Enterobacteriaceae as a Key Indicator of Huanglongbing Infection in Diaphorina citri.

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray-Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment.

The Ecological Differentiation of Particle-Attached and Free-Living Bacterial Communities in a Seasonal Flooding Lake-the Poyang Lake.

Particle-attached (PA) and free-living (FL) bacterial communities play essential roles in the biogeochemical cycling of essential nutrients in aquatic environments. However, little is known about the factors that drive the differentiation of bacterial lifestyles, especially in flooding lake systems. Here we assessed the compositional and functional similarities between the FL and PA bacterial fractions in a typical flooding lake-the Poyang Lake (PYL) of China. The results revealed that PA communities had significantly different compositions and functions from FL communities in every hydrological period, and the diversity of both PA and FL communities was affected mainly by the water regime rather than bacterial lifestyles. PA communities were more diverse and enriched with Proteobacteria and Bacteroidetes, while FL communities had more Actinobacteria. There was a higher abundance of photosynthetic and nitrogen-cycling bacterial groups in PA communities, but a higher abundance of members involved in hydrocarbon degradation, aromatic hydrocarbon degradation, and methylotrophy in FL communities. Water properties (e.g., temperature, pH, total phosphorus) significantly regulated the lifestyle variations of PA and FL bacteria in PYL. Collectively, our results have demonstrated a clear ecological differentiation of PA and FL bacterial communities in flooding lakes, suggesting that the connectivity between FL and PA bacterial fractions is water property-related rather than water regime-related.

Diet drives the gut microbiome composition and assembly processes in winter migratory birds in the Poyang Lake wetland, China.

The complex gut bacterial communities may facilitate the function, distribution, and diversity of birds. For migratory birds, long-distance traveling poses selection pressures on their gut microbiota, ultimately affecting the birds' health, fitness, ecology, and evolution. However, our understanding of mechanisms that underlie the assembly of the gut microbiome of migratory birds is limited. In this study, the gut microbiota of winter migratory birds in the Poyang Lake wetland was characterized using MiSeq sequencing of 16S rRNA genes. The sampled bird included herbivorous, carnivorous, and omnivorous birds from a total of 17 species of 8 families. Our results showed that the gut microbiota of migratory birds was dominated by four major bacterial phyla: Firmicutes (47.8%), Proteobacteria (18.2%), Fusobacteria (12.6%), and Bacteroidetes (9.1%). Dietary specialization outweighed the phylogeny of birds as an important factor governing the gut microbiome, mainly through regulating the deterministic processes of homogeneous selection and stochastic processes of homogeneous dispersal balance. Moreover, the omnivorous had more bacterial diversity than the herbivorous and carnivorous. Microbial networks for the gut microbiome of the herbivorous and carnivorous were less integrated, i.e., had lower average node degree and greater decreased network stability upon node attack removal than those of the omnivorous birds. Our findings advance the understanding of host-microbiota interactions and the evolution of migratory bird dietary flexibility and diversification.

A comparative analysis of COVID-19 vaccination certificates in 12 countries/regions around the world: Rationalising health policies for international travel and domestic social activities during the pandemic.

As the unprecedented pandemic of COVID-19 became a major barrier during the past two years, many countries were using the "COVID pass" or COVID-19 vaccination certificates in attempts to facilitate effective international travel and domestic social activities. The difficulty remained in how the "COVID pass" from different countries and regions could be mutually recognised. This study surveys the current practice of COVID-19 vaccination certificates in 12 representative countries/regions around the world and provides a comprehensive mapping of the vaccination certificates in these countries/regions. The study compares and contrasts the vaccination certificates in both format and content, including their primary purposes, international accreditation, naming conventions, recipients' personal information, and the details on vaccines and vaccination. The findings are interpreted in light of implementation practices in each country/region and discussed in relation to their various functions, as well as legal, technical, and ethical considerations. Based on the analysis and discussion recommendations are made on the practice of vaccination certificates in attempts to facilitate effective international travel and domestic social activities.

Automatic identification of respiratory events based on nasal airflow and respiratory effort of the chest and abdomen.

