Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced idiopathic effect (CPE) progression and significantly inhibited viral gene transcription of Ronny and SGI.
Lipids, the major components of biological cell membranes, play essential roles in intracellular signaling, and act as the precursors for ligands' binding to nuclear receptors (1 – 3). Lipids are involved in various cellular processes, such as autoplay (4, 5), a conserved biological pathway that delivers cytoplasmic components to lysosomes and maintains the balance between synthesis, degradation, and recycling of cellular components (4, 6).
On the other hand, different viruses demonstrate differential manipulation of cellular lipid metabolism (7 – 9). Recent research on zebrafish suggests politic acid processes antiviral activity via its inhibition of automatic flux (17).
Another report revealed that live Edwardsville tardy vaccine promoted biosynthesis of politic acid and then increased the IL-8 expression in zebrafish, and subsequently contributed to the resistance against E. tardy infection (18). Moreover, politic acid has an inhibitory effect on IFN-based anti-Hepatitis C Virus (CV) therapy (19).
Politic acid plays a significant role in cell autoplay and pathogen-host interactions. Orange-spotted grouper, Epimetheus coincides, is one of the commercially important farmed fishes in China and Southeast Asian countries.
However, outbreaks of viral and bacterial diseases always cause massive economic losses and affect the development of grouper aquaculture (20 – 23). Additionally, findings of the role of lipids on modulating cell immunity have been advanced.
Furthermore, the characterization of cellular lipid metabolism revealed the increased politic acid level in SGI infected cells (unpublished data). Although considerable progress had been made in molecular analysis of viral infection or host antiviral strategies, the functions of FAS, especially politic acid, in fish virus infection, remained mostly unclear.
SGI infection led to the accumulation of politic acid in GS cells. By suppressing cell automatic flux and the TBK1-IRF3/7 signaling pathway, politic acid finally facilitated the replication of SGI.
Thus, we speculated that SGI utilized politic acid in immune evasion processes. Our results provided new insights into the biological activity of politic acid and increased the understanding of SGI pathogenesis as well.
GS's cells were cultured in Leibniz's L-15 medium containing 10% fetal bovine serum (FBS, Rico) at 25 °C (33). Singapore grouper rhinovirus (SGI, strain A3/12/98 PPD) was propagated in GS cells and stored at 80 °C until use (34).
Cellular toxicity detection of politic acid incubation was performed as described (35). Briefly, 100 mm politic acid (Sigma-Aldrich, St Louis, MO) stocks were prepared in 0.1 M NaOH at 70 °C and filter sterilized.
Bovine serum albumin (BSA, certified fatty acid free, low antitoxin, Sigma) was dissolved in complete media to a final concentration of 1% (w/v) and sterilized using a 0.45 km non-pyogenic filter. Politic acid was added to a final concentration of 0.2, 0.4, 0.6, and 0.8 mm as different treatment levels.
Control media (carrier) contained NaOH and filtered acid-free bovine serum albumin (BSA, Sigma-Aldrich). GS's cells were pretreated with politic acid for 20 h. And then, CD was added into the culture medium at the final concentration of 5 km and co-incubated for another 4 h.
It has been reported that the biosynthesis of palmitate is catalyzed by fatty acid synthase (FAS) (38). GS's cells were transected with Fan SIRPA (Silas: 5-GGGUUCAAGUCGUUGACCAGCCUAU-3) or the same volume of negative control (NC) SIRPA for 24 h, and then infected with SGI for 24 h. Idiopathic effects (CPE) caused by SGI infection were observed under a light microscope (Mass).
The effects of Fan SIRPA on the transcriptional levels of viral genes were evaluated by qRT-PCR. WST-1 assay was performed to examine the impact of politic acid treatment on cell proliferation.
After adding 10 SL of cell proliferation reagent WST-1 (Roche) into each well and incubation at 28 °C for 4 h, the absorbance was measured in Varioskan™ LUX multimode micro plate reader (Thermos Fisher, USA) at 450/655 nm. Mock- or virus-infected cells were collected for further qRT-PCR analysis, western blot, and virus tier assay.
The total RNA's of cells were extracted using the SV Total RNA Isolation Kit (Pr omega) and reversed to synthesize the first-strand CDA using the Reverted Ace kit (Toyota). Then the mRNA transcriptional levels of FA synthesis related genes were evaluated by qRT-PCR at 12 h post-infection (p.i.).
The primers of ACC1 (Acetyl-CoA carboxylase 1), SREBP-1 (sterol regulatory element-binding protein 1), LXR (liver X receptor), and Fan gene (fatty acid synthase) are listed in Table 1. The qRT-PCR analysis was performed in the QuantStudio™ 5 Real-Time PCR System (Thermos Fisher, USA).
In brief, GS cells pretreated with politic acid or vehicle for 24 h were infected with SGI (at MOI of 0.5) and collected at 48 h p.i. The viral liters of cell Yates were evaluated using the 50% tissue culture infectious dose (Acid 50) assay (39).
The Cues were observed under a light microscope (Lacey, Germany) every day, and each sample was measured in triplicate. After the experimental treatments, cells were lased and solubilized in 40 SL of Pierce IP Lysis Buffer (Thermos Fisher Scientific), containing protease/phosphatase inhibitor cocktail.
Solubilized proteins were resolved by 6, 10, or 12% SDS-PAGE and then electrophoretically transferred to 0.2 km Trans-Blot Turbo PDF (Mini pore). The membranes were blocked with 5% skim milk or 3% bovine serum albumin (BSA) dissolved in PBS for 2 h, then incubated with different primary antibodies overnight at 4 °C.
