Immunostimulant effects of Pituitary Adenylate Cyclase-Activating Polypeptide and double-stranded (ds)RNA in Orconectes propinquus.

Disease outbreaks in crustacean aquaculture caused by opportunistic and obligate pathogens cause severe economic losses to the industry. Antibiotics are frequently used as prophylactic treatments worldwide, although its overuse and misuse has led to microbial resistance, which has driven the search for novel molecules with immunostimulant and antibacterial activities. Antimicrobial peptides (AMP) and double-stranded (ds)RNAs constitute promising immunostimulants in the fight against infectious diseases in aquaculture. Scientists have made significant progress in testing these molecules in aquatic organisms as potential candidates for replacing conventional antibiotics. However, most studies have been conducted in teleost fish, thus little is known about the immunostimulatory effects in crustaceans, especially in freshwater crayfishes. Consequently, in the present work, we evaluate the immunomodulatory effects of the AMP Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and high molecular weight (HMW) Poly (I:C) in the northern clearwater crayfish Orconectes propinquus. Two bioassays were conducted to evaluate the effects of different doses of PACAP and Poly (I:C) HMW, different administration routes, as well as the effects of the combined treatment on the crayfish immune system. Results showed the immunostimulatory role of PACAP and Poly (I:C) HMW with effects depending on the dose, the site of injection and the treatment assessed. These findings offer new insights into the crayfish immune system and contribute to the development of effective broad-spectrum immune therapies in aquaculture.

Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs.

The common field lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is used to treat streams and creeks infested with highly invasive and destructive sea lamprey (Petromyzon marinus) in the tributaries of the Great Lakes. Unfortunately, amphibian deaths have been reported following stream treatments with TFM. Larval amphibians (tadpoles) are more susceptible to TFM toxicity than adult amphibians. The aim of this study was to test the toxicity of TFM in eight new tadpole cell lines from the green frog (Lithobates clamitans), wood frog (Lithobates sylvaticus), and American toad (Anaxyrus americanus). A cell viability bioassay using two fluorescent dyes, Alamar Blue and CFDA-AM, was performed following 24-h and 72-h exposures to a range of TFM concentrations. In general, TFM exposure reduced Alamar Blue fluorescence more rapidly than CFDA-AM fluorescence in tadpole cells, suggesting that Alamar Blue is perhaps a better diagnostic indicator of cell health for acute TFM cytotoxicity. At present, the in vivo 96-h LC50s of TFM are only available for L. clamitans and they correlated well with the in vitro EC50 values for the green frog tadpole cell lines in this study. The eight tadpole cell lines with different relative sensitivities to TFM cytotoxicity could prove to be useful tools in assessing next-generation lampricides in high-throughput bioassays to ensure safety in frogs before their sea lamprey-targeted application in the field.

Hypersensitive response to interferon-stimulated gene (ISG)-inducing double-stranded RNA in American bullfrog tadpole fibroblasts.

American bullfrogs are thought to be carriers of ranaviruses and contribute to their global spread via trade. Bullfrog tadpoles succumb to ranaviral infection's more severe and deadly effects than bullfrog adults. Presently, little is known about bullfrog tadpoles' innate antiviral immunity, possible due to the lack of available bullfrog tadpole cell lines. In this study, we describe a novel bullfrog tadpole fibroblast cell line named BullTad-leg. Its general cellular attributes, gene expression and function of class-A scavenger receptors (SR-As), and responses to poly IC (a synthetic dsRNA mimicking viral dsRNAs and a potent inducer of the interferon (IFN)-mediated antiviral responses) are investigated. Its abundant expression of vimentin corroborated with the cells' fibroblast morphology. BullTad-leg cells expressed transcripts of four SR-A members: SR-AI, SCARA3, SCARA4, and SCARA5, but transcripts of MARCO, the fifth SR-A member, were not detected. BullTad-leg cells expressed functional SR-As and could bind AcLDL. BullTad-leg cells exhibited cytotoxicity in response to poly IC treatment via SR-As. Additionally, very low doses of poly IC were able to induce dose-dependent expressions of ISGs including Mx, PKR, ISG20, and IFI35. This research sheds new light on the innate immune response, particularly SR-A biology and dsRNA responsiveness, in bullfrog tadpoles.

