detected. Five of them were sited in the coding region and all synonymous, and another located in the 5' untranslated region (UTR). The following SNP analysis revealed that 2406 C/T was just a mutation. Only 417 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype (P=0.013) and allele (P=0.015). -8 A/T and 2574 C/T, 863 C/T and 1923 G/T, 863 C/T and 2574 C/T were pairwise linkage disequilibrium. None of the haplotype was associated with the resistance of grass carp to GCRV. The results indicate that CiTLR22 plays an important role in the responses to dsRNA and GCRV, and is partially inhibited by GCRV in vitro. The potential molecular marker lays foundation for the selective breeding of the GCRV-resistant grass carp.