The influence of viral infection on cell line characteristics: Lessons learned from working with new cell lines from common carp.

Several factors influence the susceptibility of cell lines to infection by different viruses. These can be related to tissue specificity of the viruses, physiological status of the cells, their differentiation level and their capacity to mount immune responses to combat viral infection. To study the influence of cell characteristics and immune responses on their susceptibility on virus infection, newly developed cell lines from common carp brain (CCAbre), fins (CCApin), gills (CCAgill), and heart (CCAcar) and the established common carp brain (CCB) cells were exposed to the carp infecting viruses cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV), and the yet not fully characterized common carp paramyxovirus (CCPV). The susceptibility of these cells to viral infection was measured by formation of a cytopathic effect (CPE), estimation of viral particles produced by the cells and presence of viral mRNA in the cells. Viral susceptibility of the cells was compared to cell characteristics, measured by mRNA expression of the epithelial cell markers cadherin 1, occludin, and cytokeratin 15 and the mesenchymal cell marker vimentin, as well as to the level of type I interferon (IFN) responses. All cell lines were susceptible to CyHV-3 and CCPV but not to CEV infection. The cell lines had different levels of type I IFN responses towards the viruses. Typically, CyHV-3 did not induce high type I IFN responses, while CCPV induced high responses in CCAbre, CCAcar, CCApin cells but no response in CCAgill cells. Consequently, the type I IFN response modulated cell susceptibility to CCPV but not to CyHV-3. Interestingly, when the three different passage levels of CCB cells were examined, the susceptibility of one passage was significantly lower for CyHV-3 and higher for CCPV infection. This coincided with a loss of epithelial markers and lower type I IFN responses. This study confirms an influence of cell characteristics and immune responses on the susceptibility of carp cell lines for virus infection. Depending on the vulnerability of the virus to type I IFN responses, cells with a lower IFN-response can be superior for replication of some viruses. Batches of CCB cells can differentiate and thus may have significantly different levels of susceptibility to certain viruses.

Koi sleepy disease as a pathophysiological and immunological consequence of a branchial infection of common carp with carp edema virus.

Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, "koi sleepy disease" (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L-1, and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L-1. At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3-4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish.

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host-pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV-1) and cyprinid herpesvirus 3 (CyHV-3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up-regulated the expression of antiviral genes vig-1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV-3 induce up-regulation of vig-1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV-3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV-infected fish, a prolonged confrontation of the host with the pathogen could be studied.

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.

Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology.

Mammalian and fish skin share protective activities against environments that are rich in infectious agents. Fish epidermis is endowed with an extrinsic barrier consisting of a mucus layer and antimicrobial peptides (AMPs). These operate together as a protective chemical shield. As these AMPs are evolutionarily well preserved and also found in higher vertebrate skin (including human epidermis), fish skin offers a unique opportunity to study the origins of innate antimicrobial defense systems. Furthermore, the broad spectrum of fish mucus antimicrobial activities renders piscine AMPs interesting to investigative dermatology, as these may become exploitable for various indications in clinical dermatology. Therefore, this article aims at casting light on fish mucus, the evolutionary relationship between human and fish AMPs, and the latter's antibacterial, antifungal, and even antiviral activities. Moreover, we develop dermatological lessons from, and sketch potential future clinical applications of, fish mucus and piscine AMPs.

Pros and cons of fish skin cells in culture: long-term full skin and short-term scale cell culture from rainbow trout, Oncorhynchus mykiss.

Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines.

'Fish matters': the relevance of fish skin biology to investigative dermatology.

Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.