Interaction mechanism of marine birnavirus (MABV) in fish cell lines

Marine birnavirus (MABV) is a member of the genus Aquabirnavirus of the family Birnaviridae. MABV is an unenveloped icosahedral virus about 60 nm in diameter with two genomes of double-stranded RNA. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered into the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. The syntheses of viral proteins pVP2, NS and VP3 and further proteolytic processing after viral infection were examined by using Western blot analysis. pVP2, NS and VP3 were detected in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 4 h after infection. At this time, VP3 underwent further proteolytic processing in the cytosolic fractions of CHSE-214 and RSBK-2 cells. The expression of pVP2, NS and VP3 increased and pVP2 and NS also underwent further proteolytic processing similar to VP3 in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 8 h after infection. The further proteolytic processing of VP3 was detected in the nuclear fractions of CHSE-214, RSBK-2, but VP3 was detected as a single band in the nuclear fraction of FHM cells. pVP2 and NS were detected as thin bands only in the nuclear fractions of CHSE-214 cells. The results of Western blot analysis demonstrated that pVP2, NS and VP3 are localized in the nuclear fraction when they were independently expressed in CHSE-214, RSBK-2, FHM and EPC cells. The expression pattern in the cytosolic fraction was identical among the four cell lines when pVP2 and NS were independently expressed. However, pVP2 and NS were not detected in the nuclear fraction of CHSE-214 cells. Further proteolytic processing of VP3 was detected in both cytosolic and nuclear fractions of RSBK-2 ,FHM and EPC cells (Low level in EPC cell), but not in CHSE-214 cells when VP3 was independently expressed. Then, the processes of preVP2 to form morphological assemblages in the presence of VP3 or the cleavage of VP3 into two proteins in CHSE-214 cells were studied. When preVP2- and VP3 were co-expressed, virion like particles (64 nm, diameter) were observed close to the nuclear membrane by electron microscopy. The co-expression of preVP2 and the cleaved VP3 proteins led to an efficient assembly of tubules (22 nm, diameter). Further important finds will be obtained by this infection system using 4 fish cell lines in the next couple of years.     

Aquatic birnavirus capsid protein,VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells

Aquatic birnavirus induces post-apoptotic necrotic cell death via a newly synthesized protein-dependent pathway. However, the involvement of viral genome-encoded protein(s) in this death process remains unknown. In the present study, we demonstrated that the submajor capsid protein, VP3, up-regulates the pro-apoptotic protein, Bad, in fish and mouse cells. Western blot analysis revealed that VP3 was expressed in CHSE-214 cells at 4 h post-infection (pi), indicating an early role during viral replication. We cloned the VP3 gene and tested its function in fish and mouse cells; VP3 overexpression induced apoptotic cell death by TUNEL assay. In addition, it up-regulated Bad gene expression in zebrafish ZLE cells by threefold at 12 h post-transfection (pt) and in mouse NIH3T3 cells by tenfold at 24 h pt. VP3 up-regulation of Bad expression altered mitochondria function, inducing mitochondrial membrane potential (MMP) loss and activating initiator caspase-9 and effector caspase-3. Furthermore, reduced Bad expression (65% reduction), MMP loss (up to 40%), and enhanced cell viability (up to 60%) upon expression of VP3 antisense RNA in CHSE-214 cells at 24 h post-IPNV infection was observed. Finally, overexpression of the anti-apoptotic gene, zfBcl-xL, reduced VP3-induced apoptotic cell death and caspase-3 activation at 24 h in fish cells. Taken together, these results suggest that aquatic birnavirus VP3 induces apoptosis via up-regulation of Bad expression and mitochondrial disruption, which activates a downstream caspase-3-mediated death pathway that is blocked by zfBcl-xL.     

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.     

Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.     

Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines growing in serum- free suspension cultures

The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant protein expression. For example, the luciferase activity was significantly higher in the rodent BHK and CHO cell lines incubated at 33 °C compared to 37 °C when harvested 19 h post-infection. At 33 °C the specific expression levels increased 10–20 fold during prolongation of the post-infection time up to 50 h. In contrast, a significant decrease in luciferase activity was observed from 26 h post-infection for cell cultures incubated at 37 °C. Only a slight temperature effect on luciferase expression was seen in the human cell line HEK293 and no effect was observed for the subclone293(EBNA). This revised version was published online in August 2006 with corrections to the Cover Date.     