Membranes were washed for 3 times in Past or Test (for phosphorylation assay) buffer subsequently. Then, secondary goat-anti-rabbit or goat-anti-mouse antibody labeled with horseradish per-oxidase was used, and bound proteins were detected with Enhanced Pre-lab cryogenic substrate Kit (Tangent; www.tiangen.com) according to the manufacturer's protocol.
Applied as a vital stain for the detection of intracellular lipid droplets, Nile Red (9-diethylamino-5H-benzo phenoxazine-5-one) is characterized by red fluorescence (excitation 515–560 nm, emission >590 nm) (40, 41). The stock solution of Nile Red was prepared in acetone (0.5 mg/mL) and stored at 4 °C, protected from light.
Finally, samples were stained with 1 mg/mL 6-diamidino-2-pheny-lindole (DAPI) and observed under fluorescence microscopy (Mass, Germany). Here, Click Palmitoylated Protein Assay Kit (Bio vision, Catalog # K452-100) was used to detect the influence of SGI infection on the cellular politic acid level.
Background Control Cells are only exposed to Click Reaction, no Click Politic Acid Label. Positive Control Cells are incubated with 1× Click Politic Acid Label and Click Reaction.
Then, cells were incubated with Fixative Solution for 15 min at room temperature, protected from light. Then 1× Permeabilization Buffer was added to the cells for 10 min incubation at RT.
After staining with Hoechst33342, the cells were washed with 500 SL of ice-cold PBS and analyzed through fluorescence observation. Luciferase activity in total cell Yates was measured by luciferase reporter assay (Pr omega, USA) using a Varioskan™ LUX multimode micro plate reader (Thermos Fisher, USA).
The evaluation of palmitoylated protein staining suggested the lipid synthesis was promoted by SGI infection in GS cells (Figure 1A). Virus infection increased the accumulation of lipid products, especially politic acid, in GS cells.
In SGI infected cells, green fluorescence was observed in cytoplasm, which suggested that palmitoylated protein was accumulated (Figure 1A). Figure 1B shows that besides politic acid, SGI infection could increase the production of other intracellular lipids.
Thus, we speculated that SGI could utilize the lipid synthesis systems and control the cellular palmitoylation for its replication. (A) Intracellular politic acid accumulation analysis using Click Palmitoylated Protein Assay.
(B) Nile red staining of the intracellular lipids with and without SGI infection and immunofluorescence assay carried out at the same time with the anti-SGIV-MCP antibody. © Evaluation of the mRNA transcription levels of fatty acid synthesis related genes after SGI infection.
GS's cells were infected with SGI (MOI: 0.5), the mRNA transcriptional levels of fatty acid synthesis related genes were evaluated by qRT-PCR at 12 h post-infection. Functioning as the catalyst in palmitate biosynthesis processes (38, 45, 46), FAS was also induced by SGI in GS cells.
The CPE of SGI infection was less severe in Fan knock down cells (Figure 2B). Similarly, the mRNA level of SGI functional genes was reduced in Fan SIRPA transfection group (Figure 2C).
These results suggested Fan gene was essential for SGI replication. An earlier report had revealed that fatty acid synthase (FAS) encoded by Fan gene was a multi-functional enzyme that catalyzed palmitate biosynthesis in a NADPH-dependent reaction (38).
However, the mechanisms of palmitate in modulating SGI infection and replication remain unclear. Functional analysis should be carried out to investigate the role of politic acid in host-virus interaction.
Evaluation of the impacts of the Fan gene knockdown on SGI replication. (A) The interfering efficiency of the Fan gene in GS cells was analyzed by qRT-PCR.
Changes of metabolic profile of GS cells in response to SGI infection were documented. Politic acid massively increased in SGI infected cells (unpublished data).
Politic acid showed no adverse effect on cell viability when the concentration was lower than 0.6 mm within 24 h incubation (Figure 3A). A microscopic evaluation of GS cells after incubation with politic acid showed that the cytoplasm of politic acid pretreated cells contained numerous different-sized fluorescent bodies (stained with Nile Red) corresponding to lipid accumulation, i.e., stenosis (Figure 3B).
In the cytoplasm of control cells (1% BSA), the presence of moderate micro- and macro-vacuolar stenosis was also observed (Figure 3B). Politic acid treatment had low cytotoxicity in GS cells.
(A) WST-1 assay suggesting the effect of intracellular fat accumulation (dose-dependent) on cellular cytotoxicity of GS cells in culture. To investigate the effects of politic acid on SGI virus infection, we evaluated the CPE progression and detected the viral gene transcription as well as the viral coat protein synthesis of SGI in infected politic acid loading cells.
Moreover, in politic acid loaded cells, the transcription level of SGIV-MCP, SGIV-ICP-18, SGIV-VP19, and SGIV-LITAF was significantly increased (Figure 4B). Virus tier assay showed increased viral replication of SGI after politic acid treatment (Figure 4D).
The results of virus tier assay also suggested that exogenous addition of politic acid enhanced SGI replication (Figure 4D). (A) The severity of CPE induced by SGI infection in politic acid incubated cells was observed under the microscope (Mass).
It has been demonstrated that politic acid can modulate autoplay in many kinds of cells. We sought to determine whether politic acid could also modify this process in GS cell (5, 47).
