Establishment of three cell lines from Chinese giant salamander and their sensitivities to the wild-type and recombinant ranavirus
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Known as lethal pathogens, Ranaviruses have been identified in diseased fish, amphibians (including Chinese giant salamander Andrias davidianus, the world’s largest amphibian) and reptiles, causing organ necrosis and systemic hemorrhage. Here, three Chinese giant salamander cell lines, thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established. Their sensitivities to ranaviruses, wild-type Andrias davidianus ranavirus (ADRV) and recombinant Rana grylio virus carrying EGFP gene (rRGV-EGFP) were tested. Temporal transcription pattern of ranavirus major capsid protein (MCP), fluorescence and electron microscopy observations showed that both the wild-type and recombinant ranavirus could replicate in the cell lines.
KeywordsReverse Transcription Polymerase Chain Reaction Kidney Cell Line Major Capsid Protein Permanent Cell Line TC199 Medium
Chinese giant salamander thymus cell line
Chinese giant salamander spleen cell line
Chinese giant salamander kidney cell line
Andria davidianus ranavirus
recombinant Rana grylio virus carrying enhanced green fluorescent protein
Major capsid protein
Fetal bovine serum
hours post infection
reverse transcription polymerase chain reaction
Introduction, methods and results
The Chinese giant salamander Andrias davidianus is the largest extant species of amphibian in the world and is often crowned as a living fossil . The wild Chinese giant salamanders are on the verge of extinction, arousing wide research interest in its conservation . Unfortunately, the prevalence of iridoviruses (including ranavirus and lymphocystis virus) infections is increasingly associated with major declines of amphibian species worldwide, causing extensive damage to aquaculture [3-7]. Ranaviruses are icosahedral DNA viruses, and are recognized as major viral pathogens of ectothermic vertebrates, namely fish, amphibians and reptiles [8-10]. Ranaviruses have been identified in diseased Chinese giant salamanders, such as Andria davidianus ranavirus (ADRV), which pose a serious threat to its population [11-14]. Another ranavirus strain, Rana grylio virus (RGV) is a pathogenic agent that causes lethal disease in cultured frogs (Rana grylio), which is the first isolated ranavirus in China . In recent years, several recombinant RGV, including ΔTK-RGV, Δ67R-RGV and i53R-RGV-lacIO were constructed in our laboratory [16-18]. Recombinant virus technology has provided novel approaches for unraveling virus-host interactions and host antiviral immune response .
Cell culture is essential for virus replication and isolation, because they are obligate intracellular agents . A cell culture-based technique has been popular for studying virus infection mechanisms, viral gene functions and virus-host interactions in aquaculture animals, and it is regarded as the “gold standard” for detection and diagnosis of viral pathogens [21,22]. Besides, the cultured cells have been used to replace live animals in testing agents in virology, pharmacology, toxicology, etc. . However, in contrast to fish, from which various cell lines have been established , few permanent cell lines have been established from the Amphibian, Order Caudata [25,26]. To our knowledge, no cell line from Chinese giant salamanders is currently available. Therefore, we attempted to establish different cell lines from the Chinese giant salamander and to investigate their sensitivities to the wild-type and recombinant ranavirus.
In the present study, one-year-old healthy Chinese giant salamanders were anesthetized, killed and wiped with 75 v/v ethanol, and the thymus, spleen and kidney tissues were removed for primary culture. All animal procedures were conducted in accordance with the recommendations in the Regulations for the Administration of Affairs Concerning Experimental Animals of China, and all efforts were made to minimize suffering.
In this study, three cell lines from the Chinese giant salamander, namely the thymus cell line (GSTC), spleen cell line (GSSC), and kidney cell line (GSKC) were developed. Among them, GSTC is the first reported cell line that was established from amphibian thymus tissue, the central immune organ of amphibians . More than 280 cell lines have been established from fishes , while there has been no report about a permanent cell line from the Chinese giant salamander. Although most of the established amphibian cell lines grow well in L15 medium [25,26], Chinese giant salamander primary cells failed to be cultivated in L15 or MEM medium. We finally chose the TC199 medium which is usually used to cultivate fish cell lines. Additionally, it takes more time for primary cells to migrate from Chinese giant salamander tissues (about a month) than fish primary cells (about one to two weeks) [24,27]. This is likely to be the reason that the Chinese giant salamander cells take longer to adapt in TC199 medium. The chromosome number in GSTC cells ranged from 28 to 102 with asymmetrical distribution, and the modal number was 50, which occupies 44% in the 100 metaphase cells.
In this study, the sensitivities of three cell lines to ADRV were compared by examining cytopathic effects and viral propagations (virus titers). The appearance of CPE in GSTC cells was faster (24 hpi) and more significant than that in GSSC and GSKC cells (appearing at 36 hpi, data not shown). The highest titer of ADRV (108.4TCID50/mL) was also detected in GSTC cells at 7 days post infection. Collectively, these results suggest that GSTC cells are most sensitive to ADRV. This research also shows that the rRGV-EGFP expressed green fluorescent protein after infecting GSTC cells, which provides a convenient way to observe the infection site and development of CPE. It suggests that the GSTC was suitable for recombinant RGV in vitro infection and gene expression. In addition, these cell lines have been used to analyse interactions between ranavirus and the host, the expression profiles and antiviral activity of amphibian immune genes as we have reported before, which confirms that the infection of GSTC cells with ADRV could induce up-regulations of major histocompatibility complex (MHC) isoforms, IFN-inducible protein 6 (IFI6) and T cell receptor beta chain (TCRβ) [29,30].
In summary, we developed three cell lines from the Chinese giant salamander, and the sensitivities of the three cell lines to amphibian ranaviruses were studied. The results show that both wild-type ranavirus ADRV and recombinant ranavirus rRGV-EGFP can replicate and cause CPE in their infected cells, and GSTC has the highest sensitivity to ADRV. Our work provides useful tools for the future study of ADRV, such as identifying ADRV-specific receptors, elucidating the infection mechanism, ADRV recombination and host-pathogen interaction research.
This work is supported by grants from the National Natural Science Foundation of China (31430091, 3141101038), the National Key Basic Research Program (2010CB126303), and the Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ12).
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