Using long, sequence-specific dsRNA to knockdown inducible protein expression and virus production via an RNAi-like mechanism.

RNA interference (RNAi) is a powerful innate immune mechanism to knock down translation of specific proteins whose machinery is conserved from plants to mammals. The template used to determine which mRNA's translation is inhibited is dsRNA, whose origin can range from viruses (long dsRNA, ∼100-1000s bp) to host (micro(mi)RNA, ∼20mers). While miRNA-mediated RNAi is well described in vertebrates, the ability of long dsRNA to guide RNAi-mediated translation inhibition in vertebrates is controversial. Indeed, as long dsRNA is so effective at inducing type I interferons (IFNs), and IFNs down-regulate RNAi machinery, it is believed that IFN-competent cells are not capable of using long dsRNA for RNAi. In the present study the ability of long, sequence specific dsRNA to knock down both host protein expression and viral replication is investigated in IFN-competent rainbow trout cells. Before exploring RNAi effects, the optimal dsRNA concentration that would funnel into RNAi without triggering the IFN response was determined. After which, the ability of sequence specific long dsRNA to target knockdown via RNAi was evaluated in: (1) uninfected host cells using inducible luciferase gene expression and (2) host cells infected with chum salmon reovirus (CSV), frog virus 3 (FV3) or viral hemorrhagic septicemia virus genotype IVa (VHSV-IVa). Induced expression studies utilized RTG-P1, a luciferase reporter cell line, and dsRNA containing luciferase sequence (dsRNA-Luc) or a mis-matched sequence (dsRNA-GFP), and subsequent luminescence intensity was measured. Anti-CSV studies used dsRNA-CSVseg7 and dsRNA-CSVseg10 to target CSV segment 7 and CSV segment 10 respectively. Inhibition of virus replication was measured by viral titration and RT-qPCR. Taking advantage of the fact that long dsRNA can accommodate more sequences than miRNAs, the antiviral capability of dsRNA molecules containing both CSV segment 7 and segment 10 simultaneously was also measured. Target sequence appears important, as dsRNA-FV3MCP did not knock down FV3 titres, and while dsRNA-VHSV-N knocked down VHSV-IVa, dsRNA-VHSV-G and dsRNA-VHSV-M did not. This is the first study in fish to provide evidence that sequence specific long dsRNA induces potent gene expression silencing and antiviral responses in vitro via an RNAi-like mechanism instead of an IFN-dependent response.

More tools for our toolkit: The application of HEL-299 cells and dsRNA-nanoparticles to study human coronaviruses in vitro.

