The innate antiviral effects of extracellular viral dsRNA in rainbow trout cells

Viral double-stranded RNA (dsRNA) molecules are a potent pathogen-associated molecular pattern and play a crucial role in the innate immune response. During a viral infection, extracellular and intracellular dsRNA can initiate pathways resulting in the production of type I interferons (IFNs) and interferon-stimulated genes (ISGs). The accumulation of ISGs within a cell results in a protective antiviral state. This study used both commercially available dsRNA (poly I:C) and in vitro transcribed dsRNA molecules, based on the viral hemorrhagic septicemia virus (VHSV) genome sequence, as stimuli to investigate the effects of these molecules on the innate immune response in rainbow trout cells. The goals of the present project were to elucidate the i) IFN ii) ISG and iii) antiviral responses of fish cells to both types of dsRNA molecules. Different lengths of poly I:C and in vitro transcribed dsRNA were used to determine potential length effects of dsRNA in fish cells. The aims of the project were achieved using a functional interferon assay, an ISG-promoter reporter system, an antiviral assay, and RT-PCR. It was found that extracellular dsRNA, either poly I:C or in vitro transcribed dsRNA, is able to induce innate antiviral responses in the fish cell line, RTG-2. Consistent with mammalian studies there was a greater magnitude of immune response when cells were stimulated with longer dsRNA molecules, demonstrating dsRNA length effects in fish cells

DsRNA-mediated antiviral immunity in fish cells: visualization, sensors, and innate immune responses

The global aquaculture industry is a multibillion dollar business that is threatened by pathogens, including a wide array of aquatic viruses. Currently there are no antiviral treatments available to combat viral outbreaks, and as such viral infection can cause vast economic loss. Two important species for aquaculture include rainbow trout (Oncorhynchus mykiss), destined for human consumption, and fathead minnow (Pimephales promelas), a common species grown for bait purposes. Double-stranded (ds)RNA is a potent immunomodulating molecule produced during viral replication; dsRNA treatment induces a robust antiviral state that makes host cells refractive to viral replication. This thesis explored dsRNA-induced innate antiviral pathways from two angles. Firstly, the differences in dsRNA-induced responses between virally-produced dsRNA, synthesized dsRNA with natural sequence variation, and synthesized dsRNA with a homogenous sequence were analyzed in rainbow trout cells. The dsRNA, regardless of source, was sensed at the cell surface by a common receptor in rainbow trout cells and induced an innate immune response and antiviral state against two aquatic viruses, viral hemorrhagic septicemia virus and infectious pancreatic necrosis virus; the virally-produced and lab-synthesized dsRNA molecules with natural sequence variation produced the most similar responses. The second angle of approach was to better understand the host’s response to exogenous dsRNA treatment. To this end, novel dsRNA sensors were identified and characterized in rainbow trout. This included class A scavenger receptors, the purported surface receptors for dsRNA, including MARCO, SCARA3, SCARA4, and SCARA5, as well as two novel cytoplasmic dsRNA sensors from the DExH/D-box family, DDX3 and DHX9. The receptors found in rainbow trout contained the same conserved domains that are found in their mammalian counterparts, a first indication of conserved functionality. Two MARCO variants were identified and found to bind to two gram-negative and one gram–positive bacteria, but surprisingly not to dsRNA. Rainbow trout DDX3 and DHX9 are both functional in their ability to bind to dsRNA. The culmination of these findings was the development of a dsRNA molecule with sequence variation that can act as a potent antiviral therapy in vitro in fathead minnow cells. The findings from this thesis demonstrate the importance of ‘natural’ dsRNA as an innate immune signalling molecule and its potential to function as a prophylactic antiviral therapeutic for fish

Extracellular dsRNA induces a type I interferon response mediated via class A scavenger receptors in a novel Chinook salmon derived spleen cell line

