Juan Carlos Espinoza

Rapid simultaneous detection and quantitation of infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) is a pathogen of great concern in the salmon industry as well as in the environment. Taking advantage of the early immunofluorescent visualization of viral proteins in infected cells, a titration method was developed. At 16 h p.i., fluorescent foci were visualized with a monoclonal antibody against VP3-structural protein of the virus. The counting of each fluorescent cell allows the quantitation of infection foci; titres expressed in fluorescent foci/ml were equivalent to plaque forming units (PFU)/ml. With slight modifications, the same method used to detect the virus in field samples, can be applied to estimate virus contents. Some of the samples used during the assays were obtained from routine screening procedures. The titres recorded from positive samples correlated well with the clinical condition of the fish. With this method, rapid diagnosis and quantitation may simultaneously be performed with the same tissue extract.

Temporal and subcellular localization of infectious pancreatic necrosis virus structural proteins.

Summary. The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.

Attachment and entry of infectious pancreatic necrosis virus (IPNV) into CHSE-214 cells

SummaryInfectious pancreatic necrosis virus (IPNV) attaches to CHSE-214 cells through two types of cell components: specific and non-specific ones. Competition experiments with inactivated IPNV showed that IPNV requires specific components to productively infect cells. Just a low amount of adsorbed IPNV enters the cell. After 20 minutes, part of the adsorbed IPNV was internalized into acid compartments. Also, the viruses adsorbed on the cell surface require similar periods of time to escape from the neutralization of antibodies.

Detection and quantification of Chilean strains of infectious pancreatic necrosis virus by real-time RT-PCR assays using segment B as a target

Infectious pancreatic necrosis virus (IPNV) is the causal agent of a highly prevalent disease that affects salmonid fish, mostly during their fresh water life period. Like many other viruses, IPNV produces highly heterogeneous populations. Therefore, diagnostic methods need to be checked constantly so that no variants of the virus escape detection. The IPNV genome is composed of two double-stranded RNA segments: A and B, polymerase chain reaction (PCR) methods normally use segment A as a target. In order to develop an optimized protocol to diagnose IPNV, we present a real-time RT-PCR (reverse transcription) technique, using primers designed to recognize segment B of the virus. To validate the ubiquity of the primers used, the IPNV isolates tested were sequenced and compared with previously published cladograms, which include a wide spectrum of genogroups. These primers made it possible to detect viral isolates belonging to genogroups 1 and 5, which were obtained from different locations linked to fish farming. As expected, we were able to detect the virus from distant Aquabirnavirus genogroups.

Infectious pancreatic necrosis virus (IPNV) does not requireacid compartments for entry into cells

SummaryThe 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 Aff1 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.

Inducers of salmon innate immunity: An in vitro and in vivo approach

ABSTRACT Maintaining fish health is one of the most important aims in aquaculture. Prevention of fish diseases therefore is crucial and can be achieved by various different strategies, including most often a combination of different methods such as optimal feed and fish density, as well as strengthening the immune system. Understanding the fish innate immune system and developing methods to activate it, in an effort to prevent infections in the first place, has been a goal in recent years. In this study we choose different inducers of the innate immune system and examined their effects in vitro on the salmon cell line CHSE‐214. We found that the butyrate derivatives 4‐phenyl butyrate (PBA) and &bgr;‐hydroxy‐&bgr;‐methyl butyrate (HMB) induce the expression of various innate immune genes differentially over 24–72 h. Similarly, lipids generated from fish oils were found to have an effect on the expression of the antimicrobial peptides cathelicidin and hepcidin, as well as iNOS and the viral receptor RIG‐1. Interestingly we found that vitamin D3, similar as in mammals, was able to increase cathelicidin expression in fish cells. The observed induction of these different innate immune factors correlated with antibacterial activity against Aeromonas salmonicida and antiviral activity against IPNV and ISAV in vitro. To relate this data to the in vivo situation we examined cathelicidin expression in juvenile salmon and found that salmon families vary greatly in their basal cathelicidin levels. Examining cathelicidin levels in families known to be resistant to IPNV showed that these QTL‐families had lower basal levels of cathelicidin in gills, than non QTL‐families. Feeding fish with HMB caused a robust increase in cathelicidin expression in gills, but not skin and this was independent of the fish being resistant to IPNV. These findings support the use of fish cell lines as a tool to develop new inducers of the fish innate immune system, but also highlight the importance of the tissue studied in vivo. Understanding the response of the innate immune system in different tissues and what effect this might have on infections and downstream cellular pathways is an interesting research topic for the future. HighlightsVitamin D3 induces cathelicidin expression in the fish cell line CHSE‐214.PBA, HMB and fish oils induce innate immune gene expression in CHSE‐214 cells.Induction is correlated with increased antibacterial and antiviral activity.Basal cathelicidin levels vary in different salmon families.HMB increases cathelicidin expression in salmon gills, but not skin in vivo.

Genotyping of Infectious Pancreatic Necrosis virus through nested PCR and possible host-specific link by genogroup

Samples of fish organs from three salmonid species present in Chile, were analyzed through the nested polymerase chain reaction (nested PCR) to detect the presence of Infectious Necrosis Pancreatic virus (IPNV) and leading to their phylogenetic classification. The technique proved to be efficient and sensitive for detection and genotypification of viral samples which could not even be isolated in cell culture. The phylogenetic analysis showed the two genogroups previously described in the country, ie., European (Genogroup 5) and American (Genogroup 1), being the IPNV that belong to Genogroup 5 the dominant one (78.8%). It is clear that the Chilean IPNV is clustered within the Genogroup 1 forming a Chilean genotype that is separated from the reference strains (e.g. WB, VR-299). It was determined that there is a statistically significant relationship between the genonogroup that a viral isolate belongs and a specific host. Most of the viruses from Genogroup 5 were detected in Salmo salar , while the ones from Genogroup 1 were detected mainly in Oncorhynchus mykiss and O. Kisutch (P