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Research Project: Reducing Impacts of Disease on Rainbow Trout Aquaculture Production

Location: Office of The Director

2021 Annual Report

Rainbow trout are an important recreational and food fish species in the U.S., and it is thus important to improve disease resistance and improve methods for combatting outbreaks of disease to increase production and profitability of U.S. aquaculture. This research plan will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Identify virulence factors in pathogenic flavobacterium species and develop strategies to control disease. • Sub-Objective 1.A: Isolate and characterize F. columnare mutants and identify virulence factors associated with disease in rainbow trout. • Sub-Objective 1.B: Develop vaccines to control columnaris disease. • Sub-Objective 1.C: Develop improved genetic manipulation techniques for F. psychrophilum, which causes bacterial cold-water disease. Objective 2: Characterize salmonid antimicrobial peptides and evaluate their biocidal effects against pathogens. • Sub-Objective 2.A: Identify new antimicrobial peptides by mining the rainbow trout big data sets and characterize their actions and regulation. • Sub-Objective 2.B: Characterize the immunomodulatory actions of new trout AMPs in vitro. • Sub-Objective 2.C: Assess effects of new rainbow trout AMPs on flavobacterial biofilms. Objective 3: Identify rhabdoviral virulence factors and develop strategies to reduce pathogenesis in salmonids. • Sub-Objective 3.A: Identify and characterize potential viral targets and strategies for vaccine development. • Sub-Objective 3.B: Characterize the involvement of stress granule formation and function in pathogen response and the establishment of protective immunity in fish cell-lines.

Objective 1: For this objective, our scientific aim is to develop genetic techniques to characterize F. psychrophilum and F. columnare virulence mechanisms. Using these genetic techniques, genes encoding specific secreted proteins and components of flavobacterial secretion systems will be mutated and the effects of these modifications on bacterial pathogenesis will be evaluated using in vitro and in vivo systems. Data from these approaches will improve understanding of host-pathogen interactions and generate attenuated bacterial strains for vaccine development. Objective 2: For this objective, our scientific aim is to identify and characterize new antimicrobial peptides (AMPs) in rainbow trout, understand their actions against flavobacterial and rhabdoviral pathogens, and ascertain their physiological control to improve health of rainbow trout. Using established in vitro systems with trout cell-lines, biological actions of AMPs will be assessed and ability of AMPs to kill important flavobacterial and rhabdoviral pathogens, and flavobacterial biofilms, will be characterized. Objective 3: For this objective, our scientific aim is to understand the innate immune response and components of virulence in two important rainbow trout rhabdoviruses (IHNV and VHSV). Work will involve sequential characterization of the effects of critical rhabdoviral proteins, and modifications to these proteins, on the stress response and host immunity using trout cell-lines, in vitro. The goal is to characterize components of rhabodoviral virulence and how these components (viral proteins) influence host response as a means to identify possible viral targets for new vaccine candidates. Developing new vaccine candidates based on improved understanding of rainbow trout antiviral immune functions should advance our abilities to combat these pathogens in rainbow trout.

