Lactococcus lactis Expressing Type I Interferon From Atlantic Salmon Enhances the Innate Antiviral Immune Response In Vivo and In Vitro.

In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.

Restricted replication of viral hemorrhagic septicemia virus (VHSV) in a birnavirus-carrier cell culture.

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are economically important pathogens of the salmonid aquaculture industry. In previous work we demonstrated that a cell line persistently infected with IPNV (EPCIPNV) exhibited antiviral activity against superinfection with the heterologous virus VHSV. This work extends our study by analyzing the replication of VHSV in the IPNV-persistently infected cells. At early and late stages of infection VHSV RNA synthesis, as well as VHSV-induced syncytia formation, were examined in EPCIPNV cultures. During the course of VHSV infection the accumulation of VHSV RNA is inhibited in EPCIPNV cells. Typical VHSV-induced membrane fusion at the late stages of infection is also absent in the IPNV carrier cultures. VHSV binding and fusion to EPCIPNV cells did not appear to be impaired, but a potent inhibitory effect on VHSV RNA synthesis is exerted at early times of infection in the IPNV carrier culture. In conclusion, the EPCIPNV cells are considered to be a useful system to study viral interference as well to analyze the mechanisms underlying the phenomenon of superinfection exclusion.

Delayed protein shut down and cytopathic changes lead to high yields of infectious pancreatic necrosis virus cultured in Asian Grouper cells.

Inactivated whole virus vaccines represent the majority of commercial preparations used to prevent infectious pancreatic necrosis (IPN) in salmonids today. The production of these vaccines requires high virus concentrations that are resource-demanding. In this study, we describe the cultivation of high yields of IPN virus in Asian Grouper strain K (AGK) cells. The mechanism by which this is achieved was investigated by comparison with commonly used salmonid cell lines (RTG-2 and CHSE-214 cells). The cells were counted before and sequentially after infection. Thereafter, protein shut down, virus yields and apoptosis were assessed. The effects of poly(I:C) pre-treatment and Mx expression on IPNV concentrations were examined and the results show that high virus yields were associated with high cell numbers per unit volume, delayed cell death and apoptosis inAGKcells while the opposite was observed in RTG-2 cells. Poly(I:C) treatment and Mx expression resulted in a dose-dependent inhibition of virus multiplication. The production capacity of AGK and CHSE-214 cells were compared and higher split ratio and shorter split interval of AGK cells documents dramatic differences in virus antigen production capacity. Collectively, the results suggest that high cell numbers and prolonged survival of AGK cells are responsible for the superior virus yields over RTG-2 and higher split ratio/shorter split interval makes AGK superior over CHSE cells.

Establishment and characterization of the epithelioma papulosum cyprini (EPC) cell line persistently infected with infectious pancreatic necrosis virus (IPNV), an aquabirnavirus.

Infectious pancreatic necrosis virus (IPNV), a type species of aquabirnaviruses in the family Birnaviridae, is an etiological agent of infectious pancreatic necrosis and has been isolated from epizootics of cultured salmonids. In the present study, an epithelioma papulosum cyprini (EPC) cell line persistently infected with IPNV (PI-EPC) was experimentally established by subculturing EPC cells surviving IPNV infection, and was characterized. PI-EPC cells were morphologically indistinguishable from EPC, but continued to grow and yield IPNV. PI-EPC cells showed no cytopathic effect due to IPNV inoculation, and susceptibility of PI-EPC cells against heterologous viruses was not different from that of EPC cells. Only one cell of 10(3.5) PI-EPC cells produced IPNV at approximately 10(0.5) 50% tissue culture infectious dose (TCID50)/cell/day, which was approximately 1,000 times lower than that of normal EPC cells. PI-EPC cells that did not yield IPNV (N-PI-EPC) were screened. The IPNV genome was detected from both PI-EPC and N-PI-EPC cells, and the IPNV VP2 structural protein was detected from both cell lines, but no other IPNV proteins were observed by Western blot analysis with anti-IPNV serum. Thus, multiplication of IPNV in PI-EPC cells was regulated by some host cell factors, except interferon.

