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A strain of Clostridium butyricum (Cb) was isolated and characterized as an immunostimulant for gibel carp. Healthy gibel carp were fed and immersed with or without Cb (group Cb + V and group V), and then were intraperitoneally injected with Carassius auratus herpesvirus (CaHV) respectively. The fish began to die at 4 days post injection (dpi). The overall survival rate of group Cb + V (45%) is more than that of group V (11%), which indicated the significant protection effect of Cb. The RT-qPCR results showed that virus major capsid protein gene (MCP) copy numbers of group Cb + V was significantly lower (P < 0.05) than that of group V at 3 dpi. Meanwhile, the expression levels of the four host innate immunity-related genes (IL11, IRF7, PKR, and Mx) were significantly higher (P < 0.01) in group Cb + V than in group V at this time point. IRF7 and PKR also had significantly higher expression levels in group Cb + V than in group V at 0 dpi, which revealed enhanced immune responses in Cb-treated fish. Treatment with Cb prompted stronger host immune responses at the early stage of virus infection, and then resulted in the survival of gibel carp. To our knowledge, it is the first report about the immune protection of fish from herpesvirus by probiotics. [ABSTRACT FROM AUTHOR]

Aquatic viruses include infected viruses in aquatic animals, plants and microorganisms, and free-floating viruses (virioplankton) in water environments. In the last three decades, a huge number of aquatic viruses, especially diverse free-floating viruses, including cyanophages, phycoviruses, archaea viruses, giant viruses, and even virophages, have been identified by virological experiments and metagenomic analyses. Based on a comprehensive introduction of aquatic virus classification and their morphological and genetic diversity, here, we summarize and outline main virus species, their evolutionary contribution to aquatic communities through horizontal gene transfer, and their ecological roles for cyanobacterial bloom termination and global biogeochemical cycling in freshwater and marine ecosystems. Thereby, some novel insights of aquatic viruses and virus-host interactions, especially their evolutionary contribution and ecological rolesin diverse aquatic communities and ecosystems, are highlighted in this review. [ABSTRACT FROM AUTHOR]

Rana grylio virus (RGV), a member of genus Ranavirus in the family Iridoviridae, is a viral pathogen infecting aquatic animal. RGV 43R has homologues only in Ranavirus and contains a transmembrane (TM) domain, but its role in RGV infection is unknown. In this study, 43R was determined to be associated with virion membrane. The transcripts encoding 43R and the protein itself appeared late in RGV-infected EPC cells and its expression was blocked by viral DNA replication inhibitor, indicating that 43R is a late expressed protein. Subcellular localization showed that 43R-EGFP fusion protein distributed in cytoplasm of EPC cells and that TM domain is essential for its distribution in cytoplasm. 43R-EGFP fusion protein colocalized with viral factories in RGV-infected cells. A recombinant RGV deleting 43R (Δ43R-RGV) was constructed by homologous recombination to investigate its role in virus infection. Compared with wild type RGV, the ability of Δ43R-RGV to induce the cytopathic effect and its virus titers were significantly reduced. Furthermore, it is revealed that 43R deletion significantly inhibited viral entry but did not influence viral DNA replication by measuring and comparing the DNA levels of RGV and Δ43R-RGV in the infected cells at the early stage of infection. RGV neutralization with anti-43R serum reduced the virus titer. Therefore, these data showed that RGV 43R is a late gene that encodes an envelope protein involved in RGV entry. [ABSTRACT FROM AUTHOR]

Crucian carp Carassius auratus herpesvirus (CaHV) was isolated from diseased crucian carp with acute gill hemorrhages and high mortality. The CaHV genome was sequenced and analyzed. The data showed that it consists of 275,348 bp and contains 150 predicted ORFs. The architecture of the CaHV genome differs from those of four cyprinid herpesviruses (CyHV1, CyHV2, SY-C1, CyHV3), with insertions, deletions and the absence of a terminal direct repeat. Phylogenetic analysis of the DNA polymerase sequences of 17 strains of Herpesvirales members, and the concatenated 12 core ORFs from 10 strains of alloherpesviruses showed that CaHV clustered together with members of the genus Cyprinivirus, family Alloherpesviridae. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture. [ABSTRACT FROM AUTHOR]

Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp ( Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein. Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100 % mortality at dilutions up to 10 in rare minnows ( Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10 % when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease. [ABSTRACT FROM AUTHOR]

