BF-2 Cell Lines Research Articles

β-sitosterol isolated from the leaves of Trema orientalis (Cannabaceae) promotes viability and proliferation of BF-2 cells.

Trema orientalis is a pioneer species in the cannabis family (Cannabaceae) that is widely distributed in Thai community forests and forest edges. The mature leaves are predominantly used as an anti-parasite treatment and feed for local freshwater fish, inspiring investigation of their phytochemical composition and bioactivity. The purpose of this work was to investigate the bioactive compounds in T. orientalis leaf extract and their cytotoxicity in the BF-2 fish cell line (ATCC CCL-91). Flash column chromatography was used to produce 25 mL fractions with a mixture solvent system comprised of hexane, diethyl ether, methanol, and acetone. All fractions were profiled with HPLC-DAD (mobile phase methanol:aqueous buffer, 60:40 v/v) and UV detection (wavelengths 256 and 365 nm). After drying, a yellowish powder was isolated from lipophilic leaf extract with a yield of 280 µg/g dry weight. Structure elucidation by nuclear magnetic resonance (NMR) indicated it to consist of pure β-sitosterol. The lipophilic extract and pure compound were evaluated for cytotoxicity using BF-2 cells. MTT assays showed both leaf extract and pure compound at 1 µg/mL to increase cell viability after 24 h treatment. The respective half maximal inhibitory concentration (IC50) values of leaf extract and β-sitosterol were 7,027.13 and 86.42 µg/ml, indicating a lack of toxicity in the BF-2 cell line. Hence, T. orientalis can serve as a source of non-toxic natural lipophilic compounds that can be useful as bioactive ingredients in supplement feed development.

Detection and characterization of a novel marine birnavirus isolated from Asian seabass in Singapore

BackgroundLates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods.MethodsIn order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2′-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses.ResultsThe LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days’ post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family.ConclusionsThese findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

Nickel nanoparticles induces cytotoxicity, cell morphology and oxidative stress in bluegill sunfish (BF-2) cells

The rationale of the current study was to assess the suitability of BF-2 cell line as a model to assess nanotoxicity in the caudal fin cells of bluegill sunfish in vitro. The current study investigates the potential toxicity, morphological changes and oxidative stress of nickel nanoparticles (Ni NPs) in bluegill sunfish cells (BF-2) using mitochondrial, neutral red uptake and lactate dehydrogenase assays. Results indicated a concentration dependent cytotoxic effect after 24 h in mitochondrial, lysosomal and lactate dehydrogenase activities. BF-2 cells morphology was altered when exposed to 30 μg ml−1 concentrations of Ni NPs for 24 h. Dose dependent increase of oxidative stress was evidenced in BF-2 cells when exposed to Ni NPs, showed significant escalation in peroxidation of lipids (LPO), protein carbonyl (PC), glutathione sulfo-transferase (GST) and glutathione peroxidase (GPX) as compared to their experimental controls. However, the catalase (CAT) and total glutathione content (TGSH) was found to decrease dose dependently in BF-2 cells exposed to Ni NPs. The current study demonstrated that BF-2 cells may serve as a sensitive indicator for aquatic contaminant evaluations in toxicological research.

In vitro characteristics of cyprinid herpesvirus 2: effect of kidney extract supplementation on growth.

Herpesviral hematopoietic necrosis caused by goldfish hematopoietic necrosis virus (now identified as cyprinid herpesvirus 2, CyHV-2) has contributed to economic losses in goldfish Carassius auratus culture and is becoming a major obstacle in Prussian carp C. gibelio aquaculture in China. Several reports have described difficulties in culturing the virus, with the total loss of infectivity within several passages in cell culture. We succeeded in propagating CyHV-2 with a high infectious titer in a RyuF-2 cell line newly derived from the fin of the Ryukin goldfish variety using culture medium supplemented with 0.2% healthy goldfish kidney extract. The addition of kidney extract to the medium enabled rapid virus growth, resulting in the completion of cytopathic effect (CPE) within 4 to 6 d at 25°C. The extract also enabled reproducible virus culture with a titer of 105-6 TCID50 ml-1. The virus cultured using this protocol showed pathogenicity in goldfish after intraperitoneal injection. The virus grew in RyuF-2 cells at 15, 20, 25, 30, and 32°C but not at 34°C or higher. Higher incubation temperatures allowed earlier development of CPE, but culture at 30 and 32°C yielded a lower virus titer than that obtained at other temperatures because of heat inactivation of the propagated virus during cultivation. Cell lines derived from goldfish and ginbuna C. langsdorfii showed high susceptibility to the virus; cell lines from carp were susceptible to the virus using a medium containing goldfish kidney extract, but EPC, FHM, and BF-2 cell lines did not produce any CPE, even in the presence of the extract.