Objective.Disease may cause changes in an individual's respiratory pattern, which can be measured as parameters for disease evaluation, usually through manually annotated polysomnographic recordings. In this study, a machine learning model based on nasal airflow and respiratory effort of the chest and abdomen is proposed to automatically identify respiratory events, including normal breathing, hypopnea and apnea.Approach.The nasal airflow and chest-abdominal respiratory effort signals were collected by polysomnography (PSG). Time/frequency domain features, fractional Fourier transform features and sample entropy were calculated to obtain feature sets. Features selected through statistical analysis were used as input variables of the machine learning model. The performance of different input combinations on different models was studied and cross-validated.Main results.The dataset included PSG sleep records of 60 patients provided by the Chinese People's Liberation Army General Hospital. The extreme gradient boosting-based model (XGBoost) performed best in several models with an accuracy of 0.807 and a F1 score of 0.807, depending on the combination of nasal airflow and two respiratory effort signals. The precision for normal breathing, hypopnea and apnea events were 0.764, 0.789 and 0.871, respectively. In addition, the recall scores were 0.833, 0.768 and 0.823 for normal breathing, hypopnea and apnea events, respectively. Moreover, it was found that the standard deviation and kurtosis of nasal airflow were the most important features of the respiratory event detection model.Significance.Since nasal airflow and respiratory effort of the chest and abdomen contain the characteristics of respiratory events, their combined use can improve the classification performance for identification of respiratory events. With this method, respiratory events can be automatically detected and labeled from the PSG records, which can be used to screen for patients with sleep apnea-hypopnea syndrome.

Developing Interpreting Competence Scales in China.

Tertiary-level interpreter training and education have developed rapidly in China, and over 200 undergraduate and over 200 postgraduate T&I programs have been launched over the past decade. Despite the rapid development, there has been no standardized framework allowing for the reliable and valid measurement of interpreting competence in China. Against this background, the China Standards of English (CSE), which are the Chinese counterpart to the Common European Framework of Reference (CEFR), were unveiled in 2018 after 4 years of government-funded research and validation. One vital component of the CSE is the descriptor-referenced interpreting competence scales. This article provides a systematic account of the design, development, and validation of the interpreting competence scales in China. Within the CSE, the construct of interpreting competence was defined according to an interactionist approach. It not only encompasses cognitive abilities, interpreting strategies, and subject-matter knowledge but also considers performance in typical communicative settings. Based on the construct definition, a corpus of relevant descriptors was built from three main sources, including: (a) interpreting training syllabuses, curricular frameworks, rating scales, and professional codes of conduct; (b) previous literature on interpreting performance assessment, competence development, and interpreter training and education; and (c) exemplar-generation data on assessing interpreting competence and typical interpreting activities, which were collected from interpreting professionals, trainers, and trainees. The corpus contains 9,208 descriptors of interpreting competence. A mixed-method survey was then conducted to analyze, scale, and validate the descriptors among 30,682 students, 5,787 teachers, and 139 interpreting professionals from 28 provinces, municipalities, and regions in China. The finalized set included 369 descriptors that reference interpreting competence. The CSE-Interpreting Competence Scales with theoretically and empirically based descriptors represent a major effort in research on interpreting competence and its assessment, and they have significant potential to be applied widely in interpreting training, research, and assessment.

Structural characteristics of gonadal development and hermaphroditic phenomenon in freshwater pearl mussel, Sinohyriopisis schlegelii (Hyriopisis schlegelii).

The period of gonads development was first studied from one to five years in the freshwater pearl mussel Hyriopisis schlegelii. It lasted for 36 months and was divided into three main stages: initiation of gonad formation, a stable growth phase, and a reproductive cell development phase. Each reproductive cycle consisted of five stages: proliferative stage (from late January to late February), growth stage (from late February to late March), maturation stage, spawning stage (from early April to late October) and recovery stage (from early November to late January). Interestingly, a hermaphroditic phenomenon was observed in this mussel for the first time, which appears during the development stage from 26 to 32 months. Male and female follicular tissues coexisted in hermaphrodite individuals with the male follicular tissue accounting for more than 90% of the whole gonad tissue. No hermaphroditic phenomenon was observed in matured gonad. We thus speculate that self-fertilization does not exist in H. schlegelii.

Transcriptome data analysis of grass carp (Ctenopharyngodon idella) infected by reovirus provides insights into two immune-related genes.

Grass carp (Ctenopharyngodon idella) was one of the economically important freshwater fish in China. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in a tremendous loss in the process of grass carp cultivation. Transcriptome analysis could provide a comprehensive understanding of the molecular mechanisms involved in specific biological processes and diseases for the resistance to reovirus infection of grass carp. In this study, the raw data from NCBI (accession number: SRA099702) were analyzed, in which, 50 significant differentially expressed genes by routine transcriptome analysis and 84 notably differentially expressed genes by co-expression network method. KEGG analysis revealed that the pathway in hemorrhagic diseases in grass carp was similar to the influenza A induced pathway. The interferon-stimulated gene ISG15 and sacsin-like gene, which were up-regulated in data (SRA099702), were also up-regulated in data (SRP049081) from a similar assay. QPCR experiment was performed to validate these up-regulated genes. The ISG15 gene was shown to be the core gene in the co-expression network. The results would enhance our understanding of the antivirus system of grass carp infected by reovirus.

