Koi herpesvirus infection in experimentally infected common carp Cyprinus carpio (Linnaeus, 1758) and three potential carrier fish species Carassius carassius (Linnaeus, 1758); Rutilus rutilus (Linnaeus, 1758); and Tinca tinca (Linnaeus, 1758) by quantitative real‐time PCR and in‐situ hybridization

Only single cells in the carrier fish species Carassius carassius (Linnaeus, 1758) for koi herpesvirus (KHV) are infected in contrast to large numbers in the susceptible species common carp Cyprinus carpio (Linnaeus 1758). Several species of the family Cyprinidae have been described as virus carrier species, showing no clinical signs of a KHV disease but able to transmit the virus to other susceptible fish. In this study, 72 common carp Cyprinus carpio (Linnaeus, 1758), 36 tench Tinca tinca (Linnaeus, 1758), 36 crucian carp Carassius carassius (Linnaeus, 1758) and 36 common roach Rutilus rutilus (Linnaeus, 1758) were experimentally infected with KHV (isolate “Israel”) by immersion and kept at 20°C. The fish were euthanized at 12 timepoints over a period of 90 days and virus DNA was quantified in tissues by a real‐time TaqMan PCR. Whereas KHV‐DNA was found in Cyprinus carpio for up to 90 days, the virus DNA was detectable only in single individuals of Rutilus rutilus, Tinca tinca and Carassius carassius for up to 25 days after experimental virus exposure. Tissue samples of Cyprinus carpio and Carassius carassius were screened by in‐situ hybridization. Positive signals were found in various organs of the common carp tested crucian carp. In the latter species a much smaller number of virus‐positive stained cells was detected compared to the infected carp.     

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full‐length complementary (c)DNA of interleukin‐1 receptor‐associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine–threonine‐specific protein kinase catalytic domain and a C‐terminal domain. The amino‐acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84·5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up‐regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up‐regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.     

The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex

In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram‐positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein–protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA‐like domains that are found in all DEAD box RNA helicases and a C‐terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C‐terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.     

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full‐length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain–domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild‐type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full‐length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an “extended” catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.     

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Background  

Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.     

Physical changes in specimens of five species of Cyprinidae preserved in ethanol and frozen

Preservation in 30% ethanol and freezing to a temperature of ?20 ± 2° C is an appropriate method for measurement of fish eggs, larvae and juveniles. Egg diameter of the common carp Cyprinus carpio increased insignificantly by 1·32% after preservation compared with live size. The total length (L_T) of 1 day post‐hatching (dph) larvae as well as the standard length (LS) of 16 dph larvae of C. carpio increased significantly (2·95 and 1·50%, respectively) after preservation. Egg diameter as well as the L_T of 1 dph larvae of barbel Barbus barbus increased significantly after preservation, by 1·74 and 1·96%, respectively over their original size. The standard length (L_S) of 14 dph larvae of B. barbus as well as juveniles of B. barbus, crucian carp Carassius carassius, common nase Chondrostoma nasus and tench Tinca tinca decreased significantly after preservation (?0·56 to ?5·54%), whereas their body mass increased significantly (11·46–18·57%). Preserved eggs of C. carpio and B. barbus were hard, round and transparent. The larvae and juveniles of examined fishes, preserved in frozen ethanol, were straight, flexible and easily measurable after 60 days. Integrity of body surface and fins, as well as preservation of colours were much better in larvae or juveniles frozen and thawed only once than in specimens frozen and thawed thrice. Cooling in 30% ethanol to a temperature of 6 ± 2° C and freezing in water to a temperature of ?20 ± 2° C are not appropriate preservation methods for eggs and larvae of C. carpio (1 and 16 dph).     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

Screening and identification of male‐specific DNA fragments in common carps Cyprinus carpio using suppression subtractive hybridization

In this study, a sex subtractive genomic DNA library was constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. Twenty‐two clones with distinguishable hybridization signals were selected and sequenced. The specific primers were designed based on the sequence data. Those primers were then used to amplify the sex‐specific fragments from the genomic DNA of male and female carp. The amplified fragments from two clones showed specificity to males but not to females, which were named as Ccmf2 [387 base pairs (bp)] and Ccmf3 (183 bp), respectively. The sex‐specific pattern was analysed in a total of 40 individuals from three other different C. carpio. stocks and grass carp Ctenopharyngodon idella using Ccmf2 and Ccmf3 as dot‐blotting probes. The results revealed that the molecular diversity exists on the Y chromosome of C. carpio. No hybridization signals, however, were detected from individuals of C. idella, suggesting that the two sequences are specific to C. carpio. No significant homologous sequences of Ccmf2 and Ccmf3 were found in GenBank. Therefore, it was interpreted that the results as that Ccmf2 and Ccmf3 are two novel male‐specific sequences; and both fragments could be used as markers to rapidly and accurately identify the genetic sex of part of C. carpio. This may provide a very efficient selective tool for practically breeding monosex female populations in aquacultural production.     