Human coronaviruses (HCoVs) are important human pathogens, as exemplified by the current SARS-CoV-2 pandemic. While the ability of type I interferons (IFNs) to limit coronavirus replication has been established, the ability of double-stranded (ds)RNA, a potent IFN inducer, to inhibit coronavirus replication when conjugated to a nanoparticle is largely unexplored. Additionally, the number of IFN competent cell lines that can be used to study coronaviruses in vitro are limited. In the present study, we show that poly inosinic: poly cytidylic acid (pIC), when conjugated to a phytoglycogen nanoparticle (pIC+NDX) is able to protect IFN-competent human lung fibroblasts (HEL-299 cells) from infection with different HCoV species. HEL-299 was found to be permissive to HCoV-229E, -OC43 and MERS-CoV-GFP but not to HCoV-NL63 or SARS-CoV-2. Further investigation revealed that HEL-299 does not contain the required ACE2 receptor to enable propagation of both HCoV-NL63 and SARS-CoV-2. Following 24h exposure, pIC+NDX was observed to stimulate a significant, prolonged increase in antiviral gene expression (IFNß, CXCL10 and ISG15) when compared to both NDX alone and pIC alone. This antiviral response translated into complete protection against virus production, for 4 days or 7 days post treatment with HCoV-229E or -OC43 when either pre-treated for 6h or 24h respectively. Moreover, the pIC+NDX combination also provided complete protection for 2d post infection when HEL-299 cells were infected with MERS-CoV-GFP following a 24h pretreatment with pIC+NDX. The significance of this study is two-fold. Firstly, it was revealed that HEL-299 cells can effectively be used as an IFN-competent model system for in vitro analysis of MERS-CoV. Secondly, pIC+NDX acts as a powerful inducer of type I IFNs in HEL-299, to levels that provide complete protection against coronavirus replication. This suggests an exciting and novel area of investigation for antiviral therapies that utilize innate immune stimulants. The results of this study will help to expand the range of available tools scientists have to investigate, and thus further understand, human coronaviruses.

TAK-242 treatment and its effect on mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice.

PURPOSE: Intervertebral disc (IVD) degeneration is accompanied by mechanical and gene expression changes to IVDs. SPARC-null mice display accelerated IVD degeneration, and treatment with (toll-like receptor 4 (TLR4) inhibitor) TAK-242 decreases proinflammatory cytokines and pain. This study examined if chronic TAK-242 treatment impacts mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice. METHODS: Male and female SPARC-null and WT mice aged 7-9 months were given intraperitoneal injections with TAK-242 or an equivalent saline vehicle for 8 weeks (3x/per week, M-W-F). L2-L5 spinal segments were tested in cyclic axial tension and compression. Gene expression analysis (RT-qPCR) was performed on male IVD tissues using Qiagen RT2 PCR arrays. RESULTS: SPARC-null mice had decreased NZ length (p = 0.001) and increased NZ stiffness (p < 0.001) compared to WT mice. NZ length was not impacted by TAK-242 treatment (p = 0.967) despite increased hysteresis energy (p = 0.024). Tensile stiffness was greater in SPARC-null mice (p = 0.018), and compressive (p < 0.001) stiffness was reduced from TAK-242 treatment in WT but not SPARC-null mice (p = 0.391). Gene expression analysis found upregulation of 13 ECM and 5 inflammatory genes in SPARC-null mice, and downregulation of 2 inflammatory genes after TAK-242 treatment. CONCLUSIONS: TAK-242 had limited impacts on SPARC-null mechanical properties and did not attenuate NZ mechanical changes associated with IVD degeneration. Expression analysis revealed an increase in ECM and inflammatory gene expression in SPARCnull mice with a reduction in inflammatory expression due to TAK-242 treatment. This study provides insight into the role of TLR4 in SPARC-null mediated IVD degeneration.

Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells.

In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.

An in vitro 3D annulus fibrosus cell culture model with type I collagen: An examination of cell-matrix interactions.

Background: Disorders of the intervertebral disc (IVD) are widely known to result in low back pain; one of the most common debilitating conditions worldwide. As a multifaceted condition, both inflammatory environment and mechanical factors can play a crucial role in IVD damage, and in particular, in the annulus fibrosus (AF), the highly collagenous outer ring of the IVD. As a result, a better understanding of how cells from the IVD, and specifically the AF, interact and respond to their environment is imperative. Goal: The goal of this study is to use collagen type I as an in vitro three-dimensional extracellular matrix for AF cells of IVD and briefly examine both the cellular and mechanical effect of exposure to an inflammatory stimulant. Methods: We utilized type I collagen as a 3D in vitro model material for culturing AF cells of Sprague Dawley rat tail IVDs. Results: We showed that the cultured cells are viable and metabolically active; these cells also induced a distinct and significant contraction on their collagen matrix. Furthermore, to demonstrate potential versatility of our model our model and its versatility, we used lipopolysaccharide (LPS), as a known inflammatory stimulant in IVDs, to manipulate the cells and their interaction. LPS treatment resulted in detectable changes to the contraction cells induced on the collagen matrix and affected the mechanical properties of these constructs.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout.

