Chunrong Yang

Evaluation of internal control genes for qRT-PCR normalization in tissues and cell culture for antiviral studies of grass carp (Ctenopharyngodon idella).

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for RNA expression. To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. Here, we cloned partial sequence of elongation factor 1α (EF1α) gene from grass carp (Ctenopharyngodon idella). The stabilities of four commonly used internal control genes encoding 18S rRNA, β-actin, EF1α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were integratedly assessed using the geNorm, NormFinder and BestKeeper programs. Integrative analyses of qRT-PCR data indicated that the stability ranking of the examined genes was 18S rRNA > EF1α > GAPDH > β-actin in gill, head kidney, heart, intestine, liver, muscle, skin, spleen, and trunk kidney tissues in untreated grass carp. When the same calculations were done in spleen tissue at different time points post grass carp reovirus (GCRV) infection, the gene ranking was 18S rRNA > β-actin > GAPDH > EF1α. The rank ordering of expression stability was EF1α > β-actin>18S rRNA > GAPDH in C. idella kidney (CIK) cell culture stimulated by poly(I:C). The recommended ranking was EF1α > GAPDH > β-actin>18S rRNA in CIK cells infected by GCRV. The results indicated that 18S rRNA was the best invariant internal control gene in individual level in grass carp, EF1α was the most suitable in CIK cell culture stimulated by poly(I:C) or infected by GCRV. As an assay, EF1α was employed to examine the changes of Toll-like receptor 3 (TLR3) and melanoma differentiation associated gene 5 (MDA5) after virus infection in CIK cells. These data laid the foundation for more precise results in qRT-PCR studies of gene expression in grass carp.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, its unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll--interleukin 1--receptor (TIR) motif. Its closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24h post-injection and lasted until the fish became moribund (P<0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella).

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases.

Identification of a retinoic acid-inducible gene I from grass carp (Ctenopharyngodon idella) and expression analysis in vivo and in vitro

RIG-I (retinoic acid inducible gene-I) is a key mediator of antiviral immunity, able to couple detection of infection by RNA and DNA viruses to the induction of interferons. In the present study, a RIG-I gene from grass carp Ctenopharyngodon idella (CiRIG-I) was isolated and characterized. The full-length cDNA of CiRIG-I was of 3198 bp and encoded a polypeptide of 947 amino acids with an estimated molecular mass of 108,730 Da and a predicted isoelectric point of 5.85, including six main overlapping structural domains: two CARDs (caspase activation and recruitment domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one DEXDc (DEAD/DEAH box helicase domain), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). The CiRIG-I mRNA was widespread expression in the tested 15 tissues by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiRIG-I expressions in spleen and liver were significantly induced following grass carp reovirus (GCRV) infection. CiRIG-I mRNA expression was rapidly and significantly up-regulated in vitro after GCRV infection, and the CiRIG-I transcripts were also significantly enhanced in vitro post the synthetic double stranded RNA polyinosinic-polycytidylic potassium salt (poly(I:C)) stimulation. These results collectively suggested that CiRIG-I was an inducible protein, involved in the antiviral innate immune defense to GCRV in grass carp, and laid the foundation for the further mechanism research of RIG-I in fishes.

Functional Characterizations of RIG-I to GCRV and Viral/ Bacterial PAMPs in Grass Carp Ctenopharyngodon idella

Background RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy. Methodology/Principal Findings To clarify the functional characterizations of RIG-I in grass carp Ctenopharyngodon idella (CiRIG-I), six representative overexpression plasmids were constructed and transfected into C. idella kidney (CIK) cell lines to obtain stably expressing recombinant proteins, respectively. A virus titer test and 96-well plate staining assay showed that all constructs exhibited the antiviral activity somewhat. The quantitative real-time RT-PCR (qRT-PCR) demonstrated that mRNA expressions of CiIPS-1, CiIFN-I and CiMx2 were regulated by not only virus (GCRV) or viral PAMP (poly(IC)) challenge but also bacterial PAMPs (LPS and PGN) stimulation in the steadily transfected cells. The results showed that the full-length CiRIG-I played a key role in RLR pathway. The repressor domain (RD) exerted an inhibitory function of the signaling channel under all utilized challenges. Caspase activation and recruitment domains (CARDs) showed a positive role in GCRV and poly(I:C) challenge. Helicase motifs were crucial for the signaling pathway upon LPS and PGN stimulation. Interestingly, ΔCARDs (CARDs deleted) showed postive modulation in RIG-I signal transduction. Conclusions/Significance The results provided some novel insights into RIG-I sensing with a strikingly broad regulation in teleosts, responding not only to the dsRNA virus or synthetic dsRNA but also bacterial PAMPs.

