Genomic and Transcriptional Profiles of Kelch-like (klhl) Gene Family in Polyploid Carassius Complex.

Genome duplication supplies raw genetic materials and has been thought to be essential for evolutionary innovation and ecological adaptation. Here, we select Kelch-like (klhl) genes to study the evolution of the duplicated genes in the polyploid Carassius complex, including amphidiploid C. auratus and amphitriploid C. gibelio. Phylogenetic, chromosomal location and read coverage analyses indicate that most of Carassius klhl genes exhibit a 2:1 relationship with zebrafish orthologs and confirm two rounds of polyploidy, an allotetraploidy followed by an autotriploidy, occurred during Carassius evolution. The lineage-specific expansion and biased retention/loss of klhl genes are also found in Carassius. Transcriptome analyses across eight adult tissues and seven embryogenesis stages reveal varied expression dominance and divergence between the two species. The expression of klhls in response to Carassius herpesvirus 2 infection shows different expression changes corresponding to distinct herpesvirus resistances in three C. gibelio gynogenetic clones. Finally, we find that most C. gibelio klhl genes possess three alleles except eight genes that have lost one or two alleles due to genome rearrangement. The allele expression bias is prosperous for Cgklhl genes and varies during embryogenesis owning to the sequential expression manner of the alleles. The current study provides global insights into the genomic and transcriptional evolution of duplicated genes in a given superfamily resulting from multiple rounds of polyploidization.

Autophagy Delays Apoptotic Cell Death Induced by Siniperca chuatsi Rhabdovirus in Epithelioma Papulosum Cyprinid Cells.

Autophagy and apoptosis are two key cell fate determination pathways, which play vital roles in the interaction between viruses and host cells. Previous research had confirmed that one strain of fish rhabdoviruses, Siniperca chuatsi rhabdovirus (SCRV), could induce apoptosis and autophagy after infection. In the current study, we continued to analyze the interaction of autophagy and apoptosis in SCRV-infected EPC cell lines after treatment with different autophagy or apoptosis inhibitors. We found that SCRV infection could activate the mitochondrial apoptotic pathway by the detection of the activities of the caspase-3 and caspase-9 and by flow cytometry analysis in JC-1-stained cells, respectively. Furthermore, no significant autophagy-related factors were disturbed in SCRV-infected cell after apoptosis inhibitor Z-VAD-FMK treatment, while autophagy inducer rapamycin could obviously delay the occurrence of CPE and cell death. Meanwhile, rapamycin was able to reduce the proportion of apoptotic cells. Besides that, rapamycin could disturb the expression of p62 and LC3B-II, and the transcription level of SCRV nucleoprotein mRNA. The progeny virus titers did not show a big difference between the rapamycin treatment or without it. Collectively, our data preliminarily confirmed that SCRV-activated autophagy could delay apoptosis in EPC cells and may not affect virus production. Further study may need to focus on the crosstalk regulation and its roles on the SCRV infection.

MicroRNA expression profiling of caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

As a member of the genus Cyprinivirus in the family Alloherpesviridae, Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss in the aquaculture industry, mainly in C. auratus gibelio and goldfish. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 remain elusive. In this study, high-throughput sequencing technology was employed to explore the miRNA expression profiles of C. auratus gibelio (GiCF) caudal fin cells in response to Cyprinid Herpesvirus-2 (CyHV-2) infection. A total of 631 novel miRNAs and 409 known miRNAs were identified. The expression levels of 7 miRNAs were found as significantly modulated (5 down-regulation and 2 up-regulation; P < 0.01, |logFC|>1, TPM>10) in CyHV-2 infected cells. 7 miRNA and their potential mRNA targets were validated by Real-time PCR (qRT-PCR), respectively. Targets prediction and functional analysis of these 7 miRNAs revealed significant enrichment for several signaling pathways, including PPAR, p53 and FoxO pathways. These studies provided more valuable basis for further study on the roles of miRNAs in CyHV-2 replication and pathogenesis.

Characterization of a Novel Megalocytivirus Isolated from European Chub (Squalius cephalus).

A novel virus from moribund European chub (Squalius cephalus) was isolated on epithelioma papulosum cyprini (EPC) cells. Transmission electron microscopic examination revealed abundant non-enveloped, hexagonal virus particles in the cytoplasm of infected EPC cells consistent with an iridovirus. Illumina MiSeq sequence data enabled the assembly and annotation of the full genome (128,216 bp encoding 108 open reading frames) of the suspected iridovirus. Maximum Likelihood phylogenetic analyses based on 25 iridovirus core genes supported the European chub iridovirus (ECIV) as being the sister species to the recently-discovered scale drop disease virus (SDDV), which together form the most basal megalocytivirus clade. Genetic analyses of the ECIV major capsid protein and ATPase genes revealed the greatest nucleotide identity to members of the genus Megalocytivirus including SDDV. These data support ECIV as a novel member within the genus Megalocytivirus. Experimental challenge studies are needed to fulfill River's postulates and determine whether ECIV induces the pathognomonic microscopic lesions (i.e., megalocytes with basophilic cytoplasmic inclusions) observed in megalocytivirus infections.

