Characterization of toll-like receptor 1 (TLR1) in turbot (Scophthalmus maximus L.).

TLRs are the first and best-characterized pattern recognition receptors conserved across all the species. Different from mammals, the TLRs in teleost fishes are very diversified due to various evolutionary mechanisms. Here, we characterized one TLR1 gene in turbot, with a 2,415 bp open reading frame (ORF), that encoding 804 amino acid residues, and have the highest similarity and identity both to Paralichthys olivaceus with 88.9% and 79.9%. In phylogenetic analysis, it was firstly clustered with P. olivaceus, and then clustered with Takifugu rubripes. TLR1 was widely expressed in all the examined healthy tissues with the highest expression level in spleen, followed by head-kidney. In addition, it was significantly regulated in gill, skin and intestine following Edwardsiella tarda and Vibrio anguillarum challenge with different expression patterns. In in vitro stimulation with pathogen-associated molecular patterns, TLR1 showed significantly strong and elevated responses to LPS, but only responded to LTA and Poly(I:C) at the highest evaluated concentration, while no response was detected using PGN stimulation. Moreover, in subcellular localization analysis, TLR1 was distributed in the cytoplasm, membrane and nucleus. Taken together, TLR1 played vital roles for host immune response to bacterial infection, only with strong binding ability to LPS and involved in the production of inflammatory cytokines. However, the specific ligand for TLR1 and its functional association with other TLRs should be further characterized in fish species.

Genetic characterization of Porcine circovirus type 2 from 2013 to 2018 in Shandong Province, China.

PCV2 belongs to the genus Circovirus, family Circoviridae, who is recognized as the causative agents of postweaning multisystemic wasting syndrome. Since being found to China in 2000, it has caused serious damage to the pig industry. In this study, we downloaded 40 PCV2 genome-wide sequences uploaded to GenBank from 2013 to 2018 in Shandong Province, including 23 uploaded by our laboratory. Construction of a genome-wide evolution tree using MEGA V5.0 software. Phylogenetic tree analysis indicated that the genotype of PCV2 in Shandong Province was: three genotypes coexisted (2a, 2b, 2d); among them, PCV2d has become the main genotype in the province due to its number and spread range. Amino acid sequence analysis of different genotypes of ORF2 showed that specific amino acid sites exist in different genotypes, with the most significant range of 81-160; different genotypes of PCV2 can be distinguished at the molecular level. This study found that due to the increase in infections of the PCV2d genotype in recent years, it may replace PCV2b as the dominant base in Shandong.

Marek's disease viruses circulating in commercial poultry in Italy in the years 2015-2018 are closely related by their meq gene phylogeny.

Marek's disease (MD) is a lymphoproliferative disease important to the poultry industry worldwide; it is caused by Gallid alphaherpesvirus 2 (GaHV-2). The virulence of GaHV-2 isolates has shifted over the years from mild to virulent, very virulent and very virulent +. Nowadays the disease is controlled by vaccination, but field strains of increased virulence are emerging worldwide. Economic losses due to MD are mostly associated with its acute form, characterized by visceral lymphomas. The present study aimed to molecularly classify a group of 13 GaHV-2 strains detected in vaccinated Italian commercial chicken flocks during acute MD outbreaks, and to scrutinize the ability of predicting GaHV-2 virulence, according to the meq gene sequence. The full-length meq genes were amplified, and the obtained amino acid (aa) sequences were analysed, focusing mainly on the number of stretches of four proline molecules (PPPP) within the transactivation domain. Phylogenetic analysis was carried out with the Maximum Likelihood method using the obtained aa sequences, and the sequences of Italian strains detected in backyard flocks and of selected strains retrieved from GenBank. All the analysed strains showed 100% sequence identity in the meq gene, which encodes a Meq protein of 339 aa. The Meq protein includes four PPPP motifs in the transactivation domain and an interruption of a PPPP motif due to a proline-to-serine substitution at position 218. These features are typically encountered in highly virulent isolates. Phylogenetic analysis revealed that the analysed strains belonged to a cluster that includes high-virulence GaHV-2 strains detected in Italian backyard flocks and a hypervirulent Polish strain. Our results support the hypothesis that the virulence of field isolates can be suggested by meq aa sequence analysis.

Multiple subtypes of TLR22 molecule from Schizothorax prenanti present the functional diversity in ligand recognition and signal activation.

