Comparative effects of Novirhabdovirus genes on modulating constitutive transcription and innate antiviral responses, in different teleost host cells systems

Authors: GORGOGLIONE, BARTOLOMEO - Wright State University; RINGIESN, JEFFREY - Wright State University; Shepherd, Brian; LEAMAN, DOUGLAS - Wright State University

Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2020
Publication Date: 7/20/2020
Citation: Gorgoglione, B., Ringiesn, J.L., Shepherd, B.S., Leaman, D.W. 2020. Comparative effects of Novirhabdovirus genes on modulating constitutive transcription and innate antiviral responses, in different teleost host cells systems. Virus Research. 17:110. https://doi.org/10.1186/s12985-020-01372-4.
DOI: https://doi.org/10.1186/s12985-020-01372-4

Interpretive Summary: Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious viruses that cause disease in a variety of fish species and affect fish production facilities and fisheries in Europe and the United States. This study assessed how genes of the IHNV and VHSV pathogens modulate certain immune responses, using fish cell lines representing different tissue types from a variety of fish species (bluegill, fathead minnow and rainbow trout). To accomplish this work, optimal protocols were developed to enable transfer (transfection) of viral genes into these different fish cell lines under controlled laboratory conditions. The impact of these transferred genes (G, M, N, NV and P) on cellular gene expression was then measured. By testing the effects of four of the major structural (N, P, M, G), and the sole nonstructural (NV), genes from VHSV-IV and IHNV in a series of experiments, we were able to gain insight into the relative abilities of each to alter general or innate immune gene expression in the different fish cell lines. Findings suggest that M and NV genes of the INHV and VHSV pathogens strongly altered cellular gene expression responses across all cell lines. In contrast, other viral genes studied, P and G, impacted cellular gene expression less strongly and in ways that were linked to the cell type or dose used for transfection. This study provides novel insights on the viral regulators of host cellular responses to infection and highlights the need for additional laboratory-based studies using more than a single virus or cell type/line. In support of this, the comparative approach used allowed a glimpse into the complex nature of novirhabdoviral strategies to impact a variety of cells and teleost hosts. Information from this work may enable identification of new viral targets that modulate the host-pathogen interaction, which could in turn be used to design more efficient vaccination strategies.

Technical Abstract: Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and causing World Organization for Animal Health notifiable diseases. This study assesses the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores specific actions of IHNV genes on modulating the interferon pathways in various teleost cell-lines. Optimized transfection protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 h post-transfection with luciferase constructs driven by SV40 or a modified ß-Actin promoter (pCAG). M was generally confirmed as the strongest constitutive transcriptional suppressor, while N mediated milder inhibitory effects. IHNV-P, but not VHSV-P, mediated stronger stimulatory patterns in fibroblastic cell types. Cell-specific effects were observed for viral -G gene, with VHSV-G exhibiting suppression in EPC and BF-2 but not in trout cells, while IHNV-G was stimulatory in RTG-2 but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV-NV compared to VHSV-NV. Modulation of innate antiviral immune responses by IHNV genes was characterized after 72 h co-transfection using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation by MAVS cotransfection. IHNV-M silenced the IFN pathway in all cell lines tested. IHNV-N showed a dose-dependent suppression of IFN, but with minor effects on MX-1. IHNV-P and -G played a minor IFN-inhibitory role, consistent and dose-dependent only for -G in rainbow trout cells. IHNV-NV mediated a consistent stimulatory effect on either IFN and Mx-1, but much less pronounced in RTgill-W1. This study provides novel insights into virus-host interaction, showing differential innate immune responses in varying cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.