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Unloaded Organ Culturing Has a Detrimental Effect on the Axial Mechanical Properties of the Intervertebral Disc.

Healthy function of intervertebral discs (IVDs) depends on their tissue mechanical properties. Native cells embedded within IVD tissues are responsible for building, maintaining, and repairing IVD structures in response to genetic, biochemical, and mechanical signals. Organ culturing provides a method for investigating how cells respond to these stimuli in their natural architectural environment. The purpose of this study was to determine how organ culturing affects the mechanical characteristics of functional spine units (FSUs) across the entire range of axial loading, including the neutral zone (NZ), using a rat tail model. Rat tail FSUs were organ cultured at 37 °C in an unloaded state in standard culture media for either 1-day (n = 8) or 6-days (n = 12). Noncultured FSUs (n = 12) were included as fresh control specimens. Axial mechanical properties were tested by applying cyclical compression and tension. A novel mathematical approach was developed to fully characterize the relationship between load, stiffness, and deformation through the entire range of loading. Culturing FSUs for 1-day did not affect any of the axial mechanical outcome measures compared to noncultured IVDs; however, culturing for 6 days increased the size of NZ by 112% and decreased the stiffness in NZ, compressive, and tensile regions by 53%, 19%, and 15%, respectively, compared to noncultured FSUs. These results highlight the importance of considering how the mechanical integrity of IVD tissues may affect the transmission of mechanical signals to cells in unloaded organ culturing experiments.

Phytoglycogen Nanoparticle Delivery System for Inorganic Selenium Reduces Cytotoxicity without Impairing Selenium Bioavailability.

PURPOSE: Selenium is an essential trace element that supports animal health through the antioxidant defense system by protecting cells from oxidative-related damage. Using inorganic selenium species, such as sodium selenite (Na Sel), as a food supplement is cost-effective; however, its limitation as a nutritional supplement is its cytotoxicity. One strategy to mitigate this problem is by delivering inorganic selenium using a nanoparticle delivery system (SeNP). METHODS: Rainbow trout intestinal epithelial cells, bovine turbinate cells and bovine intestinal myofibroblasts were treated with soluble Na Sel or SeNPs. Two SeNP formulations were tested; SeNP-Ionic where inorganic selenium was ionically bound to cationic phytoglycogen (PhG) NPs, and SeNP-Covalent, where inorganic selenium was covalently bound to PhG NPs. Selenium-induced cytotoxicity along with selenium bioavailability were measured. RESULTS: SeNPs (SeNP-Ionic or SeNP-Covalent) substantially reduced cytotoxicity in all cell types examined compared to similar doses of soluble inorganic selenium. The SeNP formulations did not affect selenium bioavailability, as selenium-induced glutathione peroxidase (GPx) activity and GPx1 transcript levels were similarly elevated whether cells were treated with soluble Na Sel or SeNPs. This was the case for all three cell types tested. CONCLUSION: Nanoparticle-assisted inorganic selenium delivery, which demonstrated equal bioavailability without causing deleterious cytotoxic side effects, has potential applications for safely supplementing animal diets with inorganic selenium at what are usually toxic doses.

Immuno-stimulatory capacity of decorin in the rat tail intervertebral disc and the mechanical consequence of resultant inflammation.

