Integrated analysis of viral miRNAs, mRNA and protein in the caudal fin cells of C. auratus gibelio with cyprinid herpesvirus 2 infection.

Cyprinid herpesvirus 2 (CyHV-2), a member of the genus Cyprinivirus in the family Alloherpesviridae, has attracted worldwide attention because it causes severe disease and high mortality in crucian carp and goldfish. In this study, we focus on mRNA, protein and viral miRNA expression profiles in C. auratus gibelio caudal fin (GiCF) cells infected with CyHV-2, using high-throughput sequence techniques and TMT-labelled analyses. The results revealed that 156 virus genes were differentially expressed during the infection. Among these differentially expressed genes, 7 viral genes were significantly up-regulated and 28 were significantly down-regulated at 96 hpi (hours post-infection) vs 48 hpi. Besides, a total of 78 viral proteins, including a large number of membrane proteins and capsid proteins associated with the viral assembly, were successfully detected by using proteome analysis. Furthermore, a total of 225,143,474 raw reads were generated from cDNA library of CyHV-2-infected GiCF cells using high-throughput sequencing technology. Following annotation and secondary structure prediction, 10 viral miRNAs were found as significantly modulated in CyHV-2-infected GiCF cells (2 down-regulated and 8 up-regulated). Finally, the CyHV-2 genes (orf19, orf23, orf118, orf121, orf127) targeted by the viral miRNA CyHV-2-KT-635 identified in this study, were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the regulation of CyHV-2-KT-635 on orf121 protein expression was verified by western blotting assay. Taken together, this study provides a valuable basis for further research on the expression of virus genes during CyHV-2 replication and the molecular mechanisms by which miRNA may regulate CyHV-2 virus.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus.

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

Detection of Betanodavirus in experimentally infected European seabass (Dicentrarchus labrax, Linnaeus 1758) using non-lethal sampling methods.

One of the major disease threats affecting the Mediterranean aquaculture industry is viral encephalopathy and retinopathy (VER). The target organs for Betanodavirus detection are the brain and eyes, obtained through lethal sampling. This study aimed to evaluate the efficacy and suitability of non-lethal samples for detecting Betanodavirus in European seabass (Dicentrarchus labrax). European seabass juveniles were infected with Betanodavirus, by either an intramuscular injection or immersion (107 TCID50 /ml and 106 TCID50 /ml, respectively), and samples collected 7, 15 and 30 days post-infection (dpi). The brain was collected as a lethal sample, and gills, caudal fin and blood as non-lethal tissues for detecting Betanodavirus by quantitative reverse transcription PCR (RT-qPCR). The presence of virus in non-lethal tissues was inconsistent, with lower viral loads than in the brain. For blood, higher viral loads were detected in intramuscular-infected fish at 15 dpi until the end of the challenge. Serum antibodies against Betanodavirus were assessed using an enzyme-linked immunosorbent assay (ELISA). Antibodies were detected as early as 7 dpi, with higher mean antibody titres at 15 and 30 dpi. The presence of Betanodavirus-specific antibodies indicates that this is a suitable evaluation method for detecting early stages of the infection.

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus.

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.

Persistent development of adomavirus and aquareovirus in a novel cell line from marbled eel with petechial skin haemorrhage.

In Taiwan, a petechial haemorrhage disease associated with mortality has affected marbled eels (Anguilla marmorata). The eels were revealed to be infected with adomavirus (MEAdoV, previously recognized as a polyoma-like virus). In this study, cell line DMEPF-5 was established from the pectoral fin of a diseased eel. DMEPF-5 was passaged >70 times and thoroughly proliferated in L-15 medium containing 2%-15% foetal bovine serum at 20-30°C. Transcripts of neural cell adhesion molecule 1 and nestin genes, and nucleic acids of MEAdoV and a novel reovirus (MERV) in the cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Phylogenetic analysis revealed that the AdoV LO8 proteins mostly relate to adenovirus adenain, whereas MERV is close to American grass carp reovirus in Aquareovirus G, based on a partial VP2 nucleotide sequence. DMEPF-5 cells are susceptible to additional viral infection. Taken together, the marbled eels with the haemorrhagic disease have coinfection with MEAdoV and MERV, and the pathogenic role of MEAdoV and MERV warrants research. DMEPF-5 has gene expression associated with mesenchymal stem and progenitor cells and is the first cell line persistently infected with adomavirus and aquareovirus. DMEPF-5 can facilitate studies of such viruses and haemorrhagic disease.

Development, characterization and virus susceptibility of a continuous cell line from the caudal fin of marbled eel (Anguilla marmorata).