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.dci.2018.08.010 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Despite increased global interest in Chinook salmon aquaculture, little is known of their viral immune defenses. This study describes the establishment and characterization of a continuous cell line derived from Chinook salmon spleen, CHSS, and its use in innate immune studies. Optimal growth was seen at 14–18 °C when grown in Leibovitz's L-15 media with 20% fetal bovine serum. DNA analyses confirmed that CHSS was Chinook salmon and genetically different from the only other available Chinook salmon cell line, CHSE-214. Unlike CHSE-214, CHSS could bind extracellular dsRNA, resulting in the rapid and robust expression of antiviral genes. Receptor/ligand blocking assays confirmed that class A scavenger receptors (SR-A) facilitated dsRNA binding and subsequent gene expression. Although both cell lines expressed three SR-A genes: SCARA3, SCARA4, and SCARA5, only CHSS appeared to have functional cell-surface SR-As for dsRNA. Collectively, CHSS is an excellent cell model to study dsRNA-mediated innate immunity in Chinook salmon.Natural Sciences and Engineering Research Council of CanadaCanada Research Counci

Common Variation in Vitamin D Pathway Genes Predicts Circulating 25-Hydroxyvitamin D Levels among African Americans

Vitamin D is implicated in a wide range of health outcomes, and although environmental predictors of vitamin D levels are known, the genetic drivers of vitamin D status remain to be clarified. African Americans are a group at particularly high risk for vitamin D insufficiency but to date have been virtually absent from studies of genetic predictors of circulating vitamin D levels. Within the Southern Community Cohort Study, we investigated the association between 94 single nucleotide polymorphisms (SNPs) in five vitamin D pathway genes (GC, VDR, CYP2R1, CYP24A1, CYP27B1) and serum 25-hydroxyvitamin D (25(OH)D) levels among 379 African American and 379 Caucasian participants. We found statistically significant associations with three SNPs (rs2298849 and rs2282679 in the GC gene, and rs10877012 in the CYP27B1 gene), although only for African Americans. A genotype score, representing the number of risk alleles across the three SNPs, alone accounted for 4.6% of the variation in serum vitamin D among African Americans. A genotype score of 5 (vs. 1) was also associated with a 7.1 ng/mL reduction in serum 25(OH)D levels and a six-fold risk of vitamin D insufficiency (<20 ng/mL) (odds ratio 6.0, p = 0.01) among African Americans. With African ancestry determined from a panel of 276 ancestry informative SNPs, we found that high risk genotypes did not cluster among those with higher African ancestry. This study is one of the first to investigate common genetic variation in relation to vitamin D levels in African Americans, and the first to evaluate how vitamin D-associated genotypes vary in relation to African ancestry. These results suggest that further evaluation of genetic contributors to vitamin D status among African Americans may help provide insights regarding racial health disparities or enable the identification of subgroups especially in need of vitamin D-related interventions

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

Examining rainbow trout vig-3 expression patterns in vitro following treatment with type I IFN, poly IC or viral infection

Interferon-stimulated genes (ISGs) are key mediators of antiviral immunity. Investigating ISGs can help create novel therapeutics to increase survival rates of farmed fish and aid in protection against pathogens. The current study investigated the induction of one such ISG, viral hemorrhagic septicemia (VHSV) – induced gene 3 (vig-3), also known as ISG15 in mammals and other fish species. Vig-3 is one of many rainbow trout ISGs whose expression patterns, induction, and role remain relatively unknown. In this study, vig-3 transcript expression was identified in a panel of tissues from healthy rainbow trout. Vig-3 transcripts were upregulated in response to recombinant type I interferon (IFN-I) treatment in rainbow trout gonadal cells (RTG-2). Vig-3 transcript and protein levels were upregulated overtime following treatment with a double-stranded (ds)RNA molecule, polyinosinic: polycytidylic acid (poly IC), and infection with two viruses, infectious pancreatic necrosis virus (IPNV) and viral hemorrhagic septicemia virus (VHSV) in two rainbow trout cell lines, RTG-2 and RTgill-W1. Vig-3 transcript expression kinetics positively corelated with viral replication kinetics. Western blot analysis demonstrated evidence of protein ISGylation, a unique feature of vig-3. Vig-3′s cellular localization was assessed using immunocytochemistry following viral infection. Vig-3 expression was cytoplasmic and increased in quantity over time, upon treatment with poly IC, or infection with IPNV or VHSV. These findings improve our understanding of the rainbow trout innate immune system and could aid in the production of fish antiviral therapeutics in the future