Evaluation of the immune response against immature viral particles of infectious pancreatic necrosis virus (IPNV): a new model to develop an attenuated vaccine.

Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish. Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response. Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.

Thermal inactivation of infectious pancreatic necrosis virus in a peptone-salt medium mimicking the water-soluble phase of hydrolyzed fish by-products.

Infectious pancreatic necrosis virus (IPNV) (serotype Sp) was exposed to temperatures between 60 and 90°C in a medium mimicking the water-soluble phase of hydrolyzed fish by-products. D values ranged from 290 to 0.5 min, and the z value was approximately 9.8°C. Addition of formic acid to create a pH 4 medium did not enhance heat inactivation. Predicted inactivation effects at different temperature-time combinations are provided.

Establishment and characterization of a fin cell line from Indian walking catfish, Clarias batrachus (L.).

A new cell line, Indian Catfish Fin, derived from the fin tissue of Indian walking catfish, Clarias batrachus, was established and characterized. The cell line grew well in Leibovitz's L-15 medium supplemented with 15% foetal bovine serum (FBS) and has been subcultured more than 110 times since its initiation in 2007. The cells were able to grow at a range of temperature from 28 to 37 °C with optimal growth at 28 °C. The cell line predominantly consists of fibroblast-like cells. The growth rate of fin cells increased as the FBS concentration increased from 2% to 20% at 28 °C with optimum growth at a concentration of 15% or 20% and poor growth at a concentration of 5%. The cells were found to be susceptible to fish nodavirus and IPNV-ab and infection was confirmed by cytopathic effect and reverse transcriptase-polymerase chain reaction. PCR amplification of mitochondrial 12S rRNA using primers specific to C. batrachus confirmed the catfish origin of the cell line. The cell line was characterized further by immunocytochemistry, transfection efficiency with pEGFP-N1 and cell cycle analysis by fluorescent-activated cell sorting.

Characterization of an infectious pancreatic necrosis (IPN) virus carrier cell culture with resistance to superinfection with heterologous viruses.

A state of persistence of a non susceptible fish cell line with infectious pancreatic necrosis virus (IPNV) was established in vitro by experimental infection. The persistently infected culture showed sustained production of infectious virus and could be continuously passaged for months. A distinct feature of this culture is that only a very small fraction of the cells harbours virus replication, in contrast to other reported IPNV-persistently infected cells from salmonid fish, where nearly all the cells express viral antigens. In spite of the small number of detectable IPNV-infected cells, the carrier culture shows resistance to superinfection with homologous as well as heterologous viruses. Temperature shift-up experiments indicate that viral interference is due to continuous replication of IPNV in the culture. Quantitation of Mx gene expression suggested that the interference phenomenon could be mediated by the activation of the interferon (IFN) system. However, conditioned medium from the IPNV-infected cell cultures only marginally protected other cells against VHSV infection, indicating that other type I IFN-independent mechanism may be underlying the resistance of the persistently infected culture to infection with heterologous viruses. Our study defines a novel in vitro model of IPNV persistence and contributes to the understanding of the widespread distribution of aquabirnaviruses in marine and fresh water environments by establishing a carrier state in non susceptible fish species.

Isolation and quantification of infectious pancreatic necrosis virus from ovarian and seminal fluids of Atlantic salmon, Salmo salar L.

Methods for the isolation and quantification of infectious pancreatic necrosis virus (IPNV) from ovarian and seminal fluids of Atlantic salmon are described. Both have utility for the non-lethal detection of IPNV in mature broodstock and for research into vertical transmission. Two experiments are described to check the efficiency of an elution method for the removal of IPNV from milt. The isolation rate for ovarian fluid of females was generally higher than that for seminal fluid of males from the same populations. In IPNV milt mixing experiments up to 99.98% of available IPNV adsorbed to Atlantic salmon spermatozoa and 20-100% of virus eluted using a variety of procedures. Titration of virus from naturally infected milt can be useful in estimating the relative vertical transmission risk from male broodstock.

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.

Establishment of embryonic cell line from sea bass (Lates calcarifer) for virus isolation.