A continuous cell line consisting mostly of epithelioid cells was established from the caudal fin of marbled eels (Anguilla marmorata) and designated as marbled eel caudal fin (MECF)-1. The cells multiplied well in Leibovitz's L-15 medium containing 2% to 15% foetal bovine serum at temperatures of 20°C to 35°C and were subcultured for >90 passages during a 5-year period from 2012 to 2017. Transcripts of ictacalcin, keratin 13, cd146, nestin, ncam1 and myod1 were demonstrated in the cells using reverse transcription polymerase chain reaction. The results indicated that MECF-1 was composed of epidermal and mesenchyme stem and progenitor cells including myoblasts. MECF-1 was susceptible to Japanese eel herpesvirus HVA980811, marbled eel polyoma-like virus (MEPyV), aquabirnavirus MEIPNV1310 and aquareovirus CSV. By contrast, MECF-1 was noted refractory to megalocytiviruses RSIV-Ku and GSIV-K1 infection. Moreover, the cells were resistant to betanodavirus infection. In conclusion, MECF-1 derived from marbled eel is suitable for studies on anguillid viruses and interaction with host cells.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.

Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF-1) cells infected with red-spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non-infected groups, 51 and 16 differentially expressed miRNAs (DE-miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE-miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE-miRNAs covered a wide range of functions, such as regulation of transcription, oxidation-reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE-miRNAs including miR-1, miR-30b, miR-150, and miR-184 on RGNNV replication were evaluated, and the results showed that over-expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection.

Multiple Passages of Grunt Fin Cells Persistently Infected with Red Seabream Iridovirus (RSIV) at 15ºC or 30ºC to Yield Uninfected Cells.

Red seabream iridovirus (RSIV), a member within genus Megalocytivirus (Iridoviridae), causes serious economic losses to marine fish aquaculture industry in East Asia. In this study, we established a Blue Striped Grunt Haemulon sciurus fin (grunt fin; GF) cell line persistently infected with RSIV (PI-GFRSIV) by subculturing GF cells that survived RSIV inoculation. PI-GFRSIV cells were morphologically indistinguishable from naive GF cells. They could stably produce RSIV at approximately 104.9 ± 0.5 genomes per microliter after 24 passages over 18 months. The optimum temperature to produce RSIV in PI-GFRSIV cells was 25°C. These cells also produced RSIV at 15, 20, and 30°C with multiple subcultures. The amount of RSIV yielded from PI-GFRSIV cells decreased gradually by multiple subculturing at 15°C or 30°C. Red seabream iridovirus was no longer detected from PI-GFRSIV cells after subcultures at these temperatures. These PI-GFRSIV cells freed from RSIV infection exhibited a level of RSIV productivity similar to those of naive GF cells after inoculation with RSIV. Therefore, we consider that these PI-GFRSIV cells were no longer infected with RSIV after multiple subculturing at 15°C or 30°C. Received October 15, 2015; accepted June 27, 2016.

Host responses of Japanese flounder Paralichthys olivaceus with lymphocystis cell formation.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD). In this study, we investigated the mechanisms of lymphocystis cell (LCC) formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected Japanese flounder, Paralichthys olivaceus were monitored by visual confirmation and real-time PCR, respectively. The gene expression changes in the fish fin were investigated by microarray experiments. LCCs firstly appeared in the fish at 21 days post infection (dpi). LCD incidence increased with time and reached 92.9% at 62 dpi. LCDV genome was firstly detected from dorsal fins at 14 dpi, and the relative amount of the genome gradually-increased until 56 dpi. Since the occurrence of LCC was approximately synchronized with increasing of the virus genome, virus replication might play important roles for LCC formation. The microarray detected a few gene expression changes until 28 dpi. However, the number of expression changed genes dramatically increased between 28 and 42 dpi in which LCCs formation was active. From the microarray data analyses, apoptosis and cell division related genes were down-regulated, whereas cell fusion and collagen related genes were up-regulated at 42 dpi. Together with the observation of morphological changes of LCCs in previous reports, it is suggested that the following steps are involved in LCC formation: the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a hyaline capsule associated with the alteration of collagen fibers.

Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio.

A continuous cell line (KF-101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF-101 cell line multiplied abundantly in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF-101 cells contain keratin, junction proteins connexin-43 and occludin, and ectodermal stem-cell marker Pax-6, but not vimentin. Furthermore, the KF-101 cells reacted with anti-human DARPP-32 and anti-human GATA-4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP-32 and GATA-4 antibodies in the KF-101 cells were the suggested phosphatase-1 inhibitor-1 and GATA-3, respectively. In addition, the KF-101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF-101 cells are suitable materials for investigating biological and virological development.

Specific regulation of the chemokine response to viral hemorrhagic septicemia virus at the entry site.

The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

New RT-qPCR assay for viral nervous necrosis virus detection in sea bass, Dicentrarchus labrax (L.): application and limits for hatcheries sanitary control.

A sensitive and quantitative one step RT-qPCR method was developed to study Viral Nervous Necrosis (VNN) virus loads in sea bass Dicentrarchus labrax (L.) in hatcheries. After determining the limits of this new method, fin tissues were identified as an interesting new simple non-invasive sample source, which might be useful for screening D. labrax (L.) in hatcheries. We observed VNN virus strain V26 associated to D. labrax (L.) eggs and it's release in tank water during spawning suggesting both vertical transmission to the eggs and the possibility of horizontal transmission by contamination of tank water. VNN is widespread in water bodies and has the ability to infect a large number of fish species, with this in mind, this PCR technique may be used for the surveillance of various fish farms.

