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1.
The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0 TCID50/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.  相似文献   

2.
Abstract. Two populations of channel catfish were examined for the presence of channel catfish virus (CCV) by use of a nucleic acid probe. In one population of 22 fish with no history of CCV, viral DNA was found in every liver. These fish had previously been examined by a technique involving co-cultivation of their leucocytes with catfish tissue culture cells. The co-cultivation method had identified virus in 10 of these fish. The second fish population consisted of 14 adults that had survived a CCV outbreak in 1980. Of the 14 fish, 11 showed positive indication of CCV DNA. The tissue distribution of the CCV differed from fish to fish. All fish from the first group and one fish from the second group showed some alterations in the DNA banding patterns expected from pure CCV DNA. This might be indicative of modifications in the genomic structure of the CCV DNA when the virus is latent in a fish.  相似文献   

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5.
Channel catfish virus (CCV) causes an acute haemorrhagic disease in channel catfish, Ictalurus punctatus (Rafinesque), fry and fingerlings. The present study describes a polymerase chain reaction (PCR)-based assay for detection of CCV DNA in the tissues of acutely infected juvenile catfish. The assay is rapid, sensitive and specifically detects CCV DNA derived from epidemiologically distinct viral isolates. The use of two independent PCR primers sets, each specific for particular CCV genes (open reading frames 8 and 59), provides a means to confirm the results and minimize false-positive results. The method identifies CCV DNA in several tissues of acutely infected fish, including the brain, blood, intestine, kidney and liver. The CCV PCR assay is useful for the diagnosis of acute CCV disease and for studies to investigate the molecular basis of CCV pathogenesis.  相似文献   

6.
A nucleic acid probe for channel catfish virus (CCV) was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment generated by digestion of CCV DNA with the restriction enzyme EcoRI. The probe was used to examine DNA isolated from tissues of fish that had been injected with CCV. Viral DNA was detected in some tissues of various injected fish. The sensitivity limit of detection was determined to be one viral DNA per cell.  相似文献   

7.
Channel catfish virus (CCV) disease is an acute haemorrhagic disease in juvenile channel catfish ( Ictalurus punctatus ). To date channel catfish is the only species affected by natural outbreaks of the CCV but juvenile large mouth bass ( Micropterus salmoides ) and silurus ( Silurus meriaionalis ) have suffered high mortalities in recent years in China. Histopathological phenomenon of sick fish is similar to CCV disease, and the identified virus was CCV. In this report, the pathogenicity of infectious CCV was examined by infection trials on the first known host species, the channel catfish and other teleosts. Our results indicated that there were higher detection rates of CCV from large mouth bass and silurus fish. Channel catfish virus did not induce mortality in other cypriniformes, but histopathological studies revealed that carp might be infected by both bathing and intraperitoneal infection. No deaths, clinical or histopathological signs, were found in the six other species exposed by immersion or injection. Experimental infection studies confirm that CCV infect not only channel catfish but also other species (large mouth bass, silutus and carp). The outbreaks of CCV disease only occurred when the cultured temperature was above 25 °C.  相似文献   

8.
斑点叉尾鮰病毒囊膜蛋白ORF6在昆虫细胞中的表达   总被引：2，自引：0，他引：2  
为构建基于杆状病毒表达系统的CCV新型亚单位疫苗,将CCV的囊膜蛋白基因ORF6克隆至杆状病毒转座载体pFastBacTM 1质粒中,并将阳性重组转座质粒转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,获得重组子rBacmid-ORF6。在脂质体介导下将该重组子转染sf9昆虫细胞,获得重组杆状病毒AC-ORF6。AC-ORF6感染的sf9昆虫细胞,经超薄切片电镜观察,可见重组杆状病毒呈多粒包埋,经间接免疫荧光、Western-blotting检测,CCV的ORF6蛋白可以在感染了AC-ORF6的sf9细胞中表达。研究表明,获得了插入ORF6基因的重组杆状病毒,并且该基因可以在重组杆状病毒介导下在昆虫细胞中表达,从而为基于CCV ORF6的杆状病毒亚单位疫苗研究奠定了基础。  相似文献   

9.
Although channel catfish virus (CCV) is thought to be vertically transmitted, no mechanism has been demonstrated for such a process. When contained in small volumes of saline, CCV associated rapidly with channel catfish leukocytes and sperm, associated slowly with a continuous cell line of channel catfish cells and did not adhere to channel catfish eggs. This virus-sperm adherence appeared to be a mechanism by which eggs might become infected with virus. However, virus-sperm adherence was not detected when sperm were added to 250 ml of CCV-containing water, indicating that sperm are probably not involved with transmitting CCV into eggs from water or ovarian fluid. Egg infections in channel catfish, if they occur, must be accomplished by mechanisms different from those thought to be involved in the infection of salmonid eggs by infectious hematopoietic necrosis virus.  相似文献   