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P<0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases.

Identification and expression profiles of grass carp Ctenopharyngodon idella tlr7 in responses to double‐stranded RNA and virus infection

The toll-like receptor 7 (tlr7) gene was identified and characterized from grass carp Ctenopharyngodon idella (designated as citlr7), and the messenger (m)RNA expression profiles were examined in vivo and in vitro. The citlr7 genomic sequence consists of 4276 nucleotides (nts), including two exons and one intron. The full length of citlr7 complementary (c)DNA sequence is 3354 nts with the longest open reading frame (ORF) of 3156 nts encoding a peptide of 1051 amino acids. citlr7 mRNA expression was high in spleen, skin and heart, and low in hepatopancreas, muscle, head kidney and trunk kidney in healthy fish. The expression of citlr7 was rapidly and significantly up-regulated at 6 h after grass carp reovirus (GCRV) injection (72·91 fold, P < 0·05), and recovered to the original level at 24 h post-injection in the spleen. The citlr7 transcript was rapidly and significantly down-regulated at the 6 h time point (0·32 fold, P < 0·05) and retrieved the normal level at 72 h post-injection in the hepatopancreas. The citlr7 transcripts were rapidly and significantly inhibited at 2 h post-GCRV infection in the C. idella kidney (CIK) cell line (0·62 fold, P < 0·05), and were rapidly and significantly elevated 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 (P < 0·05). The results imply that citlr7 is involved in the responses to double-stranded RNA and virus infection.

Identification, expression profiling of a grass carp TLR8 and its inhibition leading to the resistance to reovirus in CIK cells

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 cells 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.

Molecular characterizations of grass carp (Ctenopharyngodon idella) TBK1 gene and its roles in regulating IFN-I pathway

TANK-binding kinase 1 (TBK1), a kinase at the crossroads of multiple IFN-inducing signaling pathways, plays essential roles in both antiviral and antibacterial innate immunity in mammals. Here, TBK1 gene (10339bp) was identified and characterized from grass carp Ctenopharyngodon idella (CiTBK1). The genomic sequence is shorter than other orthologs in vertebrate, and a promoter region is found in intron 1. mRNA expression of CiTBK1 was widespread in fifteen tissues investigated, and was up-regulated post GCRV challenge in vivo and in vitro, as well as after stimulation of viral/bacterial PAMPs in vitro. CiTBK1 mediates IFN-I signal pathway through over-expression experiment. Post GCRV challenge, CiTBK1 over-expression inhibits viral infection by induction of CiIFN-I and CiMx1 mainly via CiIRF7. In CiTBK1 over-expression cells, mRNA expressions of CiIRF3, CiIRF7 and CiIFN-I were inhibited, whereas CiMx1 was facilitated after poly I:C stimulation, comparing to those in control group. The result indicated that CiMx1 expression mediated by CiTBK1 is in IFN-I independent way after poly I:C stimulation. However, over-expression of CiTBK1 diminishes LPS-induced expressions of CiIRF3 and CiIRF7 but promotes the induction of CiIFN-I and CiMx1 in comparison with the control, which suggests that CiTBK1-triggered IFN-I activation is in IRF3/IRF7-independent manner after LPS stimulation. Notably, over-expression of CiTBK1 negatively regulated PGN-induced IRF3, IRF7, IFN-I and Mx1 immune response. Taken together, CiTBK1 participates in broad antiviral and antibacterial immune responses in different manners, and keeps regulatory balance that prevents harmful effects from excessive activation.

Characterizations of two grass carp Ctenopharyngodon idella HMGB2 genes and potential roles in innate immunity.

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.