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV).

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10(5) plaque-forming units (PFU) mL(-1) VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory-reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10(3) PFU mL(-1) ) than among groups that survived exposure to VHSV (1.0-2.9 × 10(2) PFU mL(-1) ); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39-47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

BACKGROUND: Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). RESULTS: Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). CONCLUSIONS: The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

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.

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus.

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.

Establishment and characterization of fin-derived cell line from ornamental carp, Cyprinus carpio koi, for virus isolation in India.

Cyprinus carpio koi fin (CCKF) cell line was established and characterized from the caudal fin tissue of ornamental common carp, C. carpio koi. This cell line has been maintained in L-15 medium supplemented with 15% foetal bovine serum (FBS) and subcultured more than 52 times over a period of 24 mo. The CCKF cell line consisted of epithelial cells and was able to grow at temperatures between 22 and 35°C with an optimum temperature of 28°C. The growth rate of these cells increased as the proportion of FBS increased from 2 to 20% with optimum growth at the concentrations of 15% FBS. Karyotype analysis revealed that the modal chromosome number of CCKF cells was 2n = 100. Partial amplification and sequencing of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CCKF cell line originated from ornamental common carp. The CCKF cells showed strong reaction to the cytokeratin marker, indicating that it was epithelial in nature. The extracellular products of Vibrio cholerae MTCC 3904 and Aeromonas hydrophila were toxic to the CCKF cells and not susceptible to viral nervous necrosis virus (VNNV). These CCKF cells were confirmed for the absence of Mycoplasma sp. by polymerase chain reaction. Furthermore, 90% of viable cells could be effectively revived 4 mo after cryopreservation from CCKF cell population suggesting the possibility of long-term storage of the cells.

Fin and gill biopsies are effective nonlethal samples for detection of viral hemorrhagic septicemia virus genotype IVb.

Nonlethal sampling is becoming a common method to diagnose fish diseases, especially with the availability of molecular testing. Viral hemorrhagic septicemia virus (VHSV) is a viral pathogen of finfish distributed worldwide. Although VHSV has been known to occur in some parts of the world for decades, a new genotype, IVb, recently emerged in the Laurentian Great Lakes of northeastern North America. Golden shiners (Notemigonus crysoleucas; Mitchill, 1814) and fathead minnows (Pimephales promelas; Rafinesque, 1820) were exposed to VHSV-IVb doses between 10(2) and 10(6) plaque forming units per fish by intraperitoneal injection at 10°C. Both species experienced significant mortality after exposure, ranging from 38% to 52% in golden shiners and from 35% to 95% in fathead minnows. In golden shiners, a fin or gill sample was somewhat less sensitive at detecting VHSV-IVb by quantitative reverse transcription polymerase chain reaction (qRT-PCR) than a pooled organ sample (consisting of liver, anterior and posterior kidney, spleen, and heart), however the relative sensitivity increased when a fin and gill sample were tested in parallel. In fathead minnows, a fin or gill sample tested alone or in parallel was relatively more sensitive than a pooled organ sample by qRT-PCR. Specificity was 100% for all sample types in both species. The results suggest that fin and gill biopsies are useful tools to test for VHSV in live fish.

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass(Dicentrarchus labrax) during the course of an experimental challenge.

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China.

A new continuous cell line (KCF-1) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than 100 passages since the present study was initiated in March 2006. KCF-1 predominantly consisted of short fibroblast-like cells and grew well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chromosome analysis revealed that 56% of the KCF-1 cells maintained normal diploid chromosome number (2n=100) at Passage 82. Using the KCF-1 cell line, a strain of cyprinid herpesvirus 3 (designated as CyHV-3-QY08) was isolated from the diseased koi. CyHV-3-QY08 continuously propagated in the KCF-1 cells, as confirmed by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). KCF-1 cells infected with CyHV-3-QY08 produced typical cytopathic effects characterized by severe vacuolation, deformation of nuclei, and marginalization of the nuclear chromatin, which are consistent with those of previous reports. CyHV-3-QY08 was purified and subsequently analyzed by SDS-PAGE and TEM. The results showed that the purified virions contained two types of morphologies and were composed of more than 30 obvious viral polypeptides. An infectivity experiment revealed that CyHV-3-QY08 could cause 100% mortality in the infected koi. Based on the genome sequence of CyHV-3-I/U, the CyHV-3(I/U)-ORF136 homologue in CyHV-3-QY08 was cloned and sequenced. Multiple sequence alignments of CyHV-3-I/U-ORF136 homologues showed that CyHV-3-QY08 belonged to the typical Asian genotype. The CyHV-3(I/U)-ORF136 homologue seems to be a novel molecule marker, which can be used to distinguish Asia isolates from Europe-America strains.