Development and characterization of a largemouth bass cell line.

Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

Characteristics of spring viremia of carp virus strains isolated in different regions of Ukraine

The paper contains results of studies of spring viremia of carp virus (SVCV) strains isolated in specialized fish farms in different regions of Ukraine. Pathogenicity of these isolates has been determined in vitro on cell culture and in vivo on young-of-the-year (YOY) carp (Cyprinus carpio L.). These SVCV isolates showed different pathogenicity towards YOY carp and caused mortality from 35 to 60%. Reproduction of these isolates in FHM, EPC, and BF-2 cell lines caused complex degenerative processes in cells, ultimately resulting in complete monolayer destruction. During experimental infection, it was found that the disease had a systemic nature. YOY carp had signs typical of SVCV infection, and affection of internal organs by the virus resulted in fish death. Clear clinical signs of the viral infection were observed in solitary individuals only, while the majority of fish died without any visible external clinical signs. A similarity between isolated SVCV strains and the reference strain in their physical–...

Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon

Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells.

In vitro and in vivo immune responses induced by a DNA vaccine encoding the VP2 gene of the infectious pancreatic necrosis virus

The work presented here describes the construction of a plasmid encoding the VP2 gene of the infectious pancreatic necrosis virus (IPNV), its expression in BF-2 cells and an evaluation of its activity in brown trout ( Salmo trutta L) soon after injection. Preliminary experiments to evaluate the potential of the plasmid to induce neutralizing antibodies were also performed. We established a BF-2 cell line that expresses VP2 constitutively and we examined the infection of these VP2-transfected BF-2 cells with homologous and heterologous viruses. The expression kinetics of IFN, and of the IFN-induced genes Mx and ISG15, was also evaluated in brown trout over a 15 day interval, and quantified by real-time or semi-quantitative PCR. Type I IFN and Mx are markers of the non-specific innate immune response to viruses and they are involved in antiviral defences. Our results demonstrate that expression of the IPNV VP2 protein in BF-2 cells induces an antiviral state against IPNV and against the infectious haematopoietic necrosis virus (IHNV). In BF-2 infected cells, VP2 inhibited both the IPNV and IHNV-induced cytopathic effect to some extent, as well as the virus yield. In vivo, VP2 was expressed in haematopoietic tissues such as the head kidney of 7 month-old trout. In addition, it induced early immune responses and specific immunity 30 days after injection. IFN mRNA expression increased sharply on the 1st and 15th day post-injection and expression of other IFN-induced genes as Mx and ISG15 was also detected soon after vaccination of brown trout. Moreover, specific antibodies were detected 30 days after vaccination. These results suggest that the VP2 gene is a good candidate for the design of IPNV-DNA vaccines and to investigate the use of cytokines as co-stimulatory molecules.

Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa

Moritella viscosa causes winter ulcer disease in salmonids. The aim of the present work was to isolate and partially characterise an extracellular peptidase from M. viscosa, and to study its role in virulence. The peptidase, termed MvP1, was a 38-kDa metallopeptidase produced in late exponential growth. The optimum temperature for MvP1 was 40 °C, but the enzyme was active over a broad range of temperatures. MvP1 was non-lethal to salmon at concentrations up to 0.22 μg/g fish, but extracellular products were lethal to salmon. MvP1 degraded casein, gelatin and collagen from lumpfish skin. It caused considerable tissue necrosis and hemorrhages at the site of injection, and affected cell–cell adhesions in EPC and BF-2 cell lines, but was not highly cytotoxic. The peptidase partially degraded fish IgM heavy chain but was non-hemolytic. The mvp1 gene was sequenced and encoded a 734-aa polypeptide containing a signal sequence, an N-terminal propeptide, a mature peptidase domain and a C-terminal propeptide. The MvP1 propeptide undergoes both N-terminal and C-terminal processing and different C-terminal processing results in the formation of several active isoforms of the mature peptidase. The catalytic domain showed highest sequence similarity with several vibriolysins (EC 3.4.24.25) originating from Pseudoalteromonas strains, showing up to 80% aa identity. The results indicate that MvP1 is a previously unknown vibriolysin that might affect M. viscosa virulence by aiding in the invasion and dissemination of the bacterium in its host, by causing tissue destruction.