10.
Abstract Two groups of European catfish, Silurus glanis L., fingerlings were infected with channel catfish virus (CCV) by either intraperitoneal injection with 105 TCID50 of CCV, or bathing in water containing 105 TCID50 of CCV per 1·0 ml. The virus was isolated from spleen, intestine and brain of CCV-injected fish at day 1 and the titres ranged from 102·1 to 103·3 TCID50/g. However, the tissue distribution of CCV was irregular and no virus was isolated after day 3 post-exposure. In CCV-bathed fish, the virus was isolated only from the liver of one specimen at day 3 post-exposure. No clinical signs of CCV disease developed in any of the fish. Specimens in each regime from all sampling periods showed some minor histopathological changes, but there were no differences between treatments. Lesions included oedema and focal haemorrhage in the liver and the spleen was congested. Electron micrographs of tissue samples showed the presence of a few virus particles around the nuclei of kidney, spleen and intestinal cells, and in or around a myelinated nerve within the optic lobes of infected fish during the first 4 days of infection.  相似文献   

11.
A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.  相似文献   

12.
A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.  相似文献   

13.
To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.  相似文献   

14.
This study established and characterized a new cell line (MAF) from the fin of blunt snout bream (Megalobrama amblycephala), a freshwater fish cultivated in China. MAF cells proliferated well in medium 199 supplemented with 10 % fetal bovine serum at 28 °C and have been subcultured more than 95 times in almost a year. MAF cells were revived at 90–95 % viability after 3–6 months of storage in liquid nitrogen. Karyotyping indicated that the modal chromosome number of MAF cells was 48. The MAF cell line consisted predominantly of fibroblastic and epithelial-like cells from M. amblycephala, which was confirmed by immunofluorescence and mitochondrial 12s rRNA sequencing. Viral susceptibility tests showed that MAF cells were susceptible to infection by snakehead rhabdovirus, spring viremia carp virus, and channel catfish virus, which was demonstrated by the presence of cytopathic effect, high viral titers, and PCR products. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells. Cu2+ was also cytotoxic to MAF cells, and the 24-h IC50 value was 144.48 μmol/l. When MAF cells were transfected with pEGFP-N1 plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to 5 %. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies.  相似文献   

15.
Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.  相似文献   

16.
The potential use of channel catfish virus (CCV) (Ictalurid herpesvirus 1) as a vaccine vector for the channel catfish industry was investigated by inserting the Escherichia coli lacZ gene into the CCV genome and evaluating the immune response to the foreign gene product in catfish exposed to the recombinant. The recombinant virus was produced by inserting the lacZ in reading frame with the ATG start codon of the CCV thymidine kinase (TK) gene in the recombinant transfer plasmid pBSCV457 described previously. The plasmid was then cotransfected with CCV DNA in a TK gene-mediated selectable homologous recombination. The recombinant progeny were selected by resistance to 0.1 mM acycloguanosine (acyclovir) and the production of blue plaques in the presence of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). The resultant construct (CCVlacZ) was TK?, and contained the lacZ gene at both TK loci in the genome. β-Galactosidase expression in infected CCO ceils reached 0.53 μg per 106 CCO cells at 12 h post-infection. When channel catfish fingerlings were immersion exposed to CCVlacZ, these developed an antibody response to the inserted foreign gene product which peaked at approximately 15–20 days post-infection. Additionally, the anti-β-galactosidase response was significantly enhanced when the fingerlings were re-exposed to the virus 20 days after the initial exposure. These results demonstrate that foreign genes can be inserted into and expressed by CCV and that such constructs could be used as vaccine vectors.  相似文献   

17.
A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT‐PCR and viral titre assays. In contrast, PaF cells were resistant to red‐spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune‐related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen–silver pomfret interaction.  相似文献   

18.
A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n = 44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.  相似文献   

19.
Knowledge about the effects of captivity and environmental stress on fish populations is basic to culture them for ornamental, conservation or exploitation purposes. The aim of this study was to test how captivity affect morphological development of bilateral characters and whether levels of fluctuating asymmetry, morphological abnormalities and ectoparasites reflect developmental stress caused by adverse environmental conditions in two species of cyprinids, goldfish Carassius auratus and two varieties of carp Cyprinus carpio, scale and mirror carp. Samples from captivity were caught in an artificial pond (Madrid City, Spain), which presents poor environmental conditions; while samples from nature were captured in the Ebro River basin (NE Spain). Six meristic and four morphometric bilateral characters were measured and compared between the two samples. Additionally, the incidence of morphological abnormalities and ectoparasites was also recorded. Captive goldfish presented significant lower number of scales of the lateral line, scale rows above lateral line and length of pectoral and ventral fins. Captive carps showed significant shorter first barbels and longer ventral fins. Ornamental goldfish showed significant higher levels of fluctuating asymmetry than wild samples for number of branched rays of pectoral fin, lateral line, scale rows below lateral line and length of pectoral and ventral fins. Ornamental carps presented a similar trend for the same characters, except for branched rays and length of pectoral fins, together with the length of second barbels. In the artificial pond, the incidence of abnormalities was higher in carp, whereas goldfish showed a higher occurrence of ectoparasites. These facts support the hypothesis of a different species response to environmental stress. The overall results indicate that fish from the artificial pond are subjected to environmental stress caused by adverse conditions such as overcrowding and lower water quality. Moreover, estimated parameters in this study mean a useful tool to detect developmental instability in confined fish.  相似文献   

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