As shown in Figure 5, increased levels of green signal of LC3, as well as the higher levels of LC3-II and p62 in the politic acid treated group evidenced by western blot assay indicate that politic acid induces the accumulation of autophagosomes in GS cells (Figures 5A,C). Autoplay is known to be a dynamic process, so the detection of LC3-II levels is not sufficient to assess automatic activity in cells (5, 48).
To discriminate between these two possibilities, we determined the impacts of politic acid on automatic flux using chloroquine (CD), an inhibitor of autophagosome-lysosome fusion. Taken together the decreased automatic flux and the accumulation of LC3-II and p62 caused by politic acid, we subsequently concluded that politic acid decreased autoplay flux by inhibition of autophagosome–lysosome fusion step.
Politic acid impacted automatic flux in GS cells. GS's cells were transected with C1-EGFP-LC3 plasmid, then incubated with or without 0.4 mm politic acid.
(B) Representative image of phospho-Akt (Ser473) , and phospho-mTOR (pastor) detection was performed to verify AKT and motor inhibition by 1% BSA or politic acid (0.4 mm) treatment. © The expression levels of LC3, and p62 in cell Yates were evaluated after the incubation of 1% BSA or politic acid (0.4 mm) for 24 h. Western blot assay was carried out, and -tubulin was used as the internal control.
Western blot assay was carried out to detect the LC3-II and -tubulin levels in cell late. Band intensity was calculated using Image J software, and the LC3-II protein level was presented by the ratio of LC3-II/-tubulin.
To determine the regulatory effects of politic acid on the expression of interferon related molecules, we detected the transcripts of these genes in GS cells treated with different concentrations of politic acid. As shown in Figure 6, the relative expression of EcIRF3 (interferon regulatory factors 3), EcIRF7, EcISG15 (interferon-stimulated gene), EcIFP35 (Interferon-induced 35 DA protein), CMI (Interferon-induced GTP-binding protein Mx1), EcTBK1 (TANK-binding kinase 1), CRIF (TIR-domain-containing adapter-inducing interferon), and EcMDA5 (melanoma differentiation-associated protein 5) were significantly decreased in politic acid treated cells compared to the control cells, suggesting that politic acid inhibited the interferon responses (Figure 6).
Politic acid decreased the expression of interferon related cytokines or effectors. GS's cells were incubated with the indicated concentration of politic acid (0.2 or 0.4 mm) or 1% BSA, then harvested at 24 h post-incubation.
On the other hand, the effects of politic acid treatment on IFN and Ire promoter activities were evaluated using reporter gene assay. (A) Treatment of politic acid decreased promoter activities of IFN1 and Ire.
GS's cells were co-transfected with IFN1-Luc/ISRE-Luc and pRL-SV40 Vanilla luciferase vector, and treated with 1% BSA or indicated concentration of politic acid (0.2 or 0.4 mm), respectively. The promoter activity was measured using the luciferase reporter gene assay.
Furthermore, the insights of politic acid modulations were discovered by detecting its regulation of MDA5 -/TBK1-induced interferon signaling pathway. As shown in Figure 8, politic acid significantly decreased the TBK1 -induced interferon responses.
But there were no significant differences between BSA or politic acid incubated groups under MDA5 transfection. Thus, we proposed that politic acid treatment could down-regulate IFN signaling by inhibiting the TBK1-IRF3/7 pathway.
GS's cells were transected with EcTBK1 (A) or EcMDA5 (B) and then incubated with 1% BSA or politic acid at indicated concentrations (0.2 or 0.4 mm). Setting the mRNA expression level in Flag transected cells as 1-fold.
As structural elements of viral and cellular membranes, lipids were suggested to be involved in the intricate virus-cell interaction in many ways (50). Results obtained with a variety of viruses suggested that lipids played significant roles in cell metabolism, fatty acid synthesis and generation of a specific lipid micro environment enriched in phosphatidylinositol 4-phosphate (PI4P) for efficient viral replication (10, 51, 52).
Identifying the influences of bioactive lipid mediators on host inflammation, viral replication, and disease progression would lead to the discovery of lipid-active compounds as potential antiviral drugs and the development of antiviral strategies (53). Numerous studies have been cited to illustrate that viral infection was associated with lipid metabolomics (54, 55).
Politic acid, the product of the FAS-mediated biosynthesis process, was up-regulated during SGI infection indicated by the lipid metabolism profiles (unpublished data). Additionally, knockdown of the Fan gene suppressed the infection and replication of SGI in vitro.
Fan gene can be induced by white spot syndrome virus (SSV) infection in shrimps, and facilitate virion formation and viral morphogenesis (56). Moreover, our previous study reveals Fan gene is critical for Ronny replication (57).
For instance, politic acid promotes astrocytogenesis in the differentiated neural stem cells (59), and also stimulated hepatocyte proliferation (60). But the high concentration of politic acid (1 mm) significantly decreased HepG2 cell viability (61).
Accordingly, the reduction of mRNA transcriptional level of interferon related signaling molecules, including EcIRF3, EcIRF7, EcISG15, EcIFP35, and CMI, implied politic acid exerted negative regulation on interferon antiviral immune in fish cells. Further analysis showed that politic acid significantly inhibited TANK-binding kinase-1 (TBK1), but not MDA5 -inducing interferon response.
EcTBK1 was identified as a vital inhibitor in the viral infection processes of SGI, by triggering the IRF3- and IRF7-regulated interferon promoter Ire and IFN activity in our previous research (63). Thus, politic acid might restrict the interferon response through its influence on the TBK1-IRF3/7 signaling pathway in fish.