Evolutionary development has increased the diversity of genotypes and the complexity of gene functions in fish. TLR22 has been identified as a teleost-specific gene, but its functions are tremendously different among different fish species. Whether the functional diversity relates to the difference of genotypes remains poorly understand. In this study, we cloned and identified three TLR22 molecules from Schizothorax prenanti (S. prenanti), named as spTLR22-1, spTLR22-2 and spTLR22-3. The full-length coding regions of spTLR22s are 2841 bp, 2805 bp and 2868 bp and coding 946 aa, 934 aa and 955 aa, respectively. All spTLR22s are composed of multiple leucine-rich repeat (LRR) domains, a transmembrane structure and a Toll/IL-1 receptor (TIR) region. The phylogenetic analysis showed that three spTLR22s were close to Cyprinus carpio TLR22-1, TLR22-2 and TLR22-3, respectively. Among the spTLR22s, they presented not close relationship but remained to belong to TLR22 subfamily. All spTLR22s were ubiquitously expressed in all tested tissues, but the expression levels of spTLR22s were dominant in immune-related tissues, such as gill and spleen. The expression levels of spTLR22-1 and spTLR22-3 were significantly increased after treatment with bacteria, LPS and Poly(I:C). However, spTLR22-2 seems like no response to these treatments. The luciferase reporter assay demonstrated that all spTLR22s could activate NF-κB signaling pathway, but only spTLR22-1 and spTLR22-2 could activate IFN-ß signaling pathway. Interestingly, in the ligand recognition analysis, spTLR22-1 and spTLR22-3 but not spTLR22-2 had the recognized potential to Poly(I:C), and all spTLR22s could not recognize LPS. Both spTLR22-1 and spTLR22-3 significantly up-regulated the expression of anti-viral-related genes (Mx, IFN and ISG15) and down-regulated the expression of anti-inflammatory factor IL-10 after the overexpression in carp EPC cell line, but spTLR22-2 failed to impact the expression of these genes. Moreover, we found that all spTLR22s localized to the intracellular region. Taken together, our results reveal that spTLR22-1 and spTLR22-3 but not spTLR22-2 may be involved into the anti-viral immune response via IFN-ß signaling pathway, and all spTLR22s can activate NF-κB signaling pathway but only spTLR22-1 and spTLR22-3 response to the stimulation of bacteria and LPS.

Effect of CRISPR/Cas9-mediated knockout of either Mx1 or ISG15 gene in EPC cells on resistance against VHSV infection.

Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.

Identification and expression analysis of suppressors of cytokine signaling (SOCS) from soiny mullet (Liza haematocheila).

The suppressor of cytokine signaling (SOCS) family members play crucial roles in regulating immune signal pathways by acting as inhibitors of cytokine receptor signaling. In this study, 10 SOCS genes were identified in soiny mullet (Liza haematocheila), an economically important aquaculture mugilid species in China and other Asian countries. Sequence comparison showed that the sequence identity between mullet SOCSs and their counterparts from other vertebrates ranged from 38.2% to 92.5%. All mullet SOCS genes were constitutively expressed in tissues examined, but their expression patterns were different. Further, following Streptococcus dysgalactiae infection, all mullet SOCS genes exhibited distinct expression patterns in tissues. These results suggest that SOCSs are involved in immune response to bacterial infection and provide the basis for understanding the complex cytokine regulatory network of teleosts.

Molecular characterization, pathogenicity, and protection efficacy analysis of 2 wild-type lentogenic class I Newcastle disease viruses from chickens in China.

In this study, 2 wild-type Newcastle disease viruses (NDVs), designated as CK/GX/65/15 and CK/GX/26/15, were isolated from asymptomatic chickens in Guangxi province, China. They were identified as lentogenic NDV with mean death time (MDT) above 90 and intracerebral pathogenicity index (ICPI) below 0.7. The results of complete genome sequence analysis show that the 2 NDV strains are members of class I genotype 3 with the length 15,198 nt, which followed the "rule of six" and the order 3'-NP-P-M-F-HN-L-5'. In addition, 8 amino acid substitutions were identified in the functional domains of fusion protein (F) of CK/GX/65/15 and 9 in CK/GX/26/15, whose amino acid sequences of F protein cleavage site are 112E-R-Q-E-R-L117. The isolates were found to be apathogenic in specific pathogen free (SPF) chickens and ducks without morbidity or mortality. Furthermore, the protection study shows that isolates can provide the same effective protection against a major NDV virulent strain in China (class II genotype VII) as the commercial vaccine LaSota. Moreover, vaccination with isolates reduced number of chickens shedding virus compared to those vaccinated with LaSota. In conclusion, 2 wild-type NDV strains exhibited fine protection efficacy against genotype VII NDV in poultry and can be considered as candidate vaccines against NDV.