PURPOSE: Determine whether decorin is immuno-stimulatory to rat tail IVD cells and to characterize the mechanical consequence of inflammation at the whole rat tail IVD level. METHODS: Cultured rat tail annulus fibrosus (AF) cells were exposed to decorin, a resident IVD small leucine-rich proteoglycan (SLRP), with and without the presence of a toll-like receptor (TLR) 4 inhibitor, TAK-242. Resultant expression of pro-inflammatory cytokine and chemokines (MCP-1; MIP-2; RANTES; IL-6; TNFα) were quantified over 24 h. Whole rat tail IVD cultures (n = 50) were also treated with decorin (two concentrations: 0.5 and 5.0 µg/mL) with and without TAK-242 (via nucleus pulpous injection with a 33-gauge needle), and resultant mechanical properties were measured. RESULTS: AF cells exposed to decorin showed significant increases in pro-inflammatory cytokine and chemokine production; this was significantly blunted with the presence of TAK-242. Whole IVDs injected with decorin showed a dose-dependent decrease in neutral zone and tensile stiffness and an increase in neutral zone size. When TAK-242 was injected into the IVD with the decorin, mechanical stiffness was preserved and not different from sham controls (injected with PBS). CONCLUSION: AF cells are capable of detecting decorin and inducing inflammation. Decorin further resulted in a functional deterioration in IVD mechanical integrity. TAK- 242, a TLR4 inhibitor, blunted chemokine production at the cellular level and preserved mechanical stiffness in the whole IVD.

Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles.

Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

Two DExD/H-box helicases, DDX3 and DHX9, identified in rainbow trout are able to bind dsRNA.

In mammals, the multifunctional DExH/D-box helicases, DDX3 and DHX9, are nucleic acid sensors with a role in antiviral immunity; their role in innate immunity in fish is not yet understood. In the present study, full-length DDX3 and DHX9 coding sequences were identified in rainbow trout (Oncorhynchus mykiss). Bioinformatic analysis demonstrated both deduced proteins were similar to those of other species, with ~80% identity to other fish species and ~70-75% identity to mammals, and both protein sequences had conserved domains found amongst all species. Phylogenetic analysis revealed clustering of DDX3 and DHX9 with corresponding proteins from other fish. Cellular localization of overexpressed DDX3 and DHX9 was performed using GFP-tagged proteins, and endogenous DDX3 localization was measured using immunocytochemistry. In the rainbow trout gonadal cell line, RTG-2, DHX9 localized mostly to the nucleus, while DDX3 was found mainly in the cytoplasm. Tissue distribution from healthy juvenile rainbow trout revealed ubiquitous constitutive expression, highest levels of DDX3 expression were seen in the liver and DHX9 levels were fairly consistent among all tissues tested. Stimulation of RTG-2 cells revealed that DDX3 and DHX9 transcripts were both significantly upregulated by treatment with the dsRNA molecule, poly I:C. A pull-down assay suggested both proteins were able to bind dsRNA. In addition to their roles in RNA metabolism, the conserved common domains found between the rainbow trout proteins and other species having defined antiviral roles, combined with the ability for the proteins to bind to dsRNA, suggest these proteins may play an important role in fish innate antiviral immunity. Future studies on both DDX3 and DHX9 function will contribute to a better understanding of teleost immunity.

Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica).

A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.

Class A Scavenger Receptors Are Used by Frog Virus 3 During Its Cellular Entry.

Frog virus 3 (FV3) is the type species of the genus Ranavirus (family Iridoviridae). FV3 and FV3-like viruses are globally distributed infectious agents with the capacity to replicate in three vertebrate classes (teleosts, amphibians, and reptiles). At the cellular level, FV3 and FV3-like viruses can infect cells from virtually all vertebrate classes. To date, the cellular receptors that are involved in the FV3 entry process are unknown. Class A scavenger receptors (SR-As) are a family of evolutionarily conserved cell-surface receptors that bind a wide range of chemically distinct polyanionic ligands and can function as cellular receptors for other DNA viruses, including vaccinia virus and herpes simplex virus. The present study aimed to determine whether SR-As are involved in FV3 cellular entry. By using well-defined SR-A competitive and non-competitive ligand-blocking assays and absolute qPCR, we demonstrated that the SR-A competitive ligands drastically reduced the quantities of cell-associated viral loads in frog cells. Moreover, inducing the expression of a human SR-AI in an SR-A null cell line significantly increased FV3⁻cell association. Together, our results indicate that SR-As are utilized by FV3 during the cellular entry process.

