Fish Cell Cultures Research Articles

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Combined Effect of Gamma Radiation and Heavy Metals on Some Living Organisms.

The purpose of this study was to systematise scientific publications on the combined effect of gamma radiation and heavy metals on living organisms. For this purpose, the method of analysis was applied, by means of which scientific papers in PubMed, Google Scholar, and other related databases were analysed for compliance with the inclusion criteria, where the objects of research were toxic effects of radiation and heavy metals on cells and adaptation processes. The results revealed that the study of the problem was carried out on organisms such as microalgae, fungi, weed and agricultural plants, fish, laboratory rats, and human cell cultures. In most studies, an antagonistic effect between low doses of gamma radiation and heavy metal salts was reported, which was manifested by a reduction in the cytotoxicity of isolated exposure to each agent separately. However, there are studies showing additive effects, especially in heavy metals. At the molecular level, heavy metal accumulation in combination with low doses of radiation (typically defined as less than 0.1Gy or sievert) induces the expression of metallothionein proteins, which can bind free radicals. At the metabolic level, this is manifested by a decrease in lipid peroxidation products, activation of antioxidant enzymes, and a decrease in apoptosis. The study proved both a direct relationship between zinc and cadmium accumulation in cells and inhibition of caspases and an indirect one, by maintaining mitochondrial membrane integrity through metallothionein.

Essential features of in vitro fish cell culture: an overview

The reports of fish cell cultivation date back to the early 20th century, and since then, it has become a tool of great importance for different areas of ichthyology, such as immunology, toxicology, and biotechnology. The cell cultivation methodology can vary according to the species and cell type, requiring knowledge of the specificities and characteristics of the target cell for them to be efficiently cultured. In this review, we address the main characteristics of the in vitro culture of fish cells, presenting their purposes, uses, main parameters, and different characteristics necessary for the culture environment. Finally, we present the experiences in Brazil using cell culture techniques.

New parameters for in vitro development of cell lines of the species Astyanax bimaculatus (Linnaeus, 1758) and Geophagus proximus (Castelnau, 1855).

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.

Features of SARS-CoV-2 Replication in Various Types of Reptilian and Fish Cell Cultures.

SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log10 TCID50/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log10 TCID50/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises.

Immune Transcriptional Response in Head Kidney Primary Cell Cultures Isolated from the Three Most Important Species in Chilean Salmonids Aquaculture.

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.

Continuous fish muscle cell line with capacity for myogenic and adipogenic-like phenotypes

Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line (“Mack” cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 h (± 19.1 SD). After a spontaneous immortalization crisis from passages 37–43, the cells proliferated at doubling times of 24.3 h (± 4.91 SD). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 and myosin heavy chain immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O staining and quantification of neutral lipids. New qPCR primers (HPRT, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.

Establishment of cell lines from individual zebrafish embryos.

With the increasing use of fish as model species for research, cell cultures derived from caudal fin explants as well as pre-hatching stage embryos have provided powerful in vitro tools that can complement or serve as an ethically more acceptable alternative to live animal experiments. The widely-used protocols to establish these lines require, as a starting point, homogeneous pools of embryos or viable adult fish which are large enough for collecting sufficient fin tissue. This excludes the use of fish lines with adverse phenotypes or lines that exhibit mortality at early developmental stages and so can only be propagated as heterozygotes. Specifically, when no visually overt mutant phenotype is detectable for identifying homozygous mutants at early embryonic stages, it is then impossible to sort pools of embryos with the same genotypes to generate cell lines from the progeny of a heterozygote in-cross. Here, we describe a simple protocol to generate cell lines on a large scale starting from individual early embryos that can subsequently be genotyped by polymerase chain reaction. This protocol should help to establish fish cell culture models as a routine approach for the functional characterization of genetic changes in fish models such as the zebrafish. Furthermore, it should contribute to a reduction of experiments which are ethically discouraged to avoid pain and distress.

Development of a novel real-time RT-PCR method using peptide nucleic acid (PNA) probes for detecting and genotyping of viral haemorrhagic septicaemia virus (VHSV)

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. The causative agent of VHS is the VHS virus (VHSV), which has been classified into four genotypes (I–IV), based on sequence analysis of the genes encoding for nucleoprotein, glycoprotein, and non-structural viral protein. Among the various diagnostic methods, real-time reverse transcription PCR method based on TaqMan-probe (RT-qPCR) is a stable, rapid, specific, and highly sensitive method for viral gene detection. However, the currently accepted diagnostic method based on RT-qPCR can only detect viral presence and load, and does not provide information about viral genotype. Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone. PNA probes can effectively detect a target gene by amplification and a specific melting temperature signal. It was reported that PNA probes can effectively distinguish between mismatched sequences based on their different melting temperatures in amplified PCR products. The present study reports a novel real-time RT-PCR method for simultaneous detection and genotyping of VHSV using PNA probes. The newly-developed method showed a sensitivity similar to that of the infectious titre by fish cell cultures inoculated with the virus, except for genotype IVa, where viral inoculation in cell culture showed a 10-fold higher sensitivity than the novel method. The melting point analysis to distinguish the four genotypes was performed on 80 VHSV isolates representing all known genotypes, showing that this novel real-time RT-PCR can distinguish between all VHSV genotypes without the need of further sequencing.

Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production.

The use of fetal bovine serum (FBS) and the price of cell culture media are the key constraints for developing serum-free cost-effective media. This study aims to replace or reduce the typical 10% serum application in fish cell culture media by applying protein hydrolysates from insects and marine invertebrate species for the growth of Zebrafish embryonic stem cells (ESC) as the model organism. Protein hydrolysates were produced from black soldier flies (BSF), crickets, oysters, mussels, and lugworms with a high protein content, suitable functional properties, and adequate amino-acid composition, with the degree of hydrolysis from 18.24 to 33.52%. Protein hydrolysates at low concentrations from 0.001 to 0.1 mg/mL in combination with 1 and 2.5% serums significantly increased cell growth compared to the control groups (5 and 10% serums) (p < 0.05). All protein hydrolysates with concentrations of 1 and 10 mg/mL were found to be toxic to cells and significantly reduced cell growth and performance (p < 0.05). However, except for crickets, all the hydrolysates were able to restore or significantly increase cell growth and viability with 50% less serum at concentrations of 0.001, 0.01, and 0.1 mg/mL. Although cell growth was enhanced at lower concentrations of protein hydrolysates, the cell morphology was altered due to the lack of serum. The lactate dehydrogenase (LDH) activity results indicated that BSF and lugworm hydrolysates did not alter the cell membrane. In addition, light and fluorescence imaging revealed that the cell morphological features were comparable to those of the 10% serum control group. Overall, lugworm and BSF hydrolysates reduced the serum by up to 90% while preserving excellent cell health.

Quillaja saponaria (Molina) Extracts Inhibits In Vitro Piscirickettsia salmonis Infections.

Simple SummaryBacterial diseases causes massive mortalities in aquaculture and antibiotic use remains the main measure to keep these under control. Pisciricketssia salmonis, an intracellular bacterium only present in Chile, produces high mortalities in farmed salmon and is currently the main reason for using antimicrobials compared to other salmon-producing countries such as Norway. Environmental and antimicrobial resistance concerns have been raised by the local and global public and society, although no scientific evidence has demonstrated such an impact. Thus, there is a constant search for new alternatives that can complement or reduce the use of antimicrobial in intensive salmon farming. Phytochemicals such as saponins from Quillaja saponaria extracts have been proven to prevent and control diseases in other animal production systems. This study explored the safety and efficacy of quillaja extract in in vitro infections with P. salmonis. The results of this study showed a good in vitro safety and efficacy to infections. The efficacy proved to be dependent on the quantity of saponins and toxicity dependent on purification. The results showed that quillaja extracts could be potentially used as a new sustainable and eco-friendly alternative to control P. salmonis infection, contributing to decreased fish mortality, antibiotic use and antimicrobial resistance in intensive aquaculture worldwide.P. salmonis infections are the cause of major bacterial disease in salmonids in Chile, and the reason for using more antibiotics compared to other salmon-producing countries. Vaccination and antibiotics have not been efficient and new approaches are needed. The safety of Quillaja saponaria extracts was measured by cytotoxicity using flow cytometry of cytopathic and death of fish cell cultures and efficacy was assessed using in vitro infection models with pathogenic P. salmonis. Cytotoxicity was low and control of in vitro infections was achieved with all products, with protection of over 90%. Minimum inhibitory concentrations were much higher than those in the infection using cell cultures. These results suggest a dual mechanism of action where less purified extracts with a combination of saponin and non-saponin components simultaneously decrease P. salmonis infection while protecting cell lines, rather than exerting a direct antimicrobial effect. Quillaja saponins controlled in vitro infections with P. salmonis and could be considered good candidates for a new, safe and sustainable method of controlling fish bacterial infectious diseases.

Genomics for the understanding of the host-pathogen interaction: the case of the Atlantic salmon and Piscirickettsia salmonis

The pollution by industrial and municipal effluents are major sources of concerns. Fish cell cultures were applied in different strategies of the evaluation of effluents, particularly whole toxicity, toxicity identification evaluation and mode of action studies based in adverse outcome pathways. Whole effluent toxicity was evaluated using a battery of five model systems from four trophic levels: Daphnia magna was the most sensitive system, followed by the hepatoma fish cell line PLHC-1, the bacterium Allivibrio fischeri, the fibroblastic fish cell line RTG-2 and the algae Chlorella vulgaris, detecting a risk of eutrofization. The uptake of neutral red was more sensitive than the content of protein assay. The main morphological alterations observed were cell loss, hydropic degeneration, and a general loss of lysosomes and of their perinuclear distribution. The toxicity was characterized in PLHC-1 cells through toxicity identification evaluation, in which a partial reduction with graduation at pH 11, filtration, aeration and addition of thiosulfate or EDTA was shown; on the other hand, a low sorption in solid phase extraction suggested that the main responsible were not organic compounds. Consequently, it was not necessary to apply an effect directed analysis HPLC fractionation. In the chemical identification phase, Zn, Cd, As, Cu and Pb were quantified in decreasing concentrations. In the toxicity confirmation phase, a reconstituted sample and individual solutions, presented decreasing toxicity: Zn > Pb > As+5 > Cd > Cu > As+3, the global toxicity being explained by response addition. In the last step, the mode of action was investigated using five specific biomarkers. While metallothionein and succinate dehydrogenase activity were increased, no changes occurred for lysosomal function, acetylcholinesterase and EROD activities, the responsibility of the toxicity for the elements found being confirmed.

Cell-Based Fish: A Novel Approach to Seafood Production and an Opportunity for Cellular Agriculture

Cellular agriculture is defined as the production of agricultural products from cell cultures rather than from whole plants or animals. With growing interest in cellular agriculture as a means to address the public health, environmental, and animal welfare challenges of animal agriculture, the concept of producing seafood from fish cell- and tissue-cultures is emerging as a means to address similar challenges with industrial aquaculture systems and marine capture. Cell-based seafood - as opposed to animal-based seafood - can combine developments in biomedical engineering with modern aquaculture techniques. Biomedical engineering developments such as closed-system bioreactor production of land animal cells create a basis for large scale production of marine animal cells. Aquaculture techniques such as genetic modification and closed system aquaculture have achieved marked gains in production that can pave the way for innovations in cell-based seafood production. Here, we present the current state of innovation relevant to the development of cell-based seafood across multiple species as well as specific opportunities and challenges that exist for advancing this science. The authors find that the physiological properties of fish cell- and tissue- culture may be uniquely suited to cultivation in vitro. These physiological properties, including hypoxia tolerance, high buffering capacity, and low-temperature growth conditions, make marine cell culture an attractive opportunity for scale production of cell-based seafood; perhaps even more so than mammalian and avian cell cultures for cell-based meats. This, coupled with the unique capabilities of crustacean tissue-friendly scaffolding such as chitosan, a common seafood waste product and mushroom derivative, presents great promise for cell-based seafood production via bioreactor cultivation. To become fully realized, cell-based seafood research will require more understanding of fish muscle culture and cultivation; more investigation into serum-free media formulations optimized for fish cell culture; and bioreactor designs tuned to the needs of fish cells for large scale production.

Integration of fish cell cultures in the toxicological assessment of effluents

The pollution by industrial and municipal effluents are major sources of concerns. Fish cell cultures were applied in different strategies of the evaluation of effluents, particularly whole toxicity, toxicity identification evaluation and mode of action studies based in adverse outcome pathways. Whole effluent toxicity was evaluated using a battery of five model systems from four trophic levels: Daphnia magna was the most sensitive system, followed by the hepatoma fish cell line PLHC-1, the bacterium Allivibrio fischeri, the fibroblastic fish cell line RTG-2 and the algae Chlorella vulgaris, detecting a risk of eutrofization. The uptake of neutral red was more sensitive than the content of protein assay. The main morphological alterations observed were cell loss, hydropic degeneration, and a general loss of lysosomes and of their perinuclear distribution. The toxicity was characterized in PLHC-1 cells through toxicity identification evaluation, in which a partial reduction with graduation at pH 11, filtration, aeration and addition of thiosulfate or EDTA was shown; on the other hand, a low sorption in solid phase extraction suggested that the main responsible were not organic compounds. Consequently, it was not necessary to apply an effect directed analysis HPLC fractionation. In the chemical identification phase, Zn, Cd, As, Cu and Pb were quantified in decreasing concentrations. In the toxicity confirmation phase, a reconstituted sample and individual solutions, presented decreasing toxicity: Zn > Pb > As+5 > Cd > Cu > As+3, the global toxicity being explained by response addition. In the last step, the mode of action was investigated using five specific biomarkers. While metallothionein and succinate dehydrogenase activity were increased, no changes occurred for lysosomal function, acetylcholinesterase and EROD activities, the responsibility of the toxicity for the elements found being confirmed.

VER-155008 induced Hsp70 proteins expression in fish cell cultures while impeding replication of two RNA viruses

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 μM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis

The complete genome sequence of a novel mononegavirus, Lepeophtheirus salmonis negative-stranded RNA virus 1 (LsNSRV-1), obtained from a salmonid ectoparasite, Lepeophtheirus salmonis was determined. The viral genome contains five open reading frames encoding three unknown proteins (ORF I, II and III), a putative glycoprotein (G), and a large (L) protein. Phylogenetic analysis placed LsNSRV-1 in the recently established mononegaviral family Artoviridae. LsNSRV-1 showed a prevalence of around 97% and was detected in all L. salmonis developmental stages. Viral genomic and antigenomic RNA was localized to nerve tissue, connective tissue, epithelial cells of the gut, subepidermal tissue, exocrine and cement glands, as well as the testis, vas deferens and spermatophore sac of male L. salmonis and the ovaries and oocytes of females. Viral RNA was detected in both the cytoplasm and the nucleoli of infected cells, and putative nuclear export and localization signals were found within the ORF I, III and L proteins, suggesting nuclear replication of LsNSRV-1. RNA interference (RNAi) was induced twice during development by the introduction of a double-stranded RNA fragment of ORF I, resulting in a transient knockdown of viral RNA. A large variation in the knockdown level was seen in adult males and off springs of knockdown animals, whereas the RNA level was more stable in adult females. Together with the localization of viral RNA within the male spermatophore and female oocytes and the amplification of viral RNA in developing embryos, this suggests that LsNSRV-1 is transmitted both maternally and paternally. Small amounts of viral RNA were detected at the site where chalimi were attached to the skin of Atlantic salmon (Salmo salar). However, as the RNAi-mediated treatment did not result in LsNSRV-1-negative offspring and the virus failed to replicate in the tested fish cell cultures, it is difficult to investigate the influence of secreted LsNSRV-1 on the salmon immune response.

Monitoring changing cellular characteristics during the development of a fin cell line from Cyprinus carpio.

The establishment and in-depth characterization of a novel continuous cell line derived from fin tissue of common carp (Cyprinus carpio), CCApin, is reported. The cells of the cell line could be propagated in Leibovitz's L-15 medium containing 15% foetal calf serum and 0.5% carp serum for >150 passages during the last 24 months, with a stable fast growth. Furthermore, antibody staining indicated that cell types obtained in primary cultures, containing the epithelial stem-cell marker tumorprotein 63, were different from cells in long-term cell cultures, containing tight junction protein zona occludens 1 and cytokeratin 7. These observations suggest a switch of dominant cell types. Molecular analysis of gene expression profiles of caudal fin tissue and CCApin cells showed that genes relevant in epithelial cells but also in mesenchymal cells were expressed. However, during cultivation of CCApin a set of very steadily expressed, primarily mesenchymal genes like collagen 1 alpha 1, fibronectin or cadherin 2 was found. In summary, the long-term cell culture could be described as a stably growing epithelial population with some mesenchymal features. There are several application possibilities, especially for virus susceptibility studies, e.g. cyprinid herpesvirus-3 (CyHV-3). The study leads to a better understanding of molecular and physiological mechanisms of in vitro fish cell cultures.

El silenciamiento de la proteína priónica celular (PrPC) mediante RNA de interferencia (siRNA) reduce la infección por HSV-1 y HSV-2 en células SK-SY5Y

Las encefalopatías espongiformes transmisibles (EETs) son enfermedades neurodegenerativas fatales que afectan a humanos y ciertas especies animales. La hipótesis más aceptada indica que el agente infeccioso, denotado como prion y compuesto principalmente por la Proteína Priónica Scrapie (PrPSc), corresponde a una conformación anormal de una proteína codificada por el huésped denominada Proteína Priónica Celular (PrPC), cuya función es aún desconocida; sin embargo, la expresión ubicua de PrPC así como su elevado grado de conservación entre especies, sugieren un papel importante para esta proteína. En este trabajo se detectó a la PrPC en diferentes tipos celulares incluyendo un cultivo primario de células de peces (Tilapia, Oreochromis spp.). Además, basándonos en la secuencia de la PrPC humana, se diseñó un RNA de interferencia (siRNA) con el fin de silenciar el gen PRNP en células neuronales SK-SY5Y. El siRNA diseñado inhibió la expresión de PrPC a lo largo de las 96 h post-transfección y las células silenciadas fueron menos susceptibles a la infección por HSV-1 y HSV-2, en comparación con células no transfectadas con el siRNA.

Activation of an Aquareovirus, Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T.canadensis.

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22°C with Tetrahymena canadenesis and two strains of T.thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30°C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30°C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26°C but not at 30°C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal-lysosomal system and released in a more infectious form. When allowed to swim in CSV-infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T.canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.

Invasion and replication of Yersinia ruckeri in fish cell cultures

BackgroundLike many members of the Enterobacteriaceae family, Yersinia ruckeri has the ability to invade non professional phagocytic cells. Intracellular location is advantageous for the bacterium because it shields it from the immune system and can help it cross epithelial membranes and gain entry into the host. In the present manuscript, we report on our investigation regarding the mechanisms of Y. ruckeri’s invasion of host cells.ResultsA gentamycin assay was applied to two isolates, belonging to both the biotype 1 (ATCC 29473) and biotype 2 (A7959–11) and using several cell culture types: Atlantic Salmon Kidney, Salmon Head Kidney and, Chinook salmon embryos cells at both low and high passage numbers. Varying degrees of sensitivity to Y. ruckeri infection were found between the cell types and the biotype 1 strain was found to be more invasive than the non-motile biotype 2 isolate. Furthermore, the effect of six chemical compounds (Cytochalasin D, TAE 226, vinblastine, genistein, colchicine and, N-acetylcysteine), known to interfere with bacterial invasion strategies, were investigated. All of these compounds had a significant impact on the ability of the bacterium to invade host cells. Changes in the concentration of bacterial cells over time were investigated and the results suggested that neither isolate could survive intracellularly for sustained periods.ConclusionsThese results suggest that Y. ruckeri can gain entrance into host cells through several mechanisms, and might take advantage of both the actin and microtubule cytoskeletal systems.