Autoplay is an essential mechanism in cell survival under certain stress conditions, such as nutrient deprivation (64). A variety of DNA and RNA viruses induce PCD actively during infection, which is critical in the pathogenesis of viral diseases (66, 67).
Evidence also revealed that autoplay induced by Zika virus (Zika) through its inhibition of Actuator signaling in human fetal neural stem cells, resulted in the increased virus replication level and impeded neurogenic (68). In the hepatitis B virus (HBO) infection, glucosamine can act as the promoter of virus replication by inducing automatic stress through its double effects in suppressing automatic degradation and inhibiting mTORC1 signaling pathway (69).
Reports suggest that politic acid impacts on cell autoplay, the steward of cellular process (4, 5). In the current study, accumulation of intracellular LC3 was observed, and the reduction of the part (Ser473) and pastor phosphorylation also suggested politic acid is associated with cell autoplay (49).
Further study revealed the increased LC3-II and p62 proteins level after politic acid treatment. Additionally, known as the phosphatidylethanolamine conjugate of LC3-I, LC3-II is recruited to autophagosomal membranes where it binds to p62 (74).
In order to discriminate the effect of politic acid between these two possibilities, GS cells were treated with CD as described previously (74, 75). The ratio between LC3-II protein level in the presence and absence of CD, which indicates automatic flux, was significantly reduced by politic acid (5, 75).
Together, our results suggested the blockade of automatic flux by politic acid through suppressing the fusion of autophagosome-lysosome. Exploiting specific lipid requirements of pathogens, and delineating the intricate interactions of these pathogens with cellular lipids and the modification of their metabolism, might provide new approaches for antiviral therapies (50).
Our findings provide new mechanistic insights linking lipids and immunity in virus infections. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
By carried out the main experiments, analyzed the data, and drafted the manuscript. CL and FM participated in the qRT-PCR experiments and western blotting assay.
By, HQ, Oh, and Oh designed the experiments and reviewed the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The reviewer JC declared a past co-authorship with several of the authors By, SW, Oh, Oh, and HQ to the handling Editor. The multi-dimensional regulation of gene expression by fatty acids: polyunsaturated fats as nutrient sensors.
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Leon Grayer Evanston Hold Francisco De Jesús Adding V. Gregory Chin char Jacques Robert Accordingly, here we coalesce the current state of understanding of the distinct facets of lower vertebrate immune responses to Canaveral infections and underline some evasion strategies by which these pathogens circumvent these host defenses.
Infections of ectothermic vertebrates by members of the genus Rotavirus (RV; family Bromoviridae) and the resulting disease outbreaks and die-offs among wild and farmed populations have escalated at alarming rates recently and raised considerable concerns. While it is apparent that individual telecast, amphibian, and reptile species vary in their susceptibility to these pathogens, the immune and viral determinants of Canaveral diseases are at present unclear.
Thus, a more thorough examination of the ranavirus-host immune interface at the molecular and cellular levels is necessary in order to devise potential preventative measures against these viral agents. Ectothermic display poorer T lymphocyte expansion, fewer antibody isoforms, and generally a less developed immunological memory response than mammals (Robert and HTA 2009).
Antimicrobial peptides are an important element of aura innate immunity that provides protection to skin and mucosal surfaces against a variety of pathogens. These small molecules are synthesized and stored in the dermal granular glands and secreted into mucus in response to stress or injury (Rollins-Smith 2009 ; Rollins-Smith et al. 2005).
Esculentin-2P (E2P) and Ranatuerin-2P (R2P), two antimicrobial peptides isolated from Reyna pipes, are capable of inactivating both FV3 and channel catfish virus (CCV) within minutes and at temperatures as low as 0 °C. This suggests that direct interaction of these molecules with the viruses rather than inhibition of viral replication is responsible for the drop in inactivity (Chin char et al. 2001).
The ability of antimicrobial peptides to function across a broad range of temperatures presumably reflects the ectothermic nature of the host. It was postulated that the greater resistance of FV3 to inactivation reflected the difficulty of antimicrobial peptides to target the inner lipid membrane beneath the FV3 caps id.
Interestingly, all of these peptides conferred concentration-dependent inhibition of Rev plaque formation, while viral clearance coincided with increased expression of genes encoding these molecules (Yang et al. 2012). As it stands, there is substantial documentation of innate immune responses and associated inflammation to rotavirus infections across a range of poikilothermic host species (Carey et al. 1999 ; Chen and Robert 2011 ; Grayer et al. 2014 ; Ivanovich and Jacobs 2011 ; Morales et al. 2010).
Our research group has adopted and optimized the infection of Venous Levis by FV3 as a model for ranavirus-ectothermic vertebrate (particularly aura) antiviral immunity. This model pairs FV3, the best-described rotavirus at the molecular level, with Venous Levis, which possesses the most-characterized amphibian immune system.
Injection of X. Levis adults or tadpoles with FV3 followed by an assessment of the progress of infection, viral replication, and the host immune response. By this approach, we have been able to delineate the sequential progression of the innate and adaptive immune responses of adult X. Levis throughout the course of FV3 infection (Morales et al. 2010).
In X. Levis adults, biochemical and flow cliometric analyses revealed that activated mononuclear and polymorphonuclear phagocytes are recruited to, and heavily represented within peritoneal exudates as early as 1 day following i.p. Notably, the rapid accumulation of peritoneal leukocytes was concomitant with substantially elevated inflammatory gene expression.