Class A scavenger receptors mediate extracellular dsRNA sensing, leading to downstream antiviral gene expression in a novel American toad cell line, BufoTad.

Viral double-stranded (ds)RNA is a potent pathogen-associated molecular pattern (PAMP), capable of inducing a strong antiviral state within the cell, protecting the cell from virus infection. In mammals and fish, sensing extracellular dsRNA is mediated by cell-surface class A scavenger receptors (SR-As). Currently, very little is known about SR-As in amphibians, including: sequence, expression patterns and function. To this end, SR-A expression and function was studied in a novel American toad (Anaxyrus americanus) tadpole cell line called BufoTad. BufoTad was derived from a whole tadpole. The cell line exhibits a cobblestone morphology and expresses abundant levels of transcripts for cytokeratin 19, vimentin, claudin 3, chemokine receptor CXCR4, and SR-AI, one of the five members of the SR-A family, collectively suggesting that BufoTad could be endothelial-like. BufoTad cells bound acetylated LDL, whereas the Xenopus laevis kidney epithelial A6 cell line did not, suggesting functional SR-A activity in BufoTad cells. Additionally, three SR-A competitive ligands (DxSO4, fucoidan, poly inosine (pI)) completely blocked AcLDL binding in BufoTad cells, whereas their three corresponding non-competitive ligands (ChSO4, fetuin, poly cytosine (pC)) did not. A commercial dsRNA, poly IC, induced robust expression of an Mx-like gene transcript, a possible antiviral protein in BufoTad cells. Employing the same SR-A ligand blocking assay used for AcLDL blocked dsRNA-induced ISG expression. This study is the first demonstration that amphibian SR-As have functional ligand binding activities in a live biological cellular model and that sensing extracellular dsRNA in amphibian cells leads to antiviral gene expression that is mediated by class A scavenger receptors.

In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line.

The farming of baitfish, fish used by anglers to catch predatory species, is of economic and ecological importance in North America. Baitfish, including the fathead minnow (Pimephales promelas), are susceptible to infection from aquatic viruses, such as viral hemorrhagic septicemia virus (VHSV). VHSV infections can cause mass mortality events and have the potential to be spread to novel water bodies through baitfish as a vector. In this study, a novel skin cell line derived from fathead minnow (FHMskin) is described and its use as a tool to study innate antiviral immune responses and possible therapies is introduced. FHMskin grows optimally in 10% fetal bovine serum and at warmer temperatures, 25-30 °C. FHMskin is susceptible and permissive to VHSV-IVb infection, producing high viral titres of 7.35 × 107 TCID50/mL after only 2 days. FHMskin cells do not experience significant dsRNA-induced death after treatment with 50-500 ng/mL of in vitro transcribed dsRNA for 48 h and respond to dsRNA treatment by expressing high levels of three innate immune genes, viperin, ISG15, and Mx1. Pretreatment with dsRNA for 24 h significantly protected cells from VHSV-induced cell death, 500 ng/mL of dsRNA reduced cell death from 70% to less than 15% at a multiplicity of infection of 0.1. Thus, the novel cell line, FHMskin, represents a new method for producing high tires of VHSV-IVb in culture, and for studying dsRNA-induced innate antiviral responses, with future applications in dsRNA-based antiviral therapeutics.

Assessing off-target cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol using novel lake sturgeon cell lines.

