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.

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.

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.

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.