The elevated level of Arg-1 gene expression at 1 day post infection may be reflective of resident, rather than recruited inflammatory eyelid populations. Indeed, after FV3 peritoneal inoculation, we have consistently observed elevated mRNA transcripts for macrophages and granulocyte colony-stimulating factor receptors (CFR and CFR, respectively), indicative of accumulating eyelid infiltrates (L. Grayer and J. Robert, University of Rochester, unpublished data).
Notably, the elevated expression of CFR (and CFR) within peritoneal leukocytes (PLs) is typically accompanied by significantly increased expression of the M1 macrophages marker, inducible nitric oxide synthase (INS), which catalyzes the production of the antimicrobial nitric oxide by inflammatory macrophages (L. Grayer, F. DE Jesús Adding and J. Robert, University of Rochester, unpublished data). This supports the observation of decreased Arg-1 expression with the onset of an inflammatory state within the peritoneum and indicates that Arg-1 and INS functions are opposite across multiple groups of vertebrates (Joe rink et al. 2006a, b, c ; Wiegertjes and Lorenzo 2010).
Extensive immunological studies of Venous suggest that tadpoles possess a distinct immune system from that of adults. The larval system is typically more immature, particularly with regard to adaptive immunity (e.g., poor T cell and antibody responses).
In this regard, it is altogether not surprising that tadpoles are typically unable to fully control rotavirus infections and succumb to these pathogens (Bayley et al. 2013 ; Grayer et al. 2014 ; Hover man et al. 2010 ; Lands berg et al. 2013 ; Reeve et al. 2013). Indeed, several reports indicate that compared to larvae of given amphibian species, metamorphic (Brunner et al. 2004 ; Harelip et al. 2011 ; Reeve et al. 2013) and adult (Duffs et al. 2013) animals may be more susceptible to rotaviruses.
Extensive immune remodeling may render meta morphs more susceptible than larvae at critical developmental stages. In contrast to infected adult frogs, tadpoles exhibited poor and considerably delayed anti-FV3 inflammatory gene responses (DE Jesús Adding et al. 2012).
Notably, stimulation of tadpoles with heat-killed Escherichia coli readily elicits rapid induction of the above genes within 24 h, suggesting that the immune delays are FV3 specific (DE Jesús Adding et al. 2012). These inefficiencies in the tadpole innate immune response to FV3 may reflect multiple nonexclusive issues including viral immune evasion, defect(s) in the tadpole pathogen sentinel receptor system, or physiological tread offs to forego energetically costly inflammatory responses in favor of growth and development.
Thus, these modest and delayed immune responses are likely contributing factors for the characteristically higher susceptibility of aura tadpoles to FV3 infection and severe disease. Consistent with the notion that hosts mount broad inflammatory responses to rotaviruses, a comprehensive micro array analysis of axolotls (Ambystoma Mexican) infected with the Ambystoma titanium virus (ATV) revealed the up regulation of numerous hallmark pro-inflammatory and innate immune gene components in the spleens and lungs of these animals (Cotter et al. 2008).
These genes included (but were not limited to) phagocyte receptors and intracellular components, cytosine signaling molecules, complement components, NADPH oxidase subunits (eyelid enzyme catalyzing the reactive oxygen antimicrobial response), and myloperoxidase (granulocyte enzyme catalyzing the production of hydrogen peroxide) (Cotter et al. 2008). There is a substantial literature documenting innate immune and associated inflammatory responses to rotavirus infections in bony fish.
Interestingly, all four viruses elicited expression of apoptotic components and 2-microglobulin, which is critical for surface MHC class I expression and cytotoxic T cell function, suggesting that at least with respect to FV3 infection of telecasts (as compared to axolotls), the adaptive immune response may be elicited by Canaveral infections. A recent micro array study examined the transcriptional response of fathead minnow (FHM) cells following infection with either wild type (wt) FV3 or a knockout (KO) mutant lacking the truncated vIF-2 gene.
For the most part, similar genes were unregulated in cells infected with the KO mutant, but the magnitude of the induction was generally lower (Cheng et al. 2014). These animals suffered from exacerbated inflammation, necrosis, and characteristic ranavirus-induced cytoplasmic inclusions within splenic, renal, lymphoid, and hematopoietic tissues (Bollinger et al. 1999).
Similarly, whole populations of ranavirus-infected green-striped tree dragons (Jayapura splendid) exhibited systemic hemorrhaging, necrosis, granulators, and necrotic inflammation, as well as severe renal pathology, hyper anemia, and extensive hepatic damage (Behance et al. 2013), culminating in mass mortality. Consistent with these inflammatory symptoms, juvenile bass inoculated with CMBV exhibited corkscrew swimming and distended abdomens (Gilbert et al. 2000).
Indeed, the above observations are reminiscent of earlier studies of FV3 infections in rodents (Gut et al. 1981 ; Kirk et al. 1980, 1982), in which, despite the inability of FV3 to replicate at 37 °C (Albertan et al. 1973), the initial viral innocuous was responsible for extensive inflammation, necrosis, and liver damage. Mindful of the idea that X. Levis adults presumably mount effective anti-ranaviral responses leading to viral clearance, we were intrigued to find that (at least during acute infections) adults possessed significantly greater FV3 loads (1 to 2 orders of magnitude) than tadpoles, which are typically more susceptible to FV3 infections (Grayer et al. 2014).