Irf2bp2a regulates liver development via stabilizing P53 protein in zebrafish

Zebrafish irf2bp2a, an ortholog of human IRF2BP2, is specifically expressed in the developing liver at growth stage. As a multi-functional protein, the role of irf2bp2a during hepatogenesis remains unclear. Here we take advantage of an irf2bp2a knockout line to show that the deficiency of irf2bp2a can induce apoptosis of hepatic cells through aberrant p53 activation at the early stage of embryonic development. Mechanistic studies reveal that within the IRF2BP2-null hepatic cells, more MDM2 molecules, the E3 ubiquitin ligase of P53, can be sequestrated into the IRF2-MDM2 complex, which in turns stabilizes P53 protein. Moreover, irf2bp2a is demonstrated as a direct downstream target of c/ebpα. Thus, a C/ebpα-Irf2bp2a-P53 axis controls liver development in zebrafish. Overall, our findings indicate a stage-specific role of irf2bp2a on liver organogenesis by regulating p53 pathway.     

Morphological features of an endangered Japanese strain of Cyprinus carpio: reconstruction based on seven SNP markers

Morphological analyses of 183 specimens of Japanese common carp Cyprinus carpio (171 from Lake Biwa and 12 from nursery ponds) using genetic hybrid indices demonstrated that the typical native Japanese strain of C. carpio has a more elongate body, more branched dorsal‐fin rays, fewer and shorter gill rakers, more developed pneumatic bulb, more coiled pneumatic duct, longer posterior swimbladder and shorter intestine than the typical introduced C. carpio. These results provide a basis for a better understanding of the ecological characteristics and taxonomic status of the endangered Japanese strain of C. carpio.     

Spontaneous polyploidy,gynogenesis and androgenesis in second generation (F2) koi Cyprinus carpio × goldfish Carassius auratus hybrids

The objective of this study was to characterize the genetics of second generation (F₂) koi Cyprinus carpio × goldfish Carassius auratus hybrids. Spermatozoa produced by a novel, fertile F₁ male were found to be diploid by flow‐cytometric analysis. Backcross (F₁ female × C. carpio male and C. carpio female × F₁ male) juveniles were triploid, confirming that female and male F₁ hybrids both produced diploid gametes. The vast majority of surviving F₂ juveniles was diploid and small proportions were aneuploid (2·1n–2·3n and 3·1n–3·9n), triploid (3n) and tetraploid (4n). Microsatellite genotyping showed that F₂ diploids repeated either the complete maternal or the complete paternal genotype. Fish with the maternal genotype were female and fish with the paternal genotype were male. This demonstrates that F₂ diploids were the result of spontaneous gynogenesis and spontaneous androgenesis. Analysis of microsatellite inheritance and the sex ratio in F₂ crosses showed that spontaneous gynogenesis and androgenesis did not always occur in equal proportions. One cross was found to have an approximate equal number of androgenetic and gynogenetic offspring while in several other crosses spontaneous androgenesis was found to occur more frequently than spontaneous gynogenesis.     

Down-regulation of human RNA/DNA helicase SUV3 induces apoptosis by a caspase- and AIF-dependent pathway

Background information. The nuclear gene hSUV3 (human SUV3) encodes an ATP‐dependent DNA/RNA helicase. In the yeast Saccharomyces cerevisiae the orthologous Suv3 protein is localized in mitochondria, and is a subunit of the degradosome complex which regulates RNA surveillance and turnover. In contrast, the functions of human SUV3 are not known to date. Results. In the present study, we show that a fraction of human SUV3 helicase is localized in the nucleus. Using small interfering RNA gene silencing in HeLa cells, we demonstrate that down‐regulation of hSUV3 results in cell cycle perturbations and in apoptosis, which is both AIF‐ and caspase‐dependent, and proceeds with the induction of p53. Conclusions. In addition to its mitochondrial localization, human SUV3 plays an important role in the nucleus and is probably involved in chromatin maintenance.     

Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan

A monoclonal antibody (MAb) was generated against the capsid protein (ORF 72) of koi herpesvirus (KHV) isolated from diseased koi Cyprinus carpio in Taiwan. The clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using indirect enzyme-linked immunosorbent assay and Western blot assay. In addition, it detected KHV in KHV-infected cells but not in those of mock-infected cells as demonstrated by indirect immunofluorescence assay. The neutralization test showed that MAb-B2 neutralized KHV. Furthermore, we uncovered that MAb-B2 recognizes the ORF72 of KHV as revealed by liquid chromatography–tandem mass spectrometry and Western blot assays. Additionally, MAb-B2 has been used as a diagnostic tool for detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicated that MAb-B2 could be used in the development of a diagnostic kit for diagnosis of KHV infections and ORF72 protein of KHV might be a candidate for future vaccine development.