No abstract

Novelgene transfer into the fertilized eggs of gold fish 31 KREYSZIG, E., 1970: Statistische Methoden und ihre Anwendungen. 422 pp. Vandenhoek und Rupprecht, Gottingen. MCCAULEY, R.; CASSELMAN, J., 1982: Final preferendum of fish -an overestimation of the optimum temperature for growth. Abstract No. 193, 4th Congress of European Ichthyologists, Hamburg TOM, E. 0.; GULYAS, P.; OLAH, J., 1982: Effect of temperature on growth, food conversion and survival of sheatfish (Silurus ghnis L.) and common carp (Cyprinus carpi0 L.) at sublethal ammonia concentrations. Aquacultura Hungarica, Vol. III,51-56. 20.-24.9.1982. SummaryNovel gene which was microinjected into fertilized eggs of the goldfish replicated during the embryogenesis. A proportion of the novel gene has been integrated into the host DNS of the 50-day-old transgenic goldfish. Zusarnmenfassung Ubertragung von Fremdgenen in befruchtete Eier des Goldfsches (Carassius auratus L. 1758)Durch Mikroinjektion in befruchtete Eier des Goldfisches eingebrachte Fremdgene wurden w a r e n d der Embryogenese repliziert. Ein Teil der Fremd-DNS ist in die DNS der 50 Tage alten Goldfische integriert worden. RisumiTransmission des gPnes aliens dans des mufs fertilisis du Cyprin dori (Carassius auratus L. 1758) De genes nouveaux sont microinjectis dans des ceufs fenilisis des embryons de Carassius aurutus. Ces genes sont ripliquis. Un part de la DNS nouveau itait integri dans la DNS des Carassius auratus originaux i ce moment ages 50 journies.In recent years, purified genes have been microinjected into fertilized eggs or embryos of mammals, amphibians, and insects species in order to assess the fate and expression of the novel gene during the development in whole animal system [for review, see (3)]. Results of experiments performed on Xenopus Levis demonstrated that the microinjected, cloned DNA sequences could be replicated and faithfully transcribed during embryogenesis (2,7). PALMITER et al. (1982) (5) introduced into the pronuclei of fertilized eggs of mice a hybrid gene containing the promoter of the mouse metallothionein-1 gene fused to the structural gene for rat growth hormone. Some transgenic mice grew significantly larger than their littermates. Thus it is hoped to use the gene transfer method to develop rapidly growing U.S. A B E D 8 m BPJ -P R 322 -m flanking sequencea and introns m O X O M -hCH flanking sequences and introns 0 hGH cxons Fig. 1.The structure of the novel gene: (A) 9.4 kilobase pair (kb) BPVMG linear sequence as a novel gene to be microinjected into gold fish eggs consists of 69% BPV (bovine papilloma virus) transforming sequence followed by an MG hybrid gene. The MG contains an EcoRl/BglZ fragment of the promoter of the mouse MT-1 (metallothionein-1) gene fused to the BamHl site of a hGH (human growth hormone) mini-gene that includes all the coding sequence and the first of its four intervening sequences. (B) Recombinate plasmid pBPVMG-6 is an MG hybrid gene inserted into a pBR-BPV vector (modified and reconstructed according to PAVLAKIS and HAMER 198...

BackgroundA virus was isolated from diseased turbot Scophthalmus maximus in China. Biophysical and biochemical assays, electron microscopy, and genome electrophoresis revealed that the virus belonged to the genus Aquareovirus, and was named Scophthalmus maximus reovirus (SMReV). To the best of our knowledge, no complete sequence of an aquareovirus from marine fish has been determined. Therefore, the complete characterization and analysis of the genome of this novel aquareovirus will facilitate further understanding of the taxonomic distribution of aquareovirus species and the molecular mechanism of its pathogenesis.ResultsThe full-length genome sequences of SMReV were determined. It comprises eleven dsRNA segments covering 24,042 base pairs and has the largest S4 genome segment in the sequenced aquareoviruses. Sequence analysis showed that all of the segments contained six conserved nucleotides at the 5' end and five conserved nucleotides at the 3' end (5'-GUUUUA ---- UCAUC-3'). The encoded amino acid sequences share the highest sequence identities with the respective proteins of aquareoviruses in species group Aquareovirus A. Phylogenetic analysis based on the major outer capsid protein VP7 and RNA-dependent RNA polymerase were performed. Members in Aquareovirus were clustered in two groups, one from fresh water fish and the other from marine fish. Furthermore, a fusion associated small transmembrane (FAST) protein NS22, which is translated from a non-AUG start site, was identified in the S7 segment.ConclusionsThis study has provided the complete genome sequence of a novel isolated aquareovirus from marine fish. Amino acids comparison and phylogenetic analysis suggested that SMReV was a new aquareovirus in the species group Aquareovirus A. Phylogenetic analysis among aquareoviruses revealed that VP7 could be used as a reference to divide the aquareovirus from hosts in fresh water or marine. In addition, a FAST protein with a non-AUG start site was identified, which partially contributed to the cytopathic effect caused by the virus infection. These results provide new insights into the virus-host and virus-environment interactions.

Grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), is a major disease that hampers the development of grass carp aquaculture in China. The mechanism underlying GCRV infection is still largely unknown. Circular RNAs (circRNAs) are important regulators involved in various biological processes. In the present study, grass carp were infected with GCRV, and spleen samples were collected at 0 (control), 1, 3, 5, and 7 days post-infection (dpi). Samples were used to construct and sequence circRNA libraries, and a total of 5052 circRNAs were identified before and after GCRV infection, of which 41 exhibited differential expression compared with controls. Many parental genes of the differentially expressed circRNAs are involved in metal ion binding, protein ubiquitination, enzyme activity, and nucleotide binding. Moreover, 72 binding miRNAs were predicted from the differentially expressed circRNAs, of which eight targeted genes were predicted to be involved in immune responses, blood coagulation, hemostasis, and complement and coagulation cascades. Upregulation of these genes may lead to endothelial and blood cell damage and hemorrhagic symptoms. Our results indicate that an mRNA–miRNA–circRNA network may be present in grass carp infected with GCRV, providing new insight into the mechanism underlying grass carp reovirus infection.

BackgroundGrass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms.ResultsFollowing infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways.ConclusionGCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3824-1) contains supplementary material, which is available to authorized users.

BackgroundReplication and assembly of vertebrate reoviruses occur in specific intracellular compartments known as viral factories. Recently, NS88 and NS80, the nonstructural proteins from aquareoviruses, have been proposed to share common traits with µNS from orthoreoviruses, which are involved in the formation of viral factories.Methodology/Principal FindingsIn this study, the NS80 characteristics and its interactions with other viral components were investigated. We observed that the NS80 structure ensured its self-aggregation and selective recruitment of viral proteins to viral factories like structures (VFLS). The minimum amino acids (aa) of NS80 required for VFLS formation included 193 aa at the C-terminal. However, this truncated protein only contained one aa coil and located in the nucleus. Its N-terminal residual regions, aa 1–55 and aa 55–85, were required for recruiting viral nonstructural protein NS38 and structural protein VP3, respectively. A conserved N-terminal region of NS38, which was responsible for the interaction with NS80, was also identified. Moreover, the minimal region of C-terminal residues, aa 506–742 (Δ505), required for NS80 self-aggregation in the cytoplasm, and aa 550–742 (Δ549), which are sufficient for recruiting viral structure proteins VP1, VP2, and VP4 were also identified.Conclusions/SignificanceThe present study shows detailed interactions between NS80 and NS38 or other viral proteins. Sequence and structure characteristics of NS80 ensures its self-aggregation to form VFLS (either in the cytoplasm or nucleus) and recruitment of viral structural or nonstructural proteins.

Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from RGV and its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization revealed that 2L protein co-localized with viral factories in RGV infected cells. In cotransfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R. However, 2L protein did not co-localize with the major capsid protein of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability to form plaques and the virus titres were strongly reduced when expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report, to our knowledge, of colocalization between envelope proteins in iridovirus and provides new insights into the understanding of envelope proteins in iridovirus.

BackgroundGrass carp hemorrhagic disease, caused by grass carp reovirus (GCRV), is the most fatal causative agent in grass carp aquaculture. Protein-protein interactions between virus and host are one avenue through which GCRV can trigger infection and induce disease. Experimental approaches for the detection of host-virus interactome have many inherent limitations, and studies on protein-protein interactions between GCRV and its host remain rare.ResultsIn this study, based on known motif-domain interaction information, we systematically predicted the GCRV virus-host protein interactome by using motif-domain interaction pair searching strategy. These proteins derived from different domain families and were predicted to interact with different motif patterns in GCRV. JAM-A protein was successfully predicted to interact with motifs of GCRV Sigma1-like protein, and shared the similar binding mode compared with orthoreovirus. Differentially expressed genes during GCRV infection process were extracted and mapped to our predicted interactome, the overlapped genes displayed different tissue expression distributions on the whole, the overall expression level in intestinal is higher than that of other three tissues, which may suggest that the functions of these genes are more active in intestinal. Function annotation and pathway enrichment analysis revealed that the host targets were largely involved in signaling pathway and immune pathway, such as interferon-gamma signaling pathway, VEGF signaling pathway, EGF receptor signaling pathway, B cell activation, and T cell activation.ConclusionsAlthough the predicted PPIs may contain some false positives due to limited data resource and poor research background in non-model species, the computational method still provide reasonable amount of interactions, which can be further validated by high throughput experiments. The findings of this work will contribute to the development of system biology for GCRV infectious diseases, and help guide the identification of novel receptors of GCRV in its host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1500-8) contains supplementary material, which is available to authorized users.