A continuous cell line was established from blastula stage embryos of sea bass (Lates calcarifer). The sea bass embryonic cells were maintained in Leibovitz's L-15 supplemented with 15% fetal bovine serum. The embryonic cell line was sub-cultured more than 70 passages over a period of 1.5 years and is designated as Sahul Indian sea bass embryonic (SISE) cell line. The cells were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of sea bass embryonic cells increased as the FBS proportion increased from 2 to 20% at 28 degrees C with optimum growth at the concentration of 15 or 20%. Polymerase chain reaction products were obtained from embryonic cells and blastula of sea bass with primer sets of microsatellite markers of sea bass. Four fish viruses were tested on this cell line to determine its susceptibility to these viruses and this cell line was found to be susceptible to IPNV VR-299 and nodavirus, and the infection was confirmed by cytopathic effect (CPE) and RT-PCR. Further, this cell line was characterized by immunocytochemistry using confocal-laser-scanning microscopy (CFLSM), transfection with pEGFP-N1, proliferate marker (BrdU).

Characteristics of inhibition of infectious pancreatic necrosis virus (IPNV) by normal rainbow trout Oncorhynchus mykiss serum.

We studied the characteristics of rainbow trout serum (RTS) inhibitory activity against infectious pancreatic necrosis virus (IPNV). Serum inhibition was related to the serum source and host cell in which the virus had been propagated. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines, with inhibition highest in rainbow trout gonad (RTG)-2 cells. The RTS sensitivity of the virus was modified by the cell line through which the virus passed, with multiple passages through Chinook salmon embryo (CHSE)-214 cells producing a virus that was less sensitive to RTS. The RTS inhibition level was dependent on cell density: at a cell density of < or = 2 x 10(5) cells ml(-1), inhibition was insignificant (tissue culture infective dose 50% = 10(-1.1) TCID50 ml(-1) reduction); however, above a density of 3 x 10(5) cells ml(-1), the inhibition level was very high (> or = 10(-6.3) TCID50 ml(-1) reduction). The salmonid sera tested showed high inhibition, except for brook trout serum (BTS), while non-salmonid sera did not inhibit IPNV, replication on RTG-2 cells. Pretreatment of cultured cells with RTS prior to exposure did not affect inhibition of IPNV and thus did not mask a viral receptor. The RTS inhibition level was dependent on the time of serum addition, with inhibition being maintained for at least 16 h postinfection. Pretreatment of IPNV revealed that the virus is directly inhibited by RTS, and more strongly so when RTS is present during viral replication.

Genome assembly and particle maturation of the birnavirus infectious pancreatic necrosis virus.

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.

Experimental infectious pancreatic necrosis infections: propagative or point-source epidemic?

Experimentally initiated epidemics of infectious pancreatic necrosis in rainbow-trout fry were analyzed using a modification of the standard mathematical model for a simple propagative epidemic. Contrary to expectations, the value of the transmission parameter (beta) was inversely related to initial density of susceptible hosts. This anomaly can be explained if we assume that the experimental epidemics were point-source rather than propagative epidemics. The implications of this conclusion for modeling experimental and natural epidemics are discussed.

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.

Infectious pancreatic necrosis virus (IPNV) does not require acid compartments for entry into cells.

The early events in the infection of unenveloped viruses are still rather unknown and puzzling. However, as in the case of enveloped viruses, the acid pH of endosomes can be important to trigger the virus entry into the cytosol. To test if the infectious pancreatic necrosis virus (IPNV), an unenveloped virus, requires acid endosomal pH to infect cells, we assayed the effect of Bafilomycin A1 on IPNV replication. Concentrations of the antibiotic which inhibited the endosomal acidification of the host cells were unable to affect IPNV replication in CHSE-214 cells; therefore, the acid pH of endosomes seems not to be a mandatory condition for the entry of IPNV into cells.

Inhibitors of infectious pancreatic necrosis virus (IPNV) replication.

In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-beta-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC50) were 0.01 micrograms/ml and 0.5 micrograms/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC50) were 50 micrograms/ml and 100 micrograms/ml, respectively, and the concentrations that reduced [methyl-3H] thymidine incorporation by 50% (IC50) were 0.5-1 and 50 micrograms/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50-100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.