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV)

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.

Interferon mediated antiviral activity against salmonid fish viruses in BF-2 and other cell lines

Double-stranded RNA and type I interferon-like activity induce an antiviral state in vertebrate cells and in several fish cell lines by increasing the expression of proteins that inhibit virus replication. We compared the protection induced by the polyinosinic:polycytidylic acid (poly I:C) or poly I:C plus transfection agents against the infectious pancreatic necrosis virus (IPNV) and the infectious hematopoietic necrosis virus (IHNV) in BF-2 cells, with that induced in RTG-2, CHSE-214, or SAF cells. In addition, we examined the reduction in the infective titers of these viruses and the correlation with Mx protein expression as IFN marker. Furthermore, the suitability of BF-2 cells for the evaluation and optimization of immune responses in an IPNV-IHNV co-infection was assessed. The results demonstrated strong anti-IPNV and anti-IHNV activity (around 90% of infected cells surviving) in BF-2 cells transfected with poly I:C, in which a loss of 1 log 10 or 3 log 10 of the IPNV or IHNV infective titers, respectively, was observed. No antiviral activity was evident in the cells incubated with poly I:C alone. The protection recorded in the co-infection experiments was comparable with those of the single infections. The SAF cell line exhibited the lowest antiviral capacity (45%), which was also increased after transfection with poly I:C. In addition, medium from transfected BF-2 provided protection against IPNV (1 log 10 loss of infective titer) and IHNV (2 log 10 loss of infective titer) in new monolayers, indicating that these cells secreted the factors that induce antiviral activity. A correlation between antiviral activity and Mx protein expression was observed in all the cells. These results indicate that poly I:C transfection could improve IFN-like production in these cell lines. However, the antiviral effectiveness of poly I:C differed between cell lines. On the basis of our findings, we conclude that the BF-2 cell line is a useful model in which to study the role of IFN-induced cytokines in resistance against single or double infections with salmonid fish viruses.

Rapid ecotoxicological testing using transformed BF-2 cells incorporating a luminescent reporter gene

Since there is an ethical need to minimise the experimental use of higher organisms such as fish, especially those used in acute toxicity testing, fish cells are considered to be useful surrogates for fish in toxicity screening. The use of fish cell lines in conventional bioassays such as neutral red retention assays is however labour intensive, lengthy and costly. The use of luminescent reporter genes has been explored in our laboratory. In this study, a transfected BF-2 cell line (BF-2/ luc1) was used for rapid toxicity testing on selected chemicals and results were compared with those obtained with in vivo fish testing and in vitro fish cell neutral red retention assays. The effect of temperature on the sensitivity of BF-2/ luc1 was also investigated. BF-2/ luc1 cells were harvested and suspended in PBS at 2.5–3.0 × 10 6 cells/ml. Individual aliquots of the suspended cells (40 μl each) were incubated for either 0.5 or 6 h at room temperature (22 °C) in the presence or absence of the toxicants. Bioluminescence was assayed using 17.5 μl Brightglo™ luciferase reagent which lysed the cells and provided the substrate luciferin. Luminescence was measured in a luminometer (Turner TD 20/20). The EC 50 values obtained from BF-2/ luc1 cells (0.5–6 h) generally compared well with the LC 50 values (24–96 h) obtained from the in vivo fish tests on a range of species. The present study also showed that BF-2/ luc1 cell sensitivity increased significantly when incubation temperature during toxicant exposure increased from 15 to 35 °C. The use of luminescent reporter genes in monitoring fish cells offers the possible advantages of increased sensitivity over the neutral red retention assay and a more rapid test to replace stain based bioassays, and provides a rapid screening method that could reduce the need for acute fish toxicity testing.