Molecular cloning, GTP recognition mechanism and tissue-specific expression profiling of myxovirus resistance (Mx) protein in Labeo rohita (Hamilton) after Poly I:C induction.

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.

Elucidation of mechanism for host response to VHSV infection at varying temperatures in vitro and in vivo through proteomic analysis.

Seasonal temperature has a major influence on the infectivity of pathogens and the host immune system. Viral hemorrhagic septicemia virus (VHSV) is one such pathogen that only causes the mortality of fish at low temperatures. This study aims to discover the host defense mechanism and pathway for resistance to VHSV at higher temperatures. We first observed the VHSV infection patterns at low and higher temperatures in fathead minnow (FHM) cells (20 °C and 28 °C) and zebrafish (15 °C and 25 °C). In comparison to the 20 °C infection, FHM cells infected at 28 °C showed decreased apoptosis, increased cell viability, and reduced VHSV N gene expression. In zebrafish, infection at 25 °C caused no mortality and significantly reduced the N gene copy number in comparison to infection at 15 °C. To explore the antiviral infection mechanisms induced by high temperature in vitro and in vivo, the changes in the proteomic profile were measured through UPLC-MSE analysis. ACADL, PTPN6, TLR1, F7, A2M, and GLI2 were selected as high temperature-specific biomarkers in the FHM cell proteome; and MYH9, HPX, ANTXR1, APOA1, HBZ, and MYH7 were selected in zebrafish. Increased immune response, anticoagulation effects, and the formation of lymphocytes from hematopoietic stem cells were analyzed as functions that were commonly induced by high temperature in vitro and in vivo. Among these biomarkers, GLI2 was predicted as an upstream regulator. When treated with GANT58, a GLI-specific inhibitor, cell viability was further reduced due to GLI2 inhibition during VHSV infection at varying temperatures in FHM cells, and the mortality in zebrafish was induced earlier at the low temperature. Overall, this study discovered a new mechanism for VHSV infection in vitro and in vivo that is regulated by GLI2 protein.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1ß, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.

Involvement of interferon regulatory factor 3 from the barbel chub Squaliobarbus curriculus in the immune response against grass carp reovirus.

The barbel chub Squaliobarbus curriculus is an important commercial fish species in China, and has shown significant resistance to grass carp reovirus (GCRV). In this study, the cDNA sequence of interferon regulatory factors 3 (IRF3) from Squaliobarbus curriculus, designated as ScIRF3, was cloned, and its effect against GCRV was investigated. The full-length 1837 base pair (bp) cDNA of ScIRF3 contained a complete open reading frame of 1374 bp and encoded a putative polypeptide of 457 amino acid residues. The ScIRF3 protein contained conserved domains, including an N-terminal DNA-binding domain, a C-terminal IRF association domain, and a serine-rich domain. Phylogenetic analysis showed that ScIRF3 was closely clustered with IRF3s from Carassius auratus and Ctenopharyngodon idellus. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScIRF3 in Squaliobarbus curriculus were the highest in the spleen and lowest in the muscle. After GCRV infection, expression levels of both ScIRF3 and type I interferon (IFN) were initially up-regulated and subsequently down-regulated in the spleen and intestine. Correlation analysis showed that the expression level of type I IFN is significantly positively correlated with that of ScIRF3 (Pearson correlation coefficient: 0.883, P: 0.004) in the intestine. The expression level of type I IFN was also significantly up-regulated and the GCRV titer was significantly decreased (P < .05) in GCRV-infected ScIRF3-overexpressing Ctenopharyngodon idellus kidney cells. These results indicate that ScIRF3 may play a role in the type I IFN immune response against GCRV in Squaliobarbus curriculus and can also inhibit GCRV replication in Ctenopharyngodon idellus kidney cells.

Genomic characterization of a novel calicivirus, FHMCV-2012, from baitfish in the USA.

During regulatory sampling of fathead minnows (Pimephales promelas), a novel calicivirus was isolated from homogenates of kidney and spleen inoculated into bluegill fry (BF-2) cells. Infected cell cultures exhibiting cytopathic effects were screened by PCR-based methods for selected fish viral pathogens. Illumina HiSeq next generation sequencing of the total RNA revealed a novel calicivirus genome that showed limited protein sequence similarity to known homologs in a BLASTp search. The complete genome of this fathead minnow calicivirus (FHMCV) is 6564 nt long, encoding a polyprotein of 2114 aa in length. The complete polyprotein shared only 21% identity with Atlantic salmon calicivirus,followed by 11% to 14% identity with mammalian caliciviruses. A molecular detection assay (RT-PCR) was designed from this sequence for screening of field samples for FHMCV in the future. This virus likely represents a prototype species of a novel genus in the family Caliciviridae, tentatively named "Minovirus".