Lampricides are currently being applied to streams and rivers to control the population of sea lamprey, an invasive species, in the Great Lakes. The most commonly used lampricide agent used in the field is 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey in lamprey-infested rivers and streams. The specificity of TFM is due to the relative inability of sea lamprey to detoxify the agent relative to non-target fishes. There is increasing concern, however, about non-target effects on fishes, particularly threatened populations of juvenile lake sturgeon (LS; Acipenser fulvescens). There is therefore a need to develop models to better define lake sturgeon's response to TFM. Here we report the establishment of five LS cell lines derived from the liver, gill, skin and intestinal tract of juvenile LS and some of their cellular characteristics. All LS cell lines grew well at 25 °C in Leibovitz's (L)- 15 medium supplemented with 10% FBS. All cell lines demonstrated high senescence-associated ß-galactosidase activity and varying levels of Periodic acid Schiff-positive polysaccharides, indicating substantial production of glycoproteins and mucosubstances by the cells. Comparative toxicity of TFM in the five LS cell lines was assessed by two fluorescent cell viability dyes, Alamar Blue and CFDA-AM, in conditions with and without serum and at 24 or 72 h exposure. Deduced EC50 values were compared between the cell lines and to the reported in vivo LC50s. Tissues sensitive to the effects of TFM in vivo correlated with cell lines from the same tissues being most sensitive to TFM in vitro. EC50 values for the LSliver-e cells was significantly lower than the EC50 for the rainbow trout (RBT) liver cells RTL-W1, reaffirming the in vivo observation that LS was generally more TFM-sensitive than rainbow trout. Our data suggests that whole-fish sensitivity of LS to TFM is likely attributable to sensitivity at the cellular level. Thus, LS cell lines, as well as those of RBT, can be used to screen and evaluate the toxicity of the next generation of lampricides on non-target fish such as lake sturgeon.

Understanding Viral dsRNA-Mediated Innate Immune Responses at the Cellular Level Using a Rainbow Trout Model.

Viruses across genome types produce long dsRNA molecules during replication [viral (v-) dsRNA]. dsRNA is a potent signaling molecule and inducer of type I interferon (IFN), leading to the production of interferon-stimulated genes (ISGs), and a protective antiviral state within the cell. Research on dsRNA-induced immune responses has relied heavily on a commercially available, and biologically irrelevant dsRNA, polyinosinic:polycytidylic acid (poly I:C). Alternatively, dsRNA can be produced by in vitro transcription (ivt-) dsRNA, with a defined sequence and length. We hypothesized that ivt-dsRNA, containing legitimate viral sequence and length, would be a more appropriate proxy for v-dsRNA, compared with poly I:C. This is the first study to investigate the effects of v-dsRNA on the innate antiviral response and to compare v-dsRNA to ivt-dsRNA-induced responses in fish cells, specifically rainbow trout. Previously, class A scavenger receptors (SR-As) were found to be surface receptors for poly I:C in rainbow trout cells. In this study, ivt-dsRNA binding was blocked by poly I:C and v-dsRNA, as well as SR-A competitive ligands, suggesting all three dsRNA molecules are recognized by SR-As. Downstream innate antiviral effects were determined by measuring IFN and ISG transcript levels using qRT-PCR and antiviral assays. Similar to what has been shown previously with ivt-dsRNA, v-dsRNA was able to induce IFN and ISG transcript production between 3 and 24 h, and its effects were length dependent (i.e., longer v-dsRNA produced a stronger response). Interestingly, when v-dsRNA and ivt-dsRNA were length and sequence matched both molecules induced statistically similar IFN and ISG transcript levels, which resulted in similar antiviral states against two aquatic viruses. To pursue sequence effects further, three ivt-dsRNA molecules of the same length but different sequences (including host and viral sequences) were tested for their ability to induce IFN/ISG transcripts and an antiviral state. All three induced responses similarly. This study is the first of its kind to look at the effects v-dsRNA in fish cells as well as to compare ivt-dsRNA to v-dsRNA, and suggests that ivt-dsRNA may be a good surrogate for v-dsRNA in the study of dsRNA-induced responses and potential future antiviral therapies.