Isolation and Identification of the Infectious Haematopoietic Necrosis Virus (IHNV)-like Agent from Farmed Rainbow Trout (Oncorhynchus mykiss) from Iran

Summary The isolation of viral causative agent of fry trout mortality syndrome in farmed rainbow trout in Iran was discussed. Four hundred twenty samples were collected from six provinces for virological, serological and electron microscopic examinations from October 2001 till May 2002. No cytopathic effect (CPE) was observed in the cell cultures except for EPC and BF-2 cells in which signs of CPE appeared one day postinoculation during the second and third passages of two brooder ovarian fluids. Electron microscopic examination revealed the viral particles measured 67-73×128-182nm in the pelleted materials from the CPE positive in BF-2 and EPC cell lines. Indirect immunoflurecence antibody test were positive for some of smears obtained from kidney tissues of the larvae, the samples of ovarian fluids that showed CPE on EPC and BF-2 cell lines as well as CPE positive cell cultures. Using ELISA eight of all sera samples tested were positive with optical density ranged 0.316-2.268 compared to 0.06 for negative controls.

Structure-activity relationships for diorganotins, chlorinated benzenes, and chlorinated anilines established with bluegill sunfish BF-2 cells

The bluegill sunfish ( Lepomis macrochirus) BF-2 cell line, propagated at 34°C, served as target for evaluation of the acute toxicities of various classes of aquatic pollutants, using the neutral red cytotoxicity assay. For a series of chlorinated benzenes and anilines, the sequence of cytotoxicity was dependent on the degree of chlorination and on their hydrophobicity, as described by their logarithmic octanol/water partition coefficients (long P values). With increasing numbers of chlorine atoms in the ring structure or with increasing log P values, greater cytotoxicity was observed. For a series of diorganotins, the sequence of cytotoxicity was dependent on the length of the carbon chain and upon their hydrophobicity, as described by Hansch π parameters. Thus, increasing the chain length or increasing the Hansch π parameter resulted in greater cytotoxicity. Similar structure-activity relationships for these classes of test agents have been previously established using acute toxicity LC50 assays with aquatic species. The ability of the neutral red in vitro cytotoxicity assay, with cultured fish cells as the bioindicators, to mimic the acute toxicity data obtained from the LC50 assays suggests its utility as a tool for preliminary screening (tier I testing) of aquatic pollutants.

Structure-Activity Relationships for Diorganotins, Chlorinated Benzenes, and Chlorinated Anilines Established with Bluegill Sunfish BF-2 Cells

Structure-Activity Relationships for Diorganotins, Chlorinated Benzenes, and Chlorinated Anilines Established with Bluegill Sunfish BF-2 Cells. BABICH, H., AND BORENFREUND, E. (1988). Fundam. Appl. Toxicol. 10,295–301. The bluegill sunfish (Lepomis macrochirus) BF-2 cell line, propagated at 34oC, served as target for evaluation of the acute toxicities of various classes of aquatic pollutants, using the neutral red cytotoxicity assay. For a series of chlorinated benzenes and anilines, the sequence of cytotoxicity was dependent on the degree of chlorination and on their hydrophobicity, as described by their logarithmic octanol/water partition coefficients (log P values). With increasing numbers of chlorine atoms in the ring structure or with increasing log P values, greater cytotoxicity was observed. For a series of diorganotins, the sequence of cytotoxicity was dependent on the length of the carbon chain and upon their hydrophobicity, as described by Hanschπ parameters. Thus, increasing the chain length or increasing the Hanschπ parameter resulted in greater cytotoxicity. Similar structure-activity relationships for these classes of test agents have been previously established using acute toxicity LC50 assays with aquatic species. The ability of the neutral red in vitro cytotoxicity assay, with cultured fish cells as the bioindicators, to mimic the acute toxicity data obtained from the LC50 assays suggests its utility as a tool for preliminary screening (tier I testing) of aquatic pollutants.