Zebrafish anti-apoptotic gene Bcl-xL can prevent aquatic birnavirus-induced cell death in fish cells without affecting expression of viral proteins

The aquatic birnavirus induces mitochondria-mediated cell death in fish; however, the molecular mechanism remains unknown. In the present study, we demonstrated that aquatic birnavirus-induced mitochondria-mediated cell death is regulated by the anti-apoptotic Bcl-2 family member, zfBcl-xL, which is anti-apoptotic and enhances host cell viability. First, CHSE-214 cells carrying EGFP-zfBcl-xL fused genes were selected, established in culture, and used to examine the involvement of zfBcl-xL in host cell protection from the effects of viral infection. EGFP-zfBcl-xL was found to prevent infectious pancreatic necrosis virus (IPNV)-induced phosphatidylserine exposure up to 40% at 12 h and 24 h post-infection (p.i.), block IPNV-induced loss of mitochondrial membrane potential (ΔΨm), and enhance host viability at the middle and late replication stages. In addition, zfBcl-xL overexpression prevented IPNV-induced caspase-9 activation up to 25% and 85% at the middle (12 h p.i.) and late (24 h p.i.) replication stages without affecting expression of viral proteins such as VP3 (as a viral death protein) protein. In the present study, we demonstrated that aquatic birnavirus-induced cell death is prevented by the anti-apoptotic Bcl-2 family member, zfBcl-xL, which enhances host cell viability through blockage of mitochondrial disruption and caspase-9 activation.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 microm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20 degrees C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13 degrees C, up to 7 d at 20 degrees C, but it was inactivated at 37 and 56 degrees C within 24 and 1 h, respectively. Freezing at -20 degrees C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20 degrees C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10(2.5)-50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.     

Feline Calicivirus Can Tolerate Gross Changes of Its Minor Capsid Protein Expression Levels Induced by Changing Translation Reinitiation Frequency or Use of a Separate VP2-Coding mRNA

Caliciviruses use reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.     

Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces

A method for studying the behavior of viruses on surfaces has been developed and is illustrated by determining the temperatures that inactivate adsorbed viral hemorrhagic septicemia virus (VHSV) and the concentration of 1-propanol that disinfected surfaces with adsorbed VHSV and chum salmon virus (CSV). VHSV is a rhabdovirus; CSV, a reovirus, and they were detected with two fish cell lines, EPC and CHSE-214, respectively. When polystyrene tissue culture surfaces were incubated with virus, rinsed, and left to dry, they still supported the attachment and spreading of cell lines and after 7 days these cells showed the characteristic CPE of the viruses. Thus cells appeared to be infected directly from surfaces on which viruses had been adsorbed. Applying this property to 96-well plates allowed duplicate surfaces to be examined for their infectiousness or support of CPE. For each treatment 80 replicate surfaces in a 96-well plate were tested at one time and the results expressed as the number of wells showing CPE. VHSV adsorbed to polystyrene was inactivated by drying in the dark at temperatures above 14 °C, but remained infectious for at least 15 days of drying at 4 °C. For chemical sterilization of polystyrene surfaces with adsorbed virus, disinfection was achieved with 1-propanol at 40% for VHSV and at 60% for CSV. As CPE can be conveniently monitored in 96-well plates with a fluorescence plate reader, this method can be used to rapidly evaluate a variety of treatments for their ability to inactivate surface-bound viruses.     

Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.     

A recombinant CHSE-214 cell line expressing an Mx1 promoter-reporter system responds to both interferon type I and type II from salmonids and represents a versatile tool to study the IFN-system in teleost fish

A transgenic cell line for the detection of salmon interferons (IFNs) has been established. It is based on a CHSE-214 cell line containing a reporter construct expressing firefly luciferase under the control of the rainbow trout promoter for the IFN-induced Mx1 gene. This cell line, named CHSE-Mx10, showed IFN-induced luciferase expression after more than 80 passages, confirming the stability of this cell line. Interestingly, the Mx promoter was shown to respond to both salmon IFN-alpha/beta and trout IFN-gamma in a dose-dependent manner, while there was no response to TNF-alpha and IL-1beta. IFN-alpha/beta activity could be measured at a range of 9-150 U/ml, and IFN-gamma showed activity between 10 and 100 ng/ml. The reproducibility of both responses was good. The CHSE-Mx10 reporter system constitutes a versatile tool to study the induction and regulation of IFN signaling in teleost fish. A preliminary study presented herein suggests that both infectious pancreas necrosis virus (IPNV) and salmon pancreas disease virus (SPDV) may block activation of the Mx promoter in CHSE-Mx10 stimulated with IFN-alpha/beta.     

Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines

Perciforms have three forms of gonadotropin-releasing hormone (GnRH) in their brain. All three GnRHs are potent secretogogues for luteinizing hormone (LH) from the pituitary. The pivotal role of GnRH-R-GnRH interactions in reproductive homeostasis is well established; however, there is a paucity of information on how a GnRH-R responds to the three endogenous GnRH forms in a perciform species. In this study, a recombinant pituitary GnRH-R from striped bass (stb) was expressed in a mammalian cell line (COS-7) and a fish cell line (CHSE-214). Activation of the signaling pathways was monitored by reporter gene (luciferase) based assays, which were specific for cAMP-PKA or Ca 2+/calmodulin kinase (activated via c-fos promoter) signaling pathways. The stbGnRH-R expressed in two different cell lines triggered different downstream signaling in response to the treatments with chicken (c) GnRH II. Interestingly, when endogenous GnRHs were used in combinations, the luciferase activity was significantly attenuated in transfected CHSE-214 cells.     

Monitoring viral-induced cell death using electric cell-substrate impedance sensing

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.