In support of this hypothesis, we observed that although tadpoles overstimulated with recombinant X. Levis type I interferon (r XL IFN) possessed viral loads several logs lower than adults, they nonetheless succumbed to FV3 infection (Grayer et al. 2014). Indeed, as seen in rodent models of FV3, rotaviruses may trigger toxic and potentially lethal effects, irrespective of their capacity to replicate within their host cells (General et al. 1981).
Similarly, heat- and UV-inactivated FV3 elicits FHM cell apoptosis and inhibits host RNA and protein synthesis (Chin char et al. 2003 ; Rag how and Gran off 1979). If this hypothesis holds true for other members of the genus and family, we may need to consider that these viruses are more pathogenic than previously thought.
The involvement of macrophage-lineage cells in rotavirus infections may be inferred from initial studies conducted 30 years ago using rodents as models of hepatitis (Gut et al. 1981 ; Kirk et al. 1980, 1982). These studies also implicated inflammation as a contributor to FV3-mediated pathology, including extensive leukotriene release by Suffer cells (Hangman et al. 1987).
Inhibition of leukotriene synthesis within FV3-infected animals dramatically reduced vitally elicited hepatic damage (Hangman et al. 1987), suggesting that pathology was largely due to inflammatory responses. Although FV3 is not a mammalian pathogen and, except expression of select early genes (Lopez et al. 1986) does not replicate at 37 °C (Albertan et al. 1973), this work nonetheless supports the current hypothesis that, because of their high phagocyte and endoscopic activity, macrophage-lineage cells are integral targets for rotavirus infections.
In cultured rat Suffer cells, viral particles appeared in phagocyte vacuoles and endoscopic compartments promptly following FV3 infection (General et al. 1981). In line with this reasoning, it is likely that cells of the eyelid lineage serve as Canaveral targets precisely because of their high efficiency of ingestion of extracellular materials, facilitated by an array of endoscopic/phagocyte surface receptors, several of which likely recognize and bind rotaviruses.
This feature of vertebrate professional phagocytes may have been targeted as a Canaveral infection strategy and may explain why rotaviruses successfully cross host species boundaries. Instead, cell death is presumably triggered by preformed lytic factors encapsulated within FV3 virions or the expression of early viral gene products (Lopez et al. 1986).
Similar to the mRNA present within adenovirus virions (Chung et al. 2003), FV3 early gene expression at nonpermissive temperatures may also result from the release of prepackaged virulence factor-encoding mRNAs rather than from DE Nova viral transcription. Indeed, FV3 infection of mammalian cells induces rapid cellular RNA, DNA, and protein synthesis arrest (Eleazar et al. 1973).
Furthermore, factors solubilized from FV3 virions result in cellular toxicity and inhibit host macromolecular synthesis (Albertan et al. 1973 ; Kirk et al. 1972) We have demonstrated that FV3 persists within amphibian hosts for several months following the resolution of clinically apparent disease (Robert et al. 2007).
Since peritoneal leukocytes are comprised predominantly of macrophage-lineage cells, our findings not only corroborate the ranavirus-macrophage tropism, but also suggest that these terminally differentiated, long-lived populations are ideal vectors for viral dissemination, or serve as “within host” reservoirs. These FV3-infected cells exhibited small numbers of intracellular viral particles, implying that FV3 may employ mononuclear phagocytes as a reservoir for dissemination.
FV3-macrophage interaction is reminiscent of the HIV-macrophage relationship in which viral particles accumulate within the eyelid cells as a mechanism of dissemination (Cobras et al. 2009 ; Goode now et al. 2003 ; Gusset et al. 2008 ; Groot et al. 2008). The eyelid origins of these leukocytes are supported not only by their expression of macrophages inflammatory genes (TNF, IL-1, and Arginase-1; Morales et al. 2010), but also of the macrophage-lineage marker, CFR (L. Grayer, F. DE Jesús Adding and J. Robert, University of Rochester, unpublished data).
Possibly, the inability to detect FV3 genomes among peritoneal leukocytes at later times may reflect the dissemination of these cells to distal sites within the X. Levis host. Notably, this interval is shorter than the 3-week period during which viral persistence is reliably detected within peritoneal leukocyte populations.
Presumably, kidney cells are productively infected and serve as sites of active FV3 replication, indicated by high viral liters and extensive tissue damage (Grayer et al. 2014). Indeed, we have recently observed that FV3 genomic DNA may be amplified from in vitro-infected, cultured peritoneal phagocytes without detectable viral gene expression as long as several months after initial infection (L. Grayer, and J. Robert, University of Rochester, unpublished data).
We believe that the key to further delineating ranavirus-macrophage interactions and Canaveral quiescence is contingent on developing in vitro eyelid cell cultures and related reagents. We recently provided substantial support for the hypothesis that amphibian macrophages serve not only as vehicles of rotavirus dissemination within the host but also as loci of disease reactivation.
When peritoneal phagocytes were isolated from 15 ×. Levis adults 30 days post infection, only cells from one individual displayed detectable levels of FV3 DNA and expressed transcripts encoding the viral DNA polymerase and major caps id protein (J. Robert, L. Grayer, and F. DE Jesús Adding, University of Rochester, unpublished data). Further research into activation states (both classical and alternative; Affray et al. 2007 ; Narendra et al. 2007 ; Zhao et al. 2009 ; Ziegler-Heitbrock 2007) of mononuclear phagocytes will be critical to devising preventative measures and understanding the precise infection strategies of these complicated pathogens. Fig.