A new grass carp reovirus strain, tentatively named GCReV-109, was isolated in Hubei, China, and its complete genome sequence was determined. The genome contained 11 double-stranded RNA segments (S1-S11) covering 24,620 base pairs. All of the segments had conserved terminal nucleotides, with GUAA/U at the 5' end and UCAUC at the 3' end. Protein sequence comparison showed that GCReV-109 was most closely related to GCRV-GD108 and shared 96.6-99.5 % protein sequence identity but only shared 16.7-46.1 and 15.1-45.4 % identity with GCRV-873 and HGDRV, respectively. Phylogenetic analysis showed that grass carp reovirus strains in China can be divided into three genotypes. Further analysis revealed homology between the GCRV-109 VP56 and HGDRV VP55 proteins, as well as GCReV-109 NS38, GCRV-873 NS38, and HGDRV VP39. The results of these comparisons also indicated that the homology between viruses was not necessarily linked to their geographical distribution. Our study will help in recognizing and understanding the genome structure and genetic diversity of grass carp reovirus. [ABSTRACT FROM AUTHOR]

Paralichthys olivaceus rhabdovirus (PORV), which is associated with high mortality rates in flounder, was isolated in China in 2005. Here, we provide an annotated sequence record of PORV, the genome of which comprises 11,182 nucleotides and contains six genes in the order 3′-N-P-M-G-NV-L-5′. Phylogenetic analysis based on glycoprotein sequences of PORV and other rhabdoviruses showed that PORV clusters with viral haemorrhagic septicemia virus (VHSV), genus Novirhabdovirus, family Rhabdoviridae. Further phylogenetic analysis of the combined amino acid sequences of six proteins of PORV and VHSV strains showed that PORV clusters with Korean strains and is closely related to Asian strains, all of which were isolated from flounder. In a comparison in which the sequences of the six proteins were combined, PORV shared the highest identity (98.3 %) with VHSV strain KJ2008 from Korea. [ABSTRACT FROM AUTHOR]

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10 TCID ml, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10 TCID ml). The titers of RGV and SVCV in GCO were 10 TCID ml and 10 TCID ml, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection. [ABSTRACT FROM AUTHOR]

Infection with Rana grylio virus (RGV), an iridovirus isolated in China in 1995, resulted in a high mortality rate in frogs. The complete genome sequence of RGV was determined and analyzed. The genomic DNA was 105,791 bp long, with 106 open reading frames (ORFs). Dot plot analysis showed that the gene order of RGV shared colinearity with three completely sequenced ranaviruses. A phylogenetic tree was constructed based on concatenated sequences of iridovirus 26 core-gene-encoded proteins, and the result showed high bootstrap support for RGV being a member of the genus Ranavirus and that iridoviruses of other genera also clustered closely. A microRNA (miRNA) prediction revealed that RGV could encode 18 mature miRNAs, many of which were located near genes associated with virus replication. Thirty-three repeated sequences were found in the RGV genome. These results provide insight into the genetic nature of RGV and are useful for laboratory diagnosis for vertebrate iridoviruses. [ABSTRACT FROM AUTHOR]

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK–GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells. [ABSTRACT FROM AUTHOR]

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Δψm collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-κB activation and intracellular Ca2+ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells. [ABSTRACT FROM AUTHOR]

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with $$ 2n = 8st + 40t $$ . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (108.5 TCID50 ml−1), while the viral titer of frog Rana grylio virus 9807 (RGV9807) reached 103.5 TCID50 ml−1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies. [ABSTRACT FROM AUTHOR]

13R, a core gene of Andrias davidianus ranavirus (ADRV), encoded a protein containing a transmembrane domain (TMD) and a restriction endonuclease-like domain. However, the characterization and function of 13R and the protein it encodes remain unclear. In this study, Chinese giant salamander thymus cell (GSTC) was used to investigate the function of 13R. The results showed that the 13R transcripts were detected first at 8 h post-infection (hpi) by RT-PCR and the protein was detected first at 24 hpi by western blot, but the transcription was inhibited by cycloheximide and cytosine arabinofuranoside, indicating that 13R is a viral late gene. Subcellular localization showed that the 13R was co-localized with endoplasmic reticulum (ER) in the cytoplasm, while 13R deleting TMD (13RΔTM) was distributed in cytoplasm and nucleus. During ADRV infection, 13R was observed first in the cytoplasm and nucleus, and later aggregated into the viromatrix, whereas 13RΔTM remain dispersed in cytoplasm and nucleus. Western blot analysis suggested that 13R was a viral non-structural protein and its overexpression did not affect the viral titer in GSTC. All these indicated that the TMD of 13R is crucial for the co-localization into the ER and the viromatrix. [ABSTRACT FROM AUTHOR]