Mutations in voltage-gated sodium channels from pyrethroid resistant salmon lice (Lepeophtheirus salmonis).

BACKGROUND: Parasitic salmon lice (Lepeophtheirus salmonis) cause high economic losses in Atlantic salmon farming. Pyrethroids, which block arthropod voltage-gated sodium channels (Nav 1), are used for salmon delousing. However, pyrethroid resistance is common in L. salmonis. The present study characterized Nav 1 homologues in L. salmonis in order to identify channel mutations associated to resistance, called kdr (knockdown) mutations. RESULTS: Genome scans identified three L. salmonis Nav 1 homologues, LsNav 1.1, LsNav 1.2 and LsNav 1.3. Arthropod kdr mutations map to specific Nav 1 regions within domains DI-III, namely segments S5 and S6 and the linker helix connecting S4 and S5. The above channel regions were amplified by RT-PCR and sequenced in deltamethrin-susceptible and deltamethrin-resistant L. salmonis. While LsNav 1.1 and LsNav 1.2 lacked nucleotide polymorphisms showing association to resistance, LsNav 1.3 showed a non-synonymous mutation in S5 of DII occurring in deltamethrin-resistant parasites. The mutation is homologous to a previously described kdr mutation (I936V, numbering according to Musca domestica Vssc1) and was present in two pyrethroid-resistant L. salmonis strains (allele frequencies of 0.800 and 0.357), but absent in two pyrethroid-susceptible strains. CONCLUSIONS: The present study indicates that a kdr-mutation in LsNaV 1.3 may contribute to deltamethrin resistance in L. salmonis. © 2018 Society of Chemical Industry.

Three genetically distinct clades of Anaplasma phagocytophilum in Ixodes scapularis.

Human granulocytic anaplasmosis (HGA) is an emerging disease in Canada because of range expansion by the arthropod vector, Ixodes scapularis. These ticks carry the Ap-ha variant of Anaplasma phagocytophilum (Ap-ha), which has been implicated in causing HGA, and the Ap-variant 1, which is not associated with human infection. We report the detection of 13 genotypes of the ankyrin (ankA) gene among 76 infected blacklegged ticks. Haplotype network and phylogenetic analyses revealed that the ankA genotypes corresponding to the Ap-ha variant did not form a monophyletic assemblage. They formed two distinct clades (Clades I and III), one of which was genetically more similar in nucleotide and amino acid sequences to genotypes of Ap-variant 1 that comprised Clade II. Additional work is needed to explore the evolutionary history of A. phagocytophilum in North America, and to determine if there are differences in pathogenicity or clinical symptoms associated with the two divergent groups of the Ap-ha variant given the significant differences in ankA amino acid sequence.

Complement component Bf/C2b gene mediates immune responses against Aeromonas hydrophila in grass carp Ctenopharyngodon idella.

The complement system is a significant component of innate immunity. Here, we identified a Bf/C2 homolog (gcBf/C2b) in grass carp. gcBf/C2b shares a high similarity with Bf/C2b counterparts in other teleosts. gcBf/C2b transcription was widely distributed in different tissues, induced by Aeromonas hydrophila in vivo and in vitro, and affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcBf/C2b, transcript levels of all components except gcC5 were significantly enhanced, and gcBf/C2b, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after A. hydrophila challenge or stimulation with bacterial pathogen-associated molecular patterns (PAMPs). However, gcBf/C2b in interference cells down-regulated the transcript levels after A. hydrophila challenge, and gcBf/C2b induced NF-κB signaling. These findings indicate the vital role of gcBf/C2b in innate immunity in grass carp.

Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014.