Poly I:C facilitates the phosphorylation of Ctenopharyngodon idellus type I IFN receptor subunits and JAK kinase.

Members of the Janus kinase (JAK) family, JAK1 and TYK2 take part in JAK-STAT signaling pathway mediated by interferon in mammalian cells. Similar to the mammalian counterparts, fish JAK1 and TYK2 also perform their potential biological activities by phosphorylating cytokine receptors and STAT. In the present study, Ctenopharyngodon idellus JAK1 (CiJAK1) and TYK2 (CiTYK2) were cloned and identified. The full-length cDNA of CiJAK1 (KT724352.1) is 3829 bp, with an Open Reading Frame (ORF) of 3465 bp encoding a putative protein of 1154 amino acids. The full-length cDNA of CiTYK2 (KT724353.1) is 4337 bp, including an ORF of 3168 bp encoding 1055 amino acids. Structurally, both of them have B41, SH2, TyrKc and TyrKc common domains. CiJAK1 and CiTYK2 share a high degree of homology with their respective counterparts from Danio rerio and Cyprinus carpio by phylogenetic tree analysis. Polyinosinic-polycytidylic acid (Poly I:C), a synthetic dsRNA analogue, can launch the JAK-STAT antiviral signaling pathway. To elucidate the molecular mechanism of Poly I:C initiating the antiviral signaling pathway in fish, C. idellus kidney (CIK) cells were stimulated with Poly I:C and then the cell lysates were separated on 10% SDS-PAGE. The results showed that not only Poly I:C drastically increased the expression level of CiJAK1 and CiTYK2, but also it induced the phosphorylation of CiJAK1 and CiTYK2, as well as C. idellus type I IFN receptor subunits, CiCRFB1 and CiCRFB5. In detail, the levels of p-CiJAK1 and p-CiTYK2 were evidently up-regulated at 3 h post stimulation; however the phosphorylation levels of CiCRFB1 and CiCRFB5 displayed a sharp up-regulation at 12 h post stimulation of Poly I:C. As a basic mechnism of feedback regulation of JAK-STAT signaling pathway, overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2.

Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional regulatory mechanism, we deduced that CiADAR1a shared a high degree of homology with mammalian ADAR1-p110.

Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate in vitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.

Classification of Pelteobagrus fish in Poyang Lake based on mitochondrial COI gene sequence.

We use DNA molecular marker technology to correct the deficiency of traditional morphological taxonomy. Totality 770 Pelteobagrus fish from Poyang Lake were collected. After preliminary morphological classification, random selected eight samples in each species for DNA extraction. Mitochondrial COI gene sequence was cloned with universal primers and sequenced. The results showed that there are four species of Pelteobagrus living in Poyang Lake. The average of intraspecific genetic distance value was 0.003, while the average interspecific genetic distance was 0.128. The interspecific genetic distance is far more than intraspecific genetic distance. Besides, phylogenetic tree analysis revealed that molecular systematics was in accord with morphological classification. It indicated that COI gene is an effective DNA molecular marker in Pelteobagrus classification. Surprisingly, the intraspecific difference of some individuals (P. e6, P. n6, P. e5, and P. v4) from their original named exceeded species threshold (2%), which should be renewedly classified into Pelteobagrus fulvidraco. However, another individual P. v3 was very different, because its genetic distance was over 8.4% difference from original named Pelteobagrus vachelli. Its taxonomic status remained to be further studied.

Mitochondrial genome of Paracheilognathus imberbis (Cypriniformes: Cyprinidae: Acheilognathinae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Paracheilognathus imberbis (Cypriniformes: Cyprinidae: Acheilognathinae) was determined by long PCR and primer-walking methods. The complete mitochondrial genome is 16,819 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(29.73%), T(27.25%), C(26.63%) and G(17.40%), respectively. The mitogenome of P. imberbis displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.

Complete mitochondrial genome of Rhodeus lighti (Cypriniformes: Cyprinidae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Rhodeus lighti (Cypriniformes: Cyprinidae) was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,677 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A (28.87%), T (27.22%), C (26.53%) and G (17.38%). The mitogenome of R. lighti displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.

The complete mitochondrial genome of Coilia brachygnathus (Clupeiformes: Engraulidae: Coilinae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Coilia brachygnathus (Clupeiformes: Engraulidae) was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,677 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(31.21%), T(26.25%), C(27.06%), G(15.48%). The mitogenome of C. brachygnathus displayed novel gene order arrangement compared with published Coilia ectenes to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Coilinae.

Complete mitochondrial genome of Gallus domesticus (Galliformes: Phasianidae).

In this paper, the complete mitochondrial genome (mitogenome) sequence of Gallus domesticus was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,783 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(30.25%), T(23.79%), C(32.44%), G(13.52%), respectively. The mitogenome of G. domesticus displayed novel gene order arrangement compared with published Gallus gallus var. domesticus to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Gallus.

A splicing isoform of Ctenopharyngodon idella ADAR1 (CiADAR1-like): Genome organization, tissue specific expression and transcriptional regulation.

Catalyzing the deamination of adenosine to inosine in RNA, ADAR1 (adenosine deaminase that act on RNA 1) belongs to ADAR family. In our previous work, we have cloned the complete genomic sequence of ADAR1 from grass carp (Ctenopharyngodon idella), named CiADAR1. In the process, we found a splicing isoform of CiADAR1 (CiADAR1-like). CiADAR1 and CiADAR1-like are possessed by different promoters but share a common exon 2. The complete genomic CiADAR1-like has 9 exons and 8 introns. Its full-length cDNA is comprised of a 5' UTR (417 bp), a 3' UTR (118 bp) and a 3324 bp-long ORF encoding a polypeptide of 1107 amino acids. The deduced amino acid sequence of CiADAR1-like contains two Z-DNA binding domains, three dsRNA binding motifs and a truncate catalytic domain. CiADAR1-like shared higher homology with Danio rerio ADAR1 and lower homology with HsADAR1-like in phylogenetic tree. qRT-PCR showed that CiADAR1-like were ubiquitously expressed and significantly up-regulated after stimulation with Poly I:C. Its mRNA reached the peak at 12 h post-stimulation in all tested tissues. Western-blotting experiment proved CiADAR1-like was factually expressed in C. idella kidney (CIK) cells. To further study the transcriptional regulatory mechanism of CiADAR1-like, we cloned its promoter sequence. CiADAR1-like promoter is 1173 bp in length containing 3 ISRE and 8 IRF-E. Subsequently, grass carp IRF1 (CiIRF1) and IRF3 (CiIRF3) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, CiIRF1 and CiIRF3 were able to bind to CiADAR1-like promoter with high affinity in gel mobility shift assays, revealing that IRF1 and IRF3 could be the potential transcriptional regulatory factors for CiADAR1-like. In vivo, Co-transfection of pcDNA3.1-IRF1 (or pcDNA3.1-IRF3) with pGL3-CiADAR1-like promoter into CIK cells showed that both IRF1 and IRF3 significantly increased the luciferase activity, suggesting that they play a positive role in CiADAR1-like transcription.

Cloning, identification of the two cytokine receptor family B subunits CRFB1 and CRFB5 from grass carp (Ctenopharyngodon idella).

Similar to the mammalian counterparts, fish type I interferon (IFN) performs its potential biological activities via binding to the corresponding receptor on target cell membrane. Fish type I IFN receptor, a kind of enzyme-linked receptor, consists of two subunits and belongs to the class II cytokine receptor family B (CRFB). In the present study, we cloned and identified two putative grass carp (Ctenopharyngodon idella) type I interferon receptor subunits (termed CiCRFB1 and CiCRFB5) by homology cloning techniques. Phylogenetic tree analysis suggested that CiCRFB1 and CiCRFB5 shared highly homology to Danio rerio CRFB1 and CRFB5 respectively. CiCRFB1 and CiCRFB5 were up-regulated after the stimulation with Grass Carp Hemorrhagic Virus (GCHV) and Polyinosinic-polycytidylic acid (Poly I:C), indicating that they are related to the intracellular antiviral activity. In order to know more about the roles of CiCRFB1 and CiCRFB5 in the process, the extracellular domains of CiCRFB1 (CiCRFB1-EC) and CiCRFB5 (CiCRFB5-EC), as well as grass carp type I IFN (CiIFN) were expressed in Escherichia coli BL21, and purified by affinity chromatography with the Ni-NTA His-Bind resin. Cross-linking reactions were employed to analyze the affinity of the ligand (CiIFN) with the two putative receptor subunits (CiCRFB1-EC and CiCRFB5-EC). The result suggested the formation of (CiCRFB5)2 homodimer was more easily than that of (CiCRFB1)2 under the induction of CiIFN in vitro. However, CiIFN was inclined to bind to (CiCRFB1)2 homodimer. Interestingly, although CiIFN seemed unable to facilitate the formation of (CiCRFB1 + CiCRFB5) heterodimer in the absence of DSS cross linker, however it can bind to the heterodimer in the presence of DSS. This indicated that the homodimer and the heterodimer were the potential receptor for CiIFN.