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line.

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.

The replication of spring viraemia of carp virus can be regulated by reactive oxygen species and NF-κB pathway.

Different viruses could induced ROS generation to alter intracellular redox state in the host cells, and unbalanced redox state was suggested to have various effects on viral replication. In this study, we investigated the influence of reactive oxygen species (ROS) on replication of spring viraemia of carp virus (SVCV) in fish cells. After SVCV infection, there existed a time-dependent increase in ROS generation. The present results revealed that antioxidant N-acetyl-l-cysteine (NAC) resulted in a lower ROS levels and increased SVCV replication in EPC cell. In contrast, a GSH synthesis inhibitor buthionine sulfoximine (BSO) induced ROS generation and decreased SVCV replication. In addition, activation of NF-κB suppressed SVCV replication by using two inhibitors of cytokine-induced IκBα phosphorylation. More importantly, enhancement of the activity of NF-κB was found in BSO treatment, which indicated that dropped SVCV replication likely occurred via ROS activation of NF-κB. Overall, our results revealed that the SVCV infection and replication could generate ROS and be affected by the redox state, where this progression was associated with the alteration in NF-κB pathway induced by oxidative stress.

Fish miR-146a promotes Singapore grouper iridovirus infection by regulating cell apoptosis and NF-κB activation.

miR-146a was reported to participate in various pathophysiological conditions in mammals, such as inflammation and immune responses, oncogenesis and cell damage. However, its function in low vertebrates has not been well elucidated. In this study, we characterized the expression profiles and functions of miR-146a in fish cells during iridovirus infection. We found that the reported fish miR-146a genes encoded an identical mature sequence, which shared high similarity with its mammalian orthologues, suggesting a putative functional conservation of miR-146a between fish and other vertebrates. Using a well-established infection model of Singapore grouper iridovirus (SGIV) in fathead minnow cells, we found that SGIV infection induced the expression of miR-146a to a dramatic extent. More importantly, we found that miR-146a promoted SGIV propagation, as demonstrated by higher expression of viral genes and increased virus titres in miR-146a-overexpressing cells. Mechanistically, we found that miR-146a overexpression suppressed, while miR-146a knockdown promoted, NF-κB activation and SGIV-induced cell apoptosis, two major cellular events involved in SGIV infection. Our study suggested that the induction of miR-146a by SGIV infection may function through a feed-forward mechanism to promote viral infection by restraining anti-viral cellular responses.

Dexamethasone treatment decreases replication of viral hemorrhagic septicemia virus in Epithelioma papulosum cyprini cells.

The expression of Mx1 in EPC cells after treatment with poly(I:C) or infection with viral hemorrhagic septicemia virus (VHSV) was significantly suppressed by treatment with dexamethasone. However, the titer of VHSV did not increase but instead decreased after dexamethasone treatment. This suggests that dexamethasone not only downregulates type I IFN but also affects certain factors that are necessary for VHSV replication. An important effect of HSP90 on replication of RNA viruses and downregulation of HSP90 by glucocorticoids have been reported. In this study, dexamethasone downregulated HSP90α expression in EPC cells that were stimulated with poly(I:C) or infected with VHSV. Furthermore, cells treated with an HSP90 inhibitor, geldanamycin, showed significantly decreased titers of VHSV, suggesting that HSP90 may be an important host component involved in VHSV replication, and HSP90 inhibition might be one of the causes for the observed reduction in viral titer caused by dexamethasone treatment.

Molecular detection of a novel totivirus from golden shiner (Notemigonus crysoleucas) baitfish in the USA.