Outbreaks of porcine epidemic diarrhea (PED) have resulted in significant economic losses in the swine industry, and another PED outbreak occurred in 2014 in Korea. Isolating and culturing PED virus (PEDV) allow investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated two PEDV isolates (QIAP1401 and QIAP1402) from naturally infected piglets at Jeju-do, Korea. Viral propagation was confirmed in Vero cells based on cytopathic effect, immunofluorescence assay, reverse transcription-polymerase chain reaction, and electron microscopic analyses. The QIAP401 isolate propagated well in Vero cells for 70 passages, with titers of 106.5 to 107.0 50% tissue culture infectious dose/mL, which increased gradually with passaging. The nucleotide and amino acid sequences of the QIAP1401 isolate were determined and compared with those of other PEDV isolates. The QIAP1401 isolate was determined to be closely related to the USA/Minnesota271/2014 strain (> 99.9% nucleotide similarity) that was isolated in the USA in 2014. Phylogenetic analysis based on several PEDV genes suggested that a new PEDV variant is circulating in the Korean swine industry, with 93.08% similarity to the SM98 strain isolated in 1998. In addition, the QIAP1401 strain showed strong virulence in 3-day-old piglets and 11-week-old growing pigs.

Charcot-Leyden crystals: do they exist in veterinary species? A case report and literature review.

The Charcot-Leyden crystal (CLC) is a major human eosinophil protein that readily crystallizes; these crystals are common in eosinophilic diseases. Although anecdotal existence of these crystals is known in veterinary pathology, definitive reports do not exist, to our knowledge. We identified eosinophilic crystals in a laryngeal myxosarcoma from a 2-y-old, spayed female, Labrador Retriever dog that were tentatively interpreted as CLCs. However, Ziehl-Neelsen acid-fast stain was negative, arguing against CLCs. The crystals stained red with Masson trichrome, precluding collagen. Periodic acid-Schiff and alcian blue were negative. The crystals stained positively with Okajima, and no myoglobin immunoreactivity was detected, supporting their identity as hemoglobin crystals. In the absence of a hematologic abnormality, these crystals were interpreted to be abnormal hemoglobin breakdown products. Protein sequence comparison was pursued to determine whether a protein similar to CLC exists in mammals. Only 3 nonhuman primate species, the Sumatran orangutan ( Pongo abelii), rhesus macaque ( Macaca mulatta), and cynomolgus monkey ( Macaca fascicularis), had a sequence similarity of >80%. Of the crystal-forming residues, 12 of 54 (22%) were different in the Sumatran orangutan and 15 of 54 (28%) were different in the Macaca spp., which may affect the crystallization process. The lack of reports of CLCs in nonhuman species and our results collectively suggest that CLCs are human-specific.

Detection and Molecular Characterization of Foot and Mouth Disease Viruses from Outbreaks in Some States of Northern Nigeria 2013-2015.

Control measures for foot and mouth disease (FMD) in Nigeria have not been implemented due to the absence of locally produced vaccines and risk-based analysis resulting from insufficient data on the circulating FMD virus (FMDV) serotypes/strains. In 2013-2015, blood and epithelial samples were collected from reported FMD outbreaks in four states (Kaduna, Kwara, Plateau and Bauchi) in northern Nigeria. FMDV non-structural protein (NSP) seroprevalence for the outbreaks was estimated at 80% (72 of 90) and 70% (131 of 188) post-outbreak. Antibodies against FMDV serotypes O, A, SAT1, SAT2 and SAT3 were detected across the states using solid-phase competitive ELISA. FMDV genome was detected in 99% (73 of 74) of the samples from FMD-affected animals using rRT-PCR, and cytopathic effect was found in cell culture by 59% (44 of 74) of these samples. Three FMDV serotypes O, A and SAT2 were isolated and characterized. The phylogenetic assessments of the virus isolates showed that two topotypes of FMDV serotype O, East Africa-3 (EA-3) and West Africa (WA) topotypes were circulating, as well as FMDV strains belonging to the Africa genotype (G-IV) of serotype A and FMDV SAT2 topotype VII strains. While the serotype O (EA-3) strains from Nigeria were most closely related to a 1999 virus strain from Sudan, the WA strain in Nigeria shares genetic relationship with three 1988 viruses in Niger. The FMDV serotype A strains were closely related to a known virus from Cameroon, and the SAT2 strains were most closely related to virus subtypes in Libya. This study provides evidence of co-occurrence of FMDV serotypes and topotypes in West, Central, East and North Africa, and this has implication for control. The findings help filling the knowledge gap of FMDV dynamics in Nigeria and West Africa subregion to support local and regional development of vaccination-based control plans and international risk assessment.

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite).

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70-90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

Genetic diversity and phylogenetic analysis of porcine reproductive and respiratory syndrome virus in southern China from 2007 to 2014.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic variation. In this study, we characterized the genetic variation and evolutionary relationships among circulating PRRSV strains in southern China. We analyzed 29 NSP2 strains and 150 ORF5 strains from clinical samples collected in southern China during 2007-2014. The alignment results showed that the nucleotide identity similarities of the two genes among these strains were 80.5%-99.7% and 80.9%-100%, respectively. Phylogenetic analysis based on the NSP2 gene showed that highly pathogenic (HP)-PRRSV was still the dominant virus in southern China from 2013 to 2014. Compared with reference strains CH-1a and VR-2332, the field strain 131101-GD-SHC, which shared high homology with JXA1-P170, had a novel 12 amino acid deletion at position 499-510. Phylogenetic analysis based on the ORF5 gene showed that HP-PRRSV, VR2332-like strains, and QYYZ-like strains were simultaneously circulating in southern China from 2007 to 2014, suggesting that, in recent years, the type 2 PRRSV was more diverse in southern China. In conclusion, mutations in the decoy epitope and primary neutralizing epitope could be markers of viral evolution and used to study evolutionary relationships among PRRSV strains in China.

Grouper (Epinephelus coioides) BCR signaling pathway was involved in response against Cryptocaryon irritans infection.

B cell antigen receptor (BCR) plays a crucial role in B cell development and antibody production. It comprises membrane immunoglobulin non-covalently associated with CD79a/CD79b heterodimer. After B cell activation, initial extracellular signals are transduced by BCR complex and amplified by two protein tyrosine kinases, LYN and SYK, which then trigger various pathways. In the present study, we cloned grouper genes for BCR accessory molecules, EcCD79a (669 bp) and EcCD79b (639 bp), as well as two protein tyrosine kinases, EcLYN (1482 bp) and EcSYK (1854 bp). Homology analysis showed that all four molecules had a relatively high amino acid identity compared with those in other animals. Among them, they all shared the highest identity with Takifugu rubripes (EcCD79a 49%, EcCD79b 52%, EcLYN 82% and EcSYK 77%). The conserved features and important functional residues were analyzed. Together with IgM and IgT, tissue distribution analysis showed that all six molecules were mainly expressed in immune organs, particularly systematic immune organs. In groupers infected with Cryptocaryon irritans, up-regulation of EcCD79a and b, EcIgM and EcIgT were not seen in the early stage skin and gill until 14-21 days. Up-regulation of EcCD79a was seen in head kidney at most time points, while EcCD79a and b were only significantly up-regulated in day 14 spleen. Significant up-regulation of EcIgT were seen in day 21 head kidney and day 1, day14 spleen. Significant up-regulation of EcIgM were seen in day 1 head kidney and 12 h spleen. In addition, two protein kinase genes, EcLYN and EcSYK, were up-regulated in the skin at most time points, which suggested that B cells may be activated at the skin local infection site.

Molecular cloning, expression and functional characterization of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) in grass carp, Ctenopharyngodon idella.

TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5'UTR of 112 bp, an ORF of 1359 bp, and a 3'UTR of 29 bp before the poly (A) tail, was cloned from grass carp, Ctenopharyngodon idella. The TRIP gene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed that TRIP mRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 and TRIP mRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P < 0.05) after hepatocyte of C. idella was treated with 2 µg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P < 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp.

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology.

Characterization of two novel gadd45a genes in hybrid tilapia and their responses to the infection of Streptococcus agalactiae.

Diseases are one of the major challenges in tilapia aquaculture. Identification of DNA markers associated with disease resistance may facilitate the acceleration of the selection for disease resistance. Gadd45a (growth arrest and DNA damage 45 A), a stress-inducible gene in humans and mice, has not been studied in fish. We characterized the two prologues of Gadd45a genes in hybrid tilapia. Gadd45a1 and Gadd45a2 shared an identical gene structure and showed an amino acid sequence identity of 73.8%. Their expressions were detected in all 10 tissues examined, with the kidney and gill having high transcriptional expressions. The expression levels of Gadd45a1 were significantly lower than those of Gadd45a2 in all examined tissues. After a challenge with a bacterial pathogen Streptococcus agalactiae, the expressions of the two genes were up-regulated significantly in the spleen, kidney, liver and intestine. These findings suggest that the two Gadd45a genes play an important role in the resistance to S. agalactiae in tilapia. We identified 10 SNPs in the two genes. The SNP markers in the two Gadd45a genes could be used to examine whether they are associated with resistance to S. agalactiae.