Akin to many other pathogens, rotaviruses presumably overcome macrophages antimicrobial and antiviral barriers, at which point these cells become vehicles for both viral dissemination and persistence. However, exploitation of macrophage-lineage cells as vectors of viral dissemination and persistence does not appear confined to FV3, as other members of the genus Rotavirus and family Bromoviridae have also adopted this mechanism of host infiltration and immune evasion.
For example, an iridovirus-like pathogen infects shellfish kidney macrophages and is capable of down-regulating formal mistake acetate-elicited reactive oxygen production by these cells in vitro (Sick et al. 1999). Likewise, following infection with the Taiwan Grouper Rhinovirus (TGI), elevated numbers of phosphatase-positive, highly phagocyte basophilic and eosinophilic mononuclear leukocytes were detected (Chao et al. 2004).
It is probable that the strategy of invading mononuclear phagocytes as a means of immune evasion and dissemination is a distinct feature of all vertebrate rhinoviruses. This branch of antiviral immunity consists of three classes of cytokines, type I, II, and III Ions (Sadler and Williams 2008).
Interestingly, while the distinct receptor systems utilized by IFN-I and -III dictate cell specificity, both cytosine families activate the same downstream Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathways, culminating in similar antiviral outcomes (Sadler and Williams 2008) including the induction of antiviral genes such as protein kinase R (Per) and Arbovirus resistance (MX) molecules. The type II IFN systems of amphibians and reptiles remain largely characterized, whereas those of bony fish appear to be much more complex than that of mammals (You and Becomes 2011), and will not be addressed further here.
These molecules are encoded by five exon/four intron gene transcripts and signal through a receptor system composed of the interferon lambda receptor-1 (IFNR1) and interleukin-10 receptor-2 (IL-10R2; reviewed in reference Potent 2011). This is especially relevant when considering that amphibians are key evolutionary intermediates between fish and mammals and inhabit both aquatic and terrestrial habitats.
As described above, an important antiviral gene product synthesized during the interferon response is the Arbovirus resistance (MX) protein (Samuel 2001). To date the MX of most, but not all, fish species have proven ineffective in preventing infection by various members of the family Bromoviridae.
Similarly, Barracuda MX inhibits replication of the norovirus viral nervous necrosis virus (VN NV) and of Infectious pancreatic necrosis virus (IPTV) but fails to show antiviral effects against Taiwan grouper rhinovirus (TGI) (Wu et al. 2012 ; Wu and Chi 2007). Thus, the inadequacy of antiviral components such as Mx1 in dealing with foreign Canaveral isolates may culminate in a global threat represented by geographically distant rotavirus strains introduced by subclinically infected migratory hosts or imported due to international trade.
Perhaps the most commercially and aqua culturally important fish species in Southern Europe is the gilt head sea bream, at least in part because of its natural resistance to most viral pathogens (Can et al. 2006, 2009). It is noteworthy that in contrast to the mortality caused by many members of the family Bromoviridae, sea bream effectively clear LCD infections, although it is believed that they may harbor the virus non-symptomatically.
Thus, the efficacy of the telecast IFN/MX response may well dictate the susceptibility of individual fish species to highly virulent pathogens such as rhinoviruses. Since these infections involve fish skin (Leibniz 1980), systemic antiviral responses such as MX may be less important to the resolution of LCD.
In another example, Japanese flounder IFN-inducible transmembrane (IFI TM) protein is unregulated in response to Reyna Giulio virus (Rev) infections (AHU et al. 2013). Furthermore, through over expression and SIRPA knockdown studies, flounder IFITM1 was shown to play an important role in the cellular antiviral response to Rev (AHU et al. 2013).
It will be important to delineate the precise repertoire(s) of antiviral Ions present within the axolotl genome and examine the transcriptional regulation, as well as functional roles of these moieties during immune responses against rotaviruses such as ATV. Furthermore, treatment of A6 cells with r XL IFN significantly unregulated the expression of Mx1 indicating that stimulation with this cytosine elicits a cellular antiviral state (Grayer et al. 2014).
With r XL IFN exhibited significant increases of Mx1 gene expression in the spleen and peritoneal leukocytes (Grayer et al. 2014). Moreover, upon FV3 challenge, r XL IFN-treated tadpoles showed decreased viral replication and transcriptional activity (Grayer et al. 2014).
However, adding to the complexity of the interaction between FV3 and tadpoles is the fact that although r XL IFN-treated tadpoles exhibited prolonged mean survival times following FV3 inoculation and viral loads that were several logs lower, these animal nonetheless incurred extensive organ damage and succumbed to infection (Grayer et al. 2014). This is consistent with the notion (as described above) that depending on the species and/or developmental stage, rotaviruses may exhibit virulence factors independent of viral replication.
However, it is becoming evident that clearance of rotaviruses is heavily contingent on successful adaptive immune responses, which have been investigated to date almost exclusively in X. Levis. The amphibian organization and usage of the immunoglobulin (IG) heavy and light chain loci are reminiscent of their mammalian counterparts, including V-(D)-J rearrangements, class-switch recombination, somatic hypermutation, and affinity maturation (Du Pastier et al. 1989, 2000 ; CSU 1998).
As in mammals, the Venous IG class-switch from IGM to Icy (Egg analog) is thymus-dependent and requires T cell-B cell collaboration (Bloomberg et al. 1980 ; Turner and Manning 1974). In addition, administration of immune era to naturally susceptible X. Levis tadpoles immediately preceding FV3 infection confers partial, but significant passive protection against the virus (Manner et al. 2006).