During regulatory and routine surveillance sampling of apparently healthy baitfish from the state of Minnesota, a novel totivirus (tentatively named "golden shiner totivirus", GSTV) was detected in a homogenate of kidney and spleen of golden shiner (Notemigonus crysoleucas). The nearly complete genome is 7788 nt long with a complete 5' untranslated region (UTR) of 135 nt (1-135 nt position), complete open reading frames (ORFs) and a partial 3' UTR of 54 nt (7734-7788). The sequence is comprised of two ORFs (ORF1 and ORF2). The larger ORF1 encodes a 1659-aa polypeptide in frame +1 from nt position 136 to 5115 (4980 nt) with a start codon at position 136-138 and a stop codon at position 5113-5115. The ORF1 is 54 aa longer than the 1605-aa ORF1-encoded protein of a reference strain of infectious myonecrosis virus (IMNV), ID-EJ-12-1(AIC34743.1). The predicted ORF1 and ORF2 fusion protein sequence was NFQDGG. Hence, an overlapping region of 99 nt was observed, which is shorter than the 172-nt and 199-nt overlapping regions in Armigeres subalbatus totivirus (AsTV) and IMNV, respectively. GSTV formed a separate lineage based on phylogenetic analysis of ORF1-encoded major capsid protein (MCP) and ORF2-encoded RNA-dependent RNA polymerase (RdRp) sequences. Based on ORF1 MCP sequence analysis, GSTV was most closely related to IMNV, with maximum aa sequence identity of 26.42-27.86 %, followed by 26.59, 22.94 and 21.75 % for Drosophila totivirus (DTV), AsTV and Omono River virus (OMRV), respectively. Similar to ORF1, the ORF2 (RdRp) of GSTV formed a separate clade with maximum identity of 38.10 % and 38.50 % to IMNV and DTV, respectively. The virus identified here differs enough from its closest relative that it may represent a new genus in the family Totiviridae. The disease-causing potential and management impact of this novel virus is unknown at this time.

Genetic analysis of a novel nidovirus from fathead minnows.

A bacilliform virus was isolated from diseased fathead minnows (Pimephales promelas). Analysis of the complete genome coding for the polyprotein (pp1ab), spike (S), membrane (M) and nucleocapsid (N) proteins revealed that the virus was most like white bream virus (WBV), another bacilliform virus isolated from white bream (Blicca bjoerkna L.) and the type species of the genus Bafinivirus within the order Nidovirales. In addition to similar gene order and size, alignment of deduced amino acid sequences of the pp1ab, M, N and S proteins of the fathead minnow nidovirus (FHMNV) with those of WBV showed 46, 44, 39 and 15 % identities, respectively. Phylogenetic analysis using the conserved helicase domain of the replicase showed FHMNV was distinct from WBV, yet the closest relative identified to date. Thus, FHMNV appears to represent a second species in the genus Bafinivirus. A PCR assay was developed for the identification of future FHMNV-like isolates.

Loop mediated isothermal amplification combined with nucleic acid lateral flow strip for diagnosis of cyprinid herpes virus-3.

An improved loop mediated isothermal amplification (LAMP) assay for rapid, sensitive and specific detection of cyprinid herpes virus-3 (CyHV-3), also known as koi herpes virus (KHV), was developed. The lower detection limit of the CyHV-3-LAMP assay is 10 fg DNA which equivalent to 30 copies of CyHV-3 genome. Nucleic acid lateral flow assay was used for visual detection of the LAMP products. The LAMP- nucleic acid lateral flow assay relies on DNA hybridization technology and antigen-antibody reactions in combination with LAMP. For application of this assay, the biotinylated LAMP product was hybridized with a FITC-labelled specific probe for 5 min. The resulting DNA complex could be visualised as purple band at the strip test line within 5 min of sample exposure. The nucleic acid lateral flow analysis of the LAMP product was equivalent in sensitivity but more rapid than the conventional agarose gel electrophoresis. The combination of LAMP assay with the nucleic acid lateral flow analysis can simplify the diagnosis and screening of CyHV-3 as it is simple, requires very little training, does not require specialized equipment such as a thermal cycler, the results are read visually with no need to run a gel and has a high sensitivity and specificity.

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, it's 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. It's 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.

Factors affecting between-lake variation in the occurrence of epidermal papillomatosis in roach, Rutilus rutilus (L.).

The theory of island biogeography predicts that the probability of a species occupying an island depends on a dynamic equilibrium between extinction and colonization. Epidermal papillomatosis is a disease manifesting as skin tumours on fish. We studied the factors affecting the occurrence of the disease in roach, Rutilus rutilus (L.), in 34 lakes. The results of discrimination analysis suggest that maximum depth, percentage of the drainage area of the lake covered by lakes in the vicinity and altitude best identified diseased lakes. Comparison of diseased and non-diseased lakes revealed that lake area could also be regarded as a variable contributing to the occurrence of the disease. The sampling date, proportion of males and mean length of fish did not discriminate between the lakes. The probability of the disease occurring was highest in large, deep, low-altitude lakes which had a high percentage of lakes in their vicinity. Thus, the results indicate that the colonization and extinction processes probably contribute to the occurrence of papillomatosis in roach, as predicted by the theory. Furthermore, the large natural variation in the occurrence of the disease could mask possible environmental effects and between-lake comparisons should be interpreted with caution when using the fish-papillomatosis system as an indicator of environmental stress.

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus.

Double-stranded RNA-activated protein kinase (PKR) plays an important role in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels 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, GrPKZ expression was up-regulated from 24h post-injection and lasted until the fish became moribund (P<0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24h post-injection (P<0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection.