A single amino acid mutation (I1012F) of the RNA polymerase of marine viral hemorrhagic septicemia virus changes in vitro virulence to rainbow trout gill epithelial cells.

UNLABELLED: Viral hemorrhagic septicemia virus (VHSV) is separated into four different genotypes (I to IV) with different sublineages (K. Einer-Jensen, P. Ahrens, R. Forsberg, and N. Lorenzen, J. Gen. Virol. 85:1167-1179, 2004; K. Einer-Jensen, J. Winton, and N. Lorenzen, Vet. Microbiol. 106:167-178, 2005). European marine VHSV strains (of genotypes I to III) are, in general, nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge, and here we also show that genotype IVa is nonpathogenic to trout. Despite several attempts, it has not been possible to link genomic variation to in vivo virulence. In vitro virulence to gill epithelial cells (GECs) has been used as a proxy for in vivo virulence, and here we extend these studies further with the purpose of identifying residues associated with in vitro virulence. Genotype Ia (DK-3592B) and III (NO/650/07) isolates, which are pathogenic to rainbow trout (O. B. Dale, I. Orpetveit, T. M. Lyngstad, S. Kahns, H. F. Skall, N. J. Olesen, and B. H. Dannevig, Dis. Aquat. Organ. 85:93-103, 2009), were compared to two marine strains that are nonpathogenic to trout, genotypes Ib (strain 1p8 [H. F. Mortensen, O. E. Heuer, N. Lorenzen, L. Otte, and N. J. Olesen, Virus Res. 63:95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high- and low-virulence strains were identified, and reverse genetics was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able to replicate and induce a cytopathic effect in trout GECs. The other six mutated variants remained nonpathogenic. IMPORTANCE: This is the first study to clearly link in vitro virulence of viral hemorrhagic septicemia virus (VHSV) with an amino acid residue in the L protein, a site located in conserved region IV of the L protein. In vitro virulence is documented by induction of cytopathic effects and viability studies of gill epithelial cells, and the observed cellular responses to infection are associated with increased viral replication levels. There are no previous studies addressing the importance of the L protein or the RNA-dependent RNA polymerase for virus virulence in vitro or in vivo. Therefore, the findings reported here should broaden the search for pathogenicity traits in novirhabdoviruses, and there is a possibility that the polymerase participates in defining the host species virulence of various VHSV strains.

Cardiomyopathy syndrome of atlantic salmon (Salmo salar L.) is caused by a double-stranded RNA virus of the Totiviridae family.

Cardiomyopathy syndrome (CMS) of farmed and wild Atlantic salmon (Salmo salar L.) is a disease of yet unknown etiology characterized by a necrotizing myocarditis involving the atrium and the spongious part of the heart ventricle. Here, we report the identification of a double-stranded RNA virus likely belonging to the family Totiviridae as the causative agent of the disease. The proposed name of the virus is piscine myocarditis virus (PMCV). On the basis of the RNA-dependent RNA polymerase (RdRp) sequence, PMCV grouped with Giardia lamblia virus and infectious myonecrosis virus of penaeid shrimp. The genome size of PMCV is 6,688 bp, with three open reading frames (ORFs). ORF1 likely encodes the major capsid protein, while ORF2 encodes the RdRp, possibly expressed as a fusion protein with the ORF1 product. ORF3 seems to be translated as a separate protein not described for any previous members of the family Totiviridae. Following experimental challenge with cell culture-grown virus, histopathological changes are observed in heart tissue by 6 weeks postchallenge (p.c.), with peak severity by 9 weeks p.c. Viral genome levels detected by real-time reverse transcription (RT)-PCR peak earlier at 6 to 7 weeks p.c. The virus genome is detected by in situ hybridization in degenerate cardiomyocytes from clinical cases of CMS. Virus genome levels in the hearts from clinical field cases correlate well with the severity of histopathological changes in heart tissue. The identification of the causative agent for CMS is important for improved disease surveillance and disease control and will serve as a basis for vaccine development against the disease.

Differentially expressed genes following persistent infection with infectious pancreatic necrosis virus in vitro and in vivo.

The mechanisms of viral persistence of infectious pancreatic necrosis virus (IPNV) are not well understood. In this study we have used a model of IPNV persistently infected CHSE (Chinook salmon embryonic) cells as correlate of persistent infection in fish focusing on differentially expressed genes using subtractive hybridization (SSH). Selected genes were also analyzed by quantitative real-time PCR (qPCR) in persistently infected parr of Atlantic salmon. Persistent infection was established by growing CHSE cells surviving an IPNV infection. Infection in rescued cells was non-lytic with a virus yield of 10(3)-10(5) TCID(50)/ml of supernatant, resembling what can be found during a persistent infection in vivo. By comparing gene expression in persistently infected cell vs. non-infected cells we found an upregulation of genes involved in direct interaction or degradation of viral proteins, proteasome activating subunit 3, and of ATRX (X-linked alpha-thalassemia/mental retardation syndrome), a transcription repressor, which may indicate a repression of viral replication through reduced transcription. Further ephrin B1 (signal-transduction group) was found strongly up-regulated, and receptors for various ephrins are used for cell interaction and as entry points for other viruses in higher vertebrates. Endonuclease/reverse transcriptase 1 (RVT1) was also found highly up-regulated in persistently infected cells. The comparison of persistently infected cells to in vivo infected fish showed that the expression profiles found in CHSE cells give corresponding results for selected genes, as ATRX, ephrin B1 and RVT-1. We observed similar results by use of two independent methods (SSH and qPCR) for 8 out of 15 genes analyzed and the transcript profile of persistently IPNV-infected cells involve upregulation of genes encoding proteins involved in viral protein degradation and translation inhibition. The understanding is that this may contribute to keep the number of virus particles low during viral persistence.

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells.

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.

Transfection efficiency and cytotoxicity of cationic liposomes in salmonid cell lines of hepatocyte and macrophage origin.

The transfection efficiency of liposome-based DNA formulations was studied in different salmonid cell lines of hepatocyte and macrophage origin. Parallel assessment of cell viability was carried out to define the balance between transfection efficiency and toxicity. For all cell lines, transfection efficiency varied with the lipoplex charge ratio and the amount of DNA added to the liposomes. The hepatocyte-derived cell line was most readily transfected while lower transfection efficiency was observed for the macrophage cell lines. The cationic liposomes showed a dose-dependent toxicity and were found to be most toxic for cells of macrophage origin. This was in line with the observation that higher amounts of lipids were associated with the cells of macrophage origin than the hepatocytes. Complexing DNA with the liposomes reduced the toxicity for all three cell lines, most markedly, however, for macrophage cell lines. The differences in the transfection and toxicity patterns between the cell lines are probably caused by differences in membrane composition as well as differences in phagocytic activity and processing of the liposomes/lipoplexes.

Expression of luciferase in selected organs following delivery of naked and formulated DNA to rainbow trout (Oncorhynchus mykiss) by different routes of administration.

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout (Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.

Long-term stability of chitosan-based polyplexes.

PURPOSE: There is a lack of information about the long-term stability of chitosan-based polyplexes although a large amount is known as regards transfection efficiency and physicochemical characteristics. The aim of this work is to study the transfection efficiency and physicochemical properties of chitosan-based polyplexes over time when stored at different temperatures in an acetate-buffer at pH 5.5. METHODS: Aqueous samples of chitosan-based polyplexes were aged at 4 degrees C, 25 degrees C, and 45 degrees C for up to 1 year. Samples were taken at predetermined time-points and evaluated for in vitro transfection efficiency and physiochemical properties (particle size, zeta potential). RESULTS: One year of storage at 4 degrees C did not result in any major changes in the properties of the polyplexes. At 25 degrees C there were minor changes in the physicochemical characteristics of the polyplexes, and the in vitro transfection efficiency was reduced at 1 year of storage. Storage at 45 degrees C altered both the in vitro transfection efficiency and the physicochemical properties of the polyplexes after a short time. CONCLUSIONS: The biological and physicochemical stability of the chitosan-based polyplexes are maintained for 1 year of storage in acetate-buffer at 4 degrees C. The changes in the polyplex characteristics at elevated temperatures may be explained by degradation of both plasmid and chitosan.

The influence of formulation variables on in vitro transfection efficiency and physicochemical properties of chitosan-based polyplexes.

The aim of this study was to investigate how a selection of formulation variables affects the in vitro transfection efficiency and physicochemical properties (particle size, zetapotential and chitosan-plasmid association) of chitosan-based polyplexes. Experimental designs in combination with multivariate data analysis were applied to reveal the effects of the formulation variables on the responses. The following formulation variables were studied: molecular weight and degree of acetylation of chitosan, pH and ionic strength of the buffer in which chitosan was dissolved, charge ratio of polyplexes, plasmid concentration and inclusion of a coacervation agent in the plasmid solution. The in vitro transfection efficiency in Epithelioma papulosum cyprini (EPC) cells was affected by the polyplex charge ratio, the DNA concentration in the complexes as well as the molecular weight and degree of acetylation of the chitosans. Two favourable formulations were identified in a more thorough investigation. These formulations were made of SC113 (theoretical charge ratio 10) and SC214 (theoretical charge ratio 3). The size of the complexes was affected by the degree of acetylation, concentration of DNA, pH, inclusion of a coacervation agent and the charge ratio. The charge ratio, pH and ionic strength determined the zetapotential of the particles, while the charge ratio was important for the association between the plasmid and chitosan.

Immersion delivery of plasmid DNA. I. A study of the potentials of a liposomal delivery system in rainbow trout (Oncorhynchus mykiss) fry.

A successful regime for intramuscular injection of naked DNA is developed in fish, but the exploration of other ways of administration has not yet been studied in any detail. Immersion is a delivery route offering many advantages compared to conventional ways of administration. Applying cationic liposomes as a delivery system for DNA by this route, however, is met with severe toxicity problems. In this report, the underlying mechanisms of the acute toxicity were investigated in vivo and in vitro. The most critical factor was found to be the charge of the liposomes. Cationic liposomes above a certain threshold concentration had a lethal effect in rainbow trout fry. In contrast, similar concentrations of neutral or anionic liposomes were not toxic. Furthermore, large liposome-mucin complexes were formed upon addition of mucin to cationic liposomes. This was not observed with neutral or anionic liposomes. Lipoplexes were less toxic and interacted less with mucin compared to cationic liposomes. Hence, the mechanism of the acute toxicity in rainbow trout fry is suggested to be an interaction between the cationic liposomes and anionic components of gill mucin. The consequence is hypoxia and this is most likely the cause of acute toxicity observed in rainbow trout fry.

Immersion delivery of plasmid DNA. II. A study of the potentials of a chitosan based delivery system in rainbow trout (Oncorhynchus mykiss) fry.

The possibility of utilising DNA vaccines for aquaculture fish has been of growing interest in recent years and novel methods to deliver DNA to the fish are under investigation. One of the delivery methods of interest is immersion. Due to the favourable properties of chitosan in gene delivery and bioadhesion, chitosan-DNA formulations have been investigated for use for immersion delivery to fish. Initial studies on this system, however, revealed an acute toxic effect of the formulations. In this study, factors important for the acute toxicity of chitosan and chitosan formulations are identified and attempts are made to explain the underlying mechanisms for the toxicity. In vivo methods revealed that the toxicity mainly was dependent on the concentration of chitosan, but also the molecular weight and the degree of acetylation of the chitosans were of importance. Noteworthy, the toxicity of the polymer decreased dramatically when the chitosan was 'decharged' by complexation with DNA. In vitro experiments supported the in vivo observations. Most likely, the observed toxicity is caused by an electrostatic interaction between the cationic polymer and the anionic parts of the gill mucus. The result is obstructed oxygen diffusion over the gills and the fish are killed by acute hypoxia. Careful selection of chitosan type and charge of the particles may result in a potential for chitosan based immersion delivery of plasmid DNA.