Progress Report
The goal for Objective 1, was to use genetic techniques to characterize mechanisms of F. columnare and F. psychrophilum virulence and identify potential strategies to control bacterial disease in rainbow trout aquaculture. Our previous results highlighted the importance of the Flavobacterium type IX secretion system (T9SS) and the many proteins that it secretes for virulence in fish. Proteomic analyses revealed over 50 secreted F. columnare proteins as potential virulence factors and a similar number for F. psychrophilum. We isolated 33 additional mutants with single or multiple deletions of genes predicted to encode virulence factors. We also determined that it was not feasible to obtain an aroA deletion mutant. Instead, we isolated iron uptake mutants. Iron uptake is required by most bacterial pathogens for virulence, and iron uptake mutants are thus attenuated for virulence and may be effective vaccine strains. We generated many mutants lacking genes that encode virulence proteins, and proteins involved in motility and in iron uptake. We now have access to zebrafish and rainbow trout and have begun to analyze the mutants for virulence. We determined that deletion of some genes encoding motility proteins (GldJ, SprB), cytolysins (CylA), proteases, and iron uptake proteins result in reduced virulence. Ongoing experiments will identify the secreted proteins that are most important for columnaris disease. This will help us understand the disease process and may lead to vaccine strains. We continue to improve methods for gene transfer into F. psychrophilum strain CSF-259-93, the preferred model strain for studies of cold water disease in rainbow trout. We are designing an optimized plasmid to express these and additional methyltransferase genes more efficiently and improve gene transfer further. This will allow genetic experiments like those performed above for F. columnare, to identify F. psychrophilum virulence factors and to construct attenuated strains as potential vaccines. For Objective 2, initial efforts to identify new trout AMPs have resulted in identification of ~200 putative AMPs in this species. Expanding this further, we recently published a paper on the regulation and gene structure of a family of AMPs called Nk-lysins. In addition to the Nk-lysins, several other trout AMPs were synthesized as linear peptides and tested for antimicrobial activities against Flavobacterium columnare. Some peptides required solvent to solubilize, which was deleterious to the bacteria. In cases where the solvent was not a problem, the peptides displayed no activity. We redesigned the peptides to be more water soluble (hepcidin) and to include a single di-sulfide bond (Nk-lysins). Once laboratories are able to resume normal (non-essential) research activities, peptides will be distributed to cooperating scientists for testing. Using rainbow trout cell-lines, we assessed the time-course and dose response of a conserved animal AMP (cecropin) on the innate immune response of the host cells. Results created more questions than answers as a number of genes associated with the innate immune response and growth-regulating hormones were affected in ways we did not anticipate. We pivoted rapidly to determine whether similar responses were seen in cells treated with ghrelin and growth hormone, which are key growth- & immune-regulating hormones. Samples are being analyzed at the moment for expression of key immune genes. In addition to cell-lines, we are also repeating work using isolates of primary lymphoid cells in rainbow trout. For Objective 3, we were able to move the Infectious Hematopoietic Virus (IHNV) mutant protein analyses forward. We developed ten IHNV M-protein mutant constructs to assess the ability of each to impact host transcription, six of which were significantly suppressed in their anti-host functions. In combining all studies conducted to date, we have focused on just two: IHNV M 10NC (missing the first 10 and last 10 amino acids [a.a.]) and 20C (missing the last 20 a.a.) mutants for insertion into the IHNV genome for attenuation studies. We have identified NV mutants that have been earmarked for next phase testing (i.e., delta-9N missing the first 9 a.a and delta-5N20C missing the first five and last 20 a.a.). This year we succeeded in developing the proposed full length, modified IHNV genome to allow swapping in and out of mutant NV and M genes. The wild type version, containing introduced restriction sites to allow the aforementioned gene swaps, was synthesized and tested in cell culture replication studies in EPC cells. Whereas transfection of the full-length genome alone or the full-length genome along with IHNV N, P and L plasmids gave modest cytopathic effects (CPE), co-transfection with IHNV NV plasmid gave rise to significant CPE, indicating a critical role for NV in efficient viral production. We can now take advantage of our modified genome to swap the M and NV mutants described above, singly or in combination, into the backbone, develop initial recombinant viral stocks, and then assess the replication potential of the mutant, versus wild type recombinant virus as originally proposed. These studies will provide new information on IHVN replication and virulence features that will be applicable to novel vaccine candidates. We have an additional aim to identify the impact of stress granules (SG) on Viral Hemorrhagic Septicemia Virus (VHSV) infection and if they are acting in a proviral or antiviral manner and how this affects host responses to infection and vaccination. Infection with both VHSV Ia and IVb activates stress kinases PKR and or PERK to induce SG assembly, visualized by foci of SG marker, G3BP1. Removal of NV protein from VHSV IVb increased SG formation suggesting that NV inhibits SG formation. SG induced by VHSV Ia involved PKR activation, unlike IVb which required PERK activation but not PKR. Interferon (IFN) signaling during VHSV Ia infections is significantly decreased when protein kinase R (PKR) and PERK (another protein kinase) activation is inhibited suggesting a possible antiviral role of VHSV Ia induced stress granules. It remains to be determined if activation of PKR and PERK have a direct impact on IFN signaling or if the impact of PKR and PERK on IFN signaling is mediated by stress granule formation. To facilitate some of these studies, we have generated stable cells of EPC, RTG2 and RTgill expressing GFP-G3BP1 to monitor SG formation in real time following virus infection and vaccination. In a major breakthrough, we used CRISPR/Cas9 gene editing technology to generate RTG2 cells with genomic knockout of G3BP1. We are in the process of generating similar knockouts in EPC and RTgill cells. Future studies will determine how SG affect host-VHSV interactions and vaccination by identifying viral proteins involved in stress response through overexpression studies.

1. New fish antimicrobial peptides (AMPs) have been identified. AMPs are short peptides/proteins that form key components as the first-line defense of the innate immune system in many species including mammals, lower vertebrates, fish, insects and plants. As such, AMPs exhibit abilities to defend their hosts against broad spectrum of infectious microbial pathogens (bacterial, viral, fungal and parasitic) and are thought to be promising alternatives to the use of antibiotics in human health and agriculture. ARS scientists in Leetown, West Virginia, and Milwaukee, Wisconsin, identified six new AMPs in rainbow trout. These new AMPs show sequence homology to the Nk-lysin family of proteins and their patterns of gene expression were altered following challenge with aquaculture-relevant pathogens as well as by physiological stressors. Overall, these newly characterized AMPs contribute to host innate immunity and understanding their regulation may provide valuable insights into improving animal health in production systems.

Review Publications
Shepherd, B.S., Ma, H., Han, Y., Palti, Y., Gao, G., Liu, S., Wiens, G.D. 2020. Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 116 (103961).