Qiuming Liu

Cloning and expression analysis of interferon regulatory factor 7 (IRF-7) in turbot, Scophthalmus maximus.

Interferon regulatory factor (IRF) 7 is known as the master regulator of type I interferon (IFN)-dependent immune responses in mammals. In this study, the cDNA and genomic sequences of turbot (Scophthalmus maximus) IRF-7 (SmIRF-7) were cloned and found to encode a putative protein of 439 amino acids. The gene is composed of 10 exons and 9 introns similar to known IRF-7 genes of fish. The SmIRF-7 shows the highest amino acid identity of 49.0-80.3% to fish IRF-7 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) of vertebrate IRF-7. In addition, the tryptophan cluster of SmIRF-7 DBD consists of only four tryptophans, which is a characteristic unique to all fish IRF-7 members. The SmIRF-7 transcripts were expressed constitutively in all analyzed tissues of healthy turbot, with higher levels observed in immune relevant tissues. Gene expressions of SmIRF-7 and Mx were monitored over a 7-day time course by quantitative real time PCR in head kidney and muscle of turbot challenged with turbot reddish body iridovirus (TRBIV), which is a prevalent viral pathogens in farmed turbot in China. Both genes were up-regulated by TRBIV although their inducibility was much weaker in the muscle. The peak levels of SmIRF-7 transcripts were detected at day 2 post-infection in the two organs with a 12- and 4.5-fold increase, respectively. Further, the Mx showed two waves of induced expression and the maximum expression of SmIRF-7 arose earlier than the second wave of the Mx expression in both organs. These findings contribute to an understanding of functions of SmIRF-7 in antiviral response.

An IRF-3 homolog that is up-regulated by DNA virus and poly I:C in turbot, Scophthalmus maximus

In this study, we described the structure, mRNA tissue distribution and regulation of an IRF-3 gene from turbot, Scophthalmus maximus (SmIRF-3). The gene sequence of SmIRF-3 is 6077 bp long, composed of 11 exons and 10 introns similar to known IRF-3 genes of fish, and encodes a peptide of 466 amino acids. The deduced protein sequence shares the highest identity of 56.0-81.2% with fish IRF-3 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) known to be important for the functions of IRF-3 in vertebrates. Phylogenetic analysis grouped SmIRF-3 with other IRF3s of vertebrates. SmIRF-3 transcripts were detectable in limited tissue types of healthy fish, with higher expression observed in head, kidney, spleen and kidney,. The SmIRF-3 was transcriptionally up-regulated by turbot reddish body iridovirus (TRBIV) and polyinosinic:polycytidylic acid (poly I:C) in the head kidney, spleen and gills, with showing a two wave induced expression during a 7-day time course in all cases. The highest inducibility and the likely earliest increase of SmIRF-3 expression were observed in the spleen, and poly I:C was a stronger inducer. In addition, the maximal expression level of SmIRF-3 arose prior to that of the Mx in all the cases.

Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus).

Myeloid differentiation factor 88 (MyD88) is an adapter protein involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB). In this study, a full length cDNA of MyD88 was cloned from turbot, Scophthalmus maximus. It is 1619 bp in length and contains an 858-bp open reading frame that encodes a peptide of 285 amino acid residues. The putative turbot (Sm)MyD88 protein possesses a N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain known to be important for the functions of MyD88 in mammals. Phylogenetic analysis grouped SmMyD88 with other fish MyD88s. SmMyD88 mRNA was ubiquitously expressed in all examined tissues of healthy turbots, with higher levels observed in immune-relevant organs. To explore the role of SmMyD88, its gene expression profile in response to stimulation of lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV) was studied in the head kidney, spleen, gills and muscle over a 7-day time course. The results showed an up-regulation of SmMyD88 transcript levels by the three immunostimulants in all four examined tissues, with the induction by CpG-ODN strongest and initiated earliest and inducibility in the muscle very weak. Additionally, TRBIV challenge resulted in a quite high level of SmMyD88 expression in the spleen, whereas the two synthetic immunostimulants induced the higher levels in the head kidney. These data provide insights into the roles of SmMyD88 in the TLR/IL-1R signaling pathway of the innate immune system in turbot.

Characteristics of the interferon regulatory factor 5 (IRF5) and its expression in response to LCDV and poly I:C challenges in Japanese flounder, Paralichthys olivaceus

Interferon regulatory factor 5 (IRF5) has been identified as a key transcriptional mediator regulating expression of both type I interferons (IFNs) and proinflammatory cytokines. In this study, the cDNA and genomic sequences of IRF5 were isolated from Japanese flounder, Paralichthys olivaceus. The gene of Japanese flounder (Jf)IRF5 is 7326 bp long, contains 9 exons and 8 introns and encodes a putative protein of 472 amino acids. The predicted protein sequence shares 61.1-81.9% identity to fish IRF5 and possesses a DNA-binding domain (DBD), a middle region (MR), an IRF association domain (IAD), a virus activated domain (VAD) and two nuclear localization signals (NLSs) conserved in all known IRF5s. Phylogenetic analysis clustered it into the teleost IRF5 subgroup within vertebrate IRF5 group. JfIRF5 mRNA was constitutively expressed in all tissues examined, with higher levels observed in the gills and head kidney. Gene expression of JfIRF5 was analyzed over a 7-day time course in the gills, head kidney, spleen and muscle of Japanese flounders challenged with lymphocystis disease virus (LCDV) and polyinosinic:polycytidylic acid (poly I:C). The data showed that JfIRF5 expression was slightly up-regulated by LCDV, but its induction time was clearly moved up; in contrast, the induction upon poly I:C challenge started not earlier than day 2 post-injection and was stronger and more persistent with a later peak time in all four organs. The late and long-lasting inductive expression of JfIRF5 following poly I:C challenge suggests that it might be an interferon stimulated gene (ISG), the induction of which is driven by poly I:C-induced type I IFNs.