Genome organization and transcriptional regulation of Adenosine Deaminase Acting on RNA gene 1 (ADAR1) in grass carp (Ctenopharyngodon idella).

ADAR1, involved in A-to-I RNA editing, belongs to adenosine deaminase acting on RNA (ADAR) family. A-to-I RNA editing is the most widespread editing phenomenon in higher eukaryotes. In the present study, we cloned and identified the full-length cDNA, complete genomic sequence and the promoter sequence of grass carp (Ctenopharyngodon idella) ADAR1 (CiADAR1) by homology cloning strategy and genome walking. CiADAR1 full-length cDNA is comprised of a 5'UTR (43 bp), a 3'UTR (229 bp) and a 4179 bp ORF encoding a polypeptide of 1392 amino acids. The deduced amino acid sequence of CiADAR1 contains two Z-DNA binding domains, three dsRNA binding motifs and a conserved catalytic domain. The complete genomic CiADAR1 has 16 exons and 15 introns. Phylogenetic tree analysis revealed that CiADAR1 shared high homology with Danio rerio ADAR1 (DrADAR1). RT-PCR showed that CiADAR1 were ubiquitously expressed and significantly up-regulated after stimulation with poly I:C. In spleen and liver, CiADAR1 mRNA reached the peak at 12 h and maintained the highest level during 12-24 h post-injection. CiADAR1 promoter was found to be 1102 bp in length and divided into two distinct regions, the proximal region containing three putative interferon regulatory factor binding elements (IRF-E) and the distal region containing only one IRF-E. To further study the transcriptional regulatory mechanism of CiADAR1, grass carp IRF1 (CiIRF1) and IRF3 (CiIRF3) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Then, gel mobility shift assay was employed to analyze the affinity of CiADAR1 promoter sequence with CiIRF1 and CiIRF3 in vitro. The result revealed that CiIRF1 and CiIRF3 bound to CiADAR1 promoter with high affinity, indicating that IRF1 and IRF3 could be the potential transcriptional regulatory factor for CiADAR1. Co-transfection of pcDNA3.1-IRF1 (or pcDNA3.1-IRF3) with pGL3-CiADAR1 into C. idella kidney (CIK) cells showed that both IRF1 and IRF3 played a positive role in CiADAR1 transcription. In addition, the mutant assay revealed that the proximal region of CiADAR1 promoter is the main regulatory region in CiADAR1 transcription.

Ctenopharyngodon idella IRF2 plays an antagonistic role to IRF1 in transcriptional regulation of IFN and ISG genes.

Interferon Regulatory Factors (IRFs) make up a family of transcription factors involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF2 gene (termed CiIRF2, JX628585) was cloned and characterized from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiIRF2 is 1809 bp in length, with the largest open reading frame (ORF) of 981 bp encoding a putative protein of 326 amino acids. CiIRF2 contains a conserved DNA-binding domain (DBD) in N-terminal and a non-conserved C-terminal region. Protein sequence analysis revealed that CiIRF2 shares significant homology to the known IRF2 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with zebra fish IRF2. CiIRF2 was ubiquitously expressed at low level in all tested grass carp tissues and significantly up-regulated except in brain following poly I:C 6-12 h post stimulation. In order to understand fish innate immune and resistance to virus diseases, recombinant CiIRF2 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. Promoter sequences of grass carp type I IFN gene (CiIFN) and two ISG genes (CiPKR and CiPKZ) were amplified and cloned. In vitro, gel mobility shift assays were employed to analyze the interaction of CiIRF2 protein with promoters of CiIFN, CiPKR and CiPKZ respectively. The results showed that CiIRF2 bound to these promoters with high affinity by means of its DBD. Afterwards, recombinant plasmids of pGL3-CiIFN, pGL3-CiPKR and pGL3-CiPKZ were constructed and transiently co-transfected with pcDNA3.1-CiIRF2 or pcDNA3.1-CiIRF1 respectively into C. idella kidney (CIK) cells. Dual-luciferase reporter assays demonstrated that CiIRF2 down-regulates the transcription activity of CiIFN, CiPKR and CiPKZ genes in CIK cells. To further understand the function of fish IRF2, expression plasmids (pcDNA3.1-IRF2 and pcDNA3.1-IRF1) were transiently co-transfected with pGL3-IFN or pGL3-CiPKZ into CIK cells, respectively. The results revealed that CiIRF2 plays an antagonistic role to CiIRF1 in transcriptional regulation of IFN and ISG genes.