X. Laevis tadpoles express suboptimal levels of MHC class Ia protein (Du Pastier et al. 1989) and yet their melanocytes include bona fide CD8 T cells that express the pan-T Venous cell-surface marker CD5 (Jürgen et al. 1995) and exhibit fully rearranged TCR/ transcripts (Horton et al. 1998). These CD8 T cell-depleted, FV3-infected animals experienced severe edema and hemorrhaging, extensive elevation of viral loads and succumbed to infections, whereas control cohorts effectively cleared the virus (Robert et al. 2005).
Frogs re-infected with FV3 exhibit expedited viral clearance concomitant with earlier proliferation of CD5 + CD8 + melanocytes and faster infiltration (3 vs. 6 dpi) of the kidney, the central site of X. Levis -FV3 replication (Morales and Robert 2007). It cannot be excluded that this modest secondary response is an inherent property of the evolutionarily primordial amphibian adaptive immune system, considering the relatively meager degree of T cell expansion seen following immunological challenges, the absence of draining lymph nodes and the lack of white pulp-red pulp splenic organization (Du Pastier et al. 1989).
Both of these lymphocyte populations undergo unconventional differentiation pathways, exhibit unique semi-invariant T cell receptor rearrangements and are believed to participate in antimicrobial and antiviral immune responses (Bear and Porcelain 2007 ; Choir et al. 2008 ; Cohen et al. 2009 ; Le Boris et al. 2010). Notably, we have recently identified a prominent X. Levis it immune cell subset, which requires XNC10 for both its development and function (Hold et al. 2013).
Using an XNC10 betrayer as well as reverse genetics combining transgenesis and RNA interference, we determined that this it cell population is CD8 /CD4 , expresses a semi-invariant T cell receptor consisting of an invariant TCR (iV6-J1.43) combined with a limited TCR repertoire, and fails to develop in the absence of, or with diminished, XNC10 expression (Hold et al. 2013). Notably, transgenic animals with effectively RNAi-silenced thy mic and splenic XNC10 expression failed to develop this it cell subset.
Moreover, they were also significantly more susceptible to, and more readily succumbed to, FV3 infections (Hold et al. 2013) suggesting that these cells are important in anti-ranaviral defenses. It is noteworthy that deep sequencing analysis of tadpole TCR revealed that Venous larvae possess several additional predominant it cell populations (Hold et al. 2013), which are presumably Unrestricted and most likely participate in immune responses such as those against rotaviruses.
However, except the rotavirus homology of the largest subunit of eukaryotic initiation factor 2 (vIF-2), the functions of these gene products have not been determined. Activated Per subsequently phosphorylates the alpha subunit of eIF-2, an event that results in a global arrest in protein synthesis (Panniers et al. 1988 ; Rowland set al. 1988).
In addition, activated Per appears to be one of the danger signals that trigger apoptosis in virus infected cells. A similar attenuation of virulence was observed following infection of Venous Levis with a FV3 KO mutant lacking a truncated version of vIF-2 (Chen et al. 2011).
Truncated versions of vIF-2 are found in FV3, soft-shell turtle rhinovirus, and Reyna Giulio virus and are missing the N-terminal half of the native molecule. Since this region contains the VxRVDxxKGYxD motif described above, the reduction in virulence cannot be due to an effect of vIF-2 on Per, but to some element present within the C-terminal half of the protein.
Moreover, knock down of RNAS III-like protein expression using antisense morphology oligonucleotides resulted in a 40% reduction in virus yield (K. Cheng and V.G. Chin char, University of Mississippi, unpublished data) suggesting that the RNaseIII-like protein plays a role in virus replication.
In addition to the four viral gene products mentioned above, rotaviruses also contain homology of Tumor Necrosis Factor (TNF) receptor (ANFR) and outpace (Chin char et al. 2009 ; Eaton et al. 2007) and a unique virus-encoded DNA cytosine methyltransferase (Dataset). Similar to their pox virus counterparts, rotavirus ANFR could function as a decoy molecule and block protection mediated by TNF.
Although outpace is generally considered to be a protein that plays a role in viral DNA synthesis (e.g., by increasing DTP pools or blocking the incorporation of duty into DNA), a herpesvirus outpace was shown to also block antiviral immunity (Glaser et al. 2006 ; Oliver et al. 1999). Lastly, the rotavirus Dataset may play a role in immune evasion by methylating cytosine residues within CPG motifs and blocking recognition by TLR-9 or cytoplasmic DNA sensors and preventing the subsequent induction of IFN and pro-inflammatory cytokines (Grieg 2002 ; Drug et al. 2004).
While this serves as a useful starting point in the identification and characterization of Canaveral immune evasion genes, there are approximately a dozen additional Offs of unknown function, which are unique to rotaviruses. Knock-down experiments using antisense morphology oligonucleotides or SIRPA and infections using knock-out mutants will be needed to resolve the function of these unique ranavirus-specific proteins.
Clearly, rotaviruses encode many putative gene products, which represent both potential virulence factors and promising targets for future therapeutic interventions. While it is easy to dismiss lower vertebrate immune systems as functionally analogous to those of mammals, there is a growing literature suggesting otherwise.
It stands to reason that rotavirus pathogens have co-evolved with these unique immune systems, thus we must garner greater insights into both to fully understand either. Open Access publication was made possible through grants provided by the University of Tennessee (Institute of Agriculture, Office of Research and Engagement, and Department of Forestry, Wildlife and Fisheries), Washington State University Libraries, Gordon State College (Office of Academic Affairs), the Association of Reptilian and Amphibian Veterinarians, and the Amphibian and Reptile Conservancy.
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