Molecular cloning and expression analysis of interferon regulatory factor 8 (IRF8) in turbot, Scophthalmus maximus

Interferon regulatory factor 8 (IRF8) in mammals is known to be involved in antiviral response. In this study, the gene of IRF8 was cloned from the turbot (Scophthalmus maximus) fish and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and turbot reddish body irrdovirus (TRBIV) challenges was studied. Turbot (Sm)IRF8 gene is 4363bp long, comprises nine exons and eight introns and encodes a putative 420 amino acid (aa) protein. The predicted protein sequence possesses a DNA binding domain (DBD), an IRF association domain (IAD) and a nuclear localization signal (NLS). Constitutive expression of SmIRF8 was detectable in all tested organs, with higher levels observed in the spleen, kidney and head kidney. SmIRF8 transcript levels were up-regulated by both poly I:C and TRBIV treatments in the spleen, head kidney, gills and muscle in an early phase of a 7-day time course and the poly I:C was a quicker inducer. In both challenge cases, the highest and earliest inductions were detected in the spleen, while the induction in the muscle was quite faint. These results provide insights into the role of SmIRF8 in antiviral response.

Molecular cloning and expression analysis of interferon stimulated gene 15 (ISG15) in turbot, Scophthalmus maximus.

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity.

Cloning and expression analysis of a Toll-like receptor 21 (TLR21) gene from turbot, Scophthalmus maximus

&NA; Toll‐like receptor 21 (TLR21) is a non‐mammalian TLR recognizing unmethylated CpG DNA and considered as a functional homolog of mammalian TLR9. In the present study, a TLR21 gene was cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (Sm)TLR21 gene is an intronless gene with a length of 3527 bp and encodes a peptide of 984 amino acids. The deduced protein possesses a signal peptide sequence, a leucine‐rich repeat (LRR) domain composed of 16 LRR motifs, a transmembrane (TM) region and a Toll/interleukin‐1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost TLR21s. Quantitative real‐time PCR (qPCR) analysis demonstrated the constitutive expression of SmTLR21 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid‐rich tissues like spleen and head kidney. Further, upon stimulation with polyinosinic: polycytidylic acid [poly(I:C)], turbot reddish body iridovirus (TRBIV) and CpG oligodeoxynucleotides (CpG‐ODN) 2395, the SmTLR21 mRNA expression was up‐regulated in the gills, head kidney, spleen and muscle. The maximum increases of SmTLR21 transcript levels ranged from 1.3 to 8.1‐fold and appeared at 3 h to 5 day post‐injection depending on different organs and stimuli. These findings suggest that SmTLR21 may play an important role in the immune responses to the infections of a broad range of pathogens that include RNA and DNA viruses and bacteria. HighlightsA TLR21 gene was cloned from turbot.SmTLR21 transcripts distributed in all tissues examined with higher levels observed in lymphomyeloid rich tissues.SmTLR21 expression was up‐regulated by poly(I:C) and TRBIV in immune and non‐immune organs.CpG‐ODN 2395 up‐regulated SmTLR21 expression only in a late phase of the treatment in some organs.

Cloning and expression study of an IRF4a gene and its two transcript variants in turbot, Scophthalmus maximus

ABSTRACT Interferon regulatory factor 4 (IRF4) is known to be involved in antiviral response as well as regulation of functional and developmental processes in lymphomyeloid cell lineages in mammals. In this study, the gene of IRF4a and its two transcript variants (named IRF4a1 and ‐2) were cloned from turbot, Scophthalmus maximus, the tissue distributions and in vivo immune responsive expression patterns of the two transcripts were subsequently examined. The Scophthalmus maximus (Sm)IRF4a gene is 8367 nucleotide (nt) in length, consisting of eight exons and seven introns. The SmIRF4a1 transcript is 3185 nt long, containing an open reading frame (ORF) of 1401 nt that encodes a polypeptide of 466 amino acids (aa). The SmIRF4a2 transcript is 2265 nt long and identical with the SmIRF4a1 from position 1 to 1171, containing an ORF of 1164 nt that encodes a truncated protein of 387 aa as a result of a frame shift in exon 6 which introduces a premature stop codon. The deduced aa sequence of SmIRF4a1 posses a DNA‐binding domain (DBD), a nuclear localization signal (NLS), a serine‐rich domain (SRD) and an IRF association domain (IAD), while SmIRF4a2 lacks the C‐terminal 52 residues of the IAD and the downstream C‐terminal extension, instead, they are replaced by a 8‐aa segment although the three upstream domains are intact. Quantitative real‐time PCR analysis revealed a broad tissue expression for both SmIRF4a1 and ‐2 with the former showing a significantly higher expression in all examined tissues except skin. Expressions of two transcript variants after stimulation with polyinosinic:polycytidylic acid [poly(I:C)] and turbot reddish body iridovirus (TRBIV) were tested in gills, spleen, head kidney and muscle. A two‐wave of induced expression pattern was observed for both transcripts with either stimulus treatment during a 7‐day time course. SmIRF4a2 responded more promptly to the stimuli and showed a higher level of inducibility in the early phase while SmIRF4a1 was strongly detected in the later phase. These data suggest an important role of SmIRF4a2 in the fast immune response under a background of SmIRF4a1‐dominant antiviral response in the IRF4a system of turbot. HighlightsAn IRF4a gene and its two mRNA splicing variants were isolated from turbot.SmIRF4a1 was a dominant form in most organs except skin.Double expression peaks were detected for the two SmIRF4as upon treatment with viral stimuli.SmIRF4a2 responded more promptly to the viral stimuli with a higher inducibility in the early phase.