Texas A&M University-San Antonio Digital Commons @ Texas A&M University- San Antonio Biology Faculty Publications College of Arts and Sciences 2008 Transcriptional Response of Mexican Axolotls to Ambystoma Tigrinum Virus (ATV) Infection J. D. Cotter A. Storfer Robert B. Page Texas A&M University-San Antonio, [email protected] C. K. Beachy S. R. Voss Follow this and additional works at: https://digitalcommons.tamusa.edu/bio_faculty Part of the Biology Commons Repository Citation Cotter, J. D.; Storfer, A.; Page, Robert B.; Beachy, C. K.; and Voss, S. R., "Transcriptional Response of Mexican Axolotls to Ambystoma Tigrinum Virus (ATV) Infection" (2008). Biology Faculty Publications. 14. https://digitalcommons.tamusa.edu/bio_faculty/14 This Article is brought to you for free and open access by the College of Arts and Sciences at Digital Commons @ Texas A&M University- San Antonio. 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BMC Genomics BioMed Central Research article Open Access Transcriptional response of Mexican axolotls to Ambystoma tigrinum virus (ATV) infection Jennifer D Cotter*1, Andrew Storfer1, Robert B Page2, Christopher K Beachy3 and S Randal Voss2 Address: 1School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA, 2Department of Biology and Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington KY 40506, USA and 3Department of Biology, Minot State University, Minot ND 58707, USA Email: Jennifer D Cotter* - [email protected]; Andrew Storfer - [email protected]; Robert B Page - [email protected]; Christopher K Beachy - [email protected]; S Randal Voss - [email protected] * Corresponding author Published: 20 October 2008 Received: 3 January 2008 Accepted: 20 October 2008 BMC Genomics 2008, 9:493 doi:10.1186/1471-2164-9-493 This article is available from: http://www.biomedcentral.com/1471-2164/9/493 © 2008 Cotter et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV). Result: At 0, 24, 72, and 144 hours post-infection, spleen and lung samples were removed for estimation of host mRNA abundance and viral load. A total of 158 up-regulated and 105 down- regulated genes were identified across all time points using statistical and fold level criteria. The presumptive functions of these genes suggest a robust innate immune and antiviral gene expression response is initiated by A. mexicanum as early as 24 hours after ATV infection. At 24 hours, we observed transcript abundance changes for genes that are associated with phagocytosis and cytokine signaling, complement, and other general immune and defense responses. By 144 hours, we observed gene expression changes indicating host-mediated cell death, inflammation, and cytotoxicity. Conclusion: Although A. mexicanum appears to mount a robust innate immune response, we did not observe gene expression changes indicative of lymphocyte proliferation in the spleen, which is associated with clearance of Frog 3 iridovirus in adult Xenopus. We speculate that ATV may be especially lethal to A. mexicanum and related tiger salamanders because they lack proliferative lymphocyte responses that are needed to clear highly virulent iridoviruses. Genes identified from this study provide important new resources to investigate ATV disease pathology and host- pathogen dynamics in natural populations. Background have been incredibly useful for discovery of genes associ- Emerging infectious diseases (EIDs) pose a serious threat ated with host response and variation in resistance or sus- to the health, stability, and persistence of human and ceptibility to a variety of pathogens [5-7]. The advent of wildlife populations [1-4]. Genetic and genomic tools genomic tools such as microarray analysis has offered new Page 1 of 19 (page number not for citation purposes) BMC Genomics 2008, 9:493 http://www.biomedcentral.com/1471-2164/9/493 insights into host-pathogen systems. Additionally, their The tiger salamander species complex includes A. mexica- application to genomic response to host disease response num (Mexican axolotl), a model organism with a growing allows rapid characterization of candidate genes for fur- genomic and informatics resource base [20]. The immune ther research into control and eradication methods. system of the Mexican axolotl has been extensively stud- ied using several classical approaches. Relative to other EIDs are a leading hypothesis for the global decline of vertebrate models, the axolotl immune response has been amphibians and two pathogens in particular, Batra- described as immunodeficient [21,22]. There are several chochytrium dendrobatidis and Ranaviruses have been reasons for this characterization, including: production of implicated in worldwide epizootics. Although studies are only two immunoglobulin (Ig) classes, only one of which beginning to investigate possible mechanisms of resist- regulates the humoral response and neither of which is ance to these pathogens [8], in general, very little is known anamnestic [23,24]; no response to soluble antigens [25]; about the immune response of amphibians to EIDs. This poor mixed lymphocyte reactions [26,27]; and lack of cel- is because most natural amphibian species are not used as lular cooperation during the humoral immune response laboratory models and we lack fundamental molecular as indicated by enhanced humoral immunity following tools to investigate disease pathology and host-pathogen thymectomy or X-ray irradiation [28,29]. Weak immune interactions at the molecular level for all but a few species responses are known for salamanders in general, and the (e.g., Ambystoma tigrinum spp., Xenopus spp.). Mexican axolotl and related tiger salamanders are espe- cially susceptible to ATV infections with high observed Over the last 15 years, Ranavirus infections have been mortality rates both in the laboratory and in the field. associated with marked increases in morbidity and mor- Indeed, an outbreak of ATV in 2003 at the Indiana Axolotl tality in fish, reptiles, and amphibians [9]. Ranaviruses are Colony significantly reduced adult stocks before the virus globally-distributed double-stranded, methylated DNA was contained. By way of comparison, adult Xenopus effec- viruses of fish, amphibians and reptiles and are impli- tively clear close-related FV3 Ranavirus with an immune cated in amphibian epizootics worldwide [9-11]. Both response that includes an early T-cell proliferative phase encapsulated and non-encapsulated forms can be infec- in the spleen [30]. tious. The virus enters the cell via receptor mediated endo- cytosis or via fusion with the plasma membrane; and To further investigate the axolotl immune response to DNA and RNA synthesis occur in the nucleus, while pro- ATV, we used an Affymetrix custom microarray to identify tein synthesis occurs at morphologically specific assembly genes that were significantly, differentially expressed in sites in the cytoplasm [9]. In North America, ranaviruses the spleen. We then compared these genes to a list of have been isolated from the majority of recent docu- genes associated with regeneration that were previously mented amphibian epizootics [12], including from tiger identified from A. mexicanum using the same microarray salamander (Ambystoma tigrinum) epizootics in Sas- platform. We reasoned that such a comparison would katchewan, Canada [13], Arizona [14], North Dakota, allow us to filter gene expression responses of humoral Utah, and Colorado, USA [15,16]. The viral variant that cells induced generally in response to injury and stress infects tiger salamanders, ATV, is transmitted either via from those expressed specifically in response to ATV infec- direct contact with an infected animal or immersion in tion. Also, this comparison would potentially identify water that contains virus and infected individuals exhibit gene expression signatures associated with cell prolifera- systemic hemorrhaging, edema, ulceration, and necrosis tion in response to ATV, as we have previously identified of the integument and internal organs [13,17,18]. In cases many cell proliferation probe sets on the Ambystoma gene- where ATV infection leads to mortality, it usually occurs chip that are differentially regulated during spinal cord within 2–3 weeks of exposure, with animals displaying regeneration [31]. The genes that we describe provide symptoms often between 8–10 days post-exposure. Thus, mechanistic insights and new tools to investigate sala- ATV can rapidly overwhelm the tiger salamander immune mander antiviral responses in the laboratory and in natu- response. However, mortality is not always a pathological ral populations. endpoint because virulence and resistance are known to vary among ATV strains and tiger salamander popula- Methods tions, respectively, as indicated by