Infectious hematopoietic necrosis virus (IHNV) persistence in Sockeye Salmon: influence on brain transcriptome and subsequent response to the viral mimic poly(I:C)

Abstract

Background: Sockeye Salmon are an iconic species widely distributed throughout the North Pacific. A devastating pathogen of Sockeye Salmon is infectious hematopoietic necrosis virus (IHNV, genus Novirhabdovirus, family Rhabdoviridae). It has been postulated that IHNV is maintained in salmon populations by persisting over the life of its host and/or by residing in natural reservoirs other than its susceptible hosts. Herein we demonstrate the presence of IHNV in the brain of Sockeye Salmon that survived an experimentally-induced outbreak, suggesting the presence of viral persistence in this susceptible species. To understand the viral persistent state in Sockeye Salmon we profiled the transcriptome to evaluate the host response in asymptomatic carriers and to determine what effects (if any) IHNV exposure may have on subsequent virus challenges.

Results: A laboratory disease model to simulate a natural IHNV outbreak in Sockeye Salmon resulted in over a third of the population incurring acute IHN disease and mortality during the first four months after initial exposure. Nine months post IHNV exposure, despite the absence of disease and mortality, a small percentage (<4 %) of the surviving population contained IHNV in brain. Transcriptome analysis in brain of asymptomatic virus carriers and survivors without virus exhibited distinct transcriptional profiles in comparison to naïve fish. Characteristic for carriers was the up-regulation of genes involved in antibody production and antigen presentation. In both carriers and survivors a down-regulation of genes related to cholesterol biosynthesis, resembling an antiviral mechanism observed in higher vertebrates was revealed along with differences in nervous system development. Moreover, following challenge with poly(I:C), survivors and carriers displayed an elevated antiviral immune response in comparison to naïve fish.

Conclusions: IHN virus persistence was identified in Sockeye Salmon where it elicited a unique brain transcriptome profile suggesting an ongoing adaptive immune response. IHNV carriers remained uncompromised in mounting efficient innate antiviral responses when exposed to a viral mimic. The capacity of IHNV to reside in asymptomatic hosts supports a virus carrier hypothesis and if proven infectious, could have significant epidemiological consequences towards maintaining and spreading IHNV among susceptible host populations.

Fig. 1

Cumulative mortality after IHNV challenge. Sockeye Salmon were exposed to waterborne IHNV (challenged fish) or left unhandled (naïve fish). Randomly selected dead fish sampled between 19 and 138 days post challenge (dpc) were tested for the presence of virus in the anterior kidney by cell culture assay. Brains of surviving fish were screened by RT-rPCR for the presence of IHNV at indicated time points. The percent of virus-positive fish is denoted in parentheses and is calculated from the number of positive detections out of the total number of fish tested (italicized numbers separated by a slash). The injection of poly(I:C) occurred at 271 dpc

Fig. 2

Overview of microarray analysis in brain of Sockeye Salmon. a) A two-way ANOVA (p ≤ 0.01) using expression data of the six groups naïve, survivors, carriers with and without poly(I:C) injection was performed. The IHNV status is indicated for each group. All samples (n) were collected 3 d post injection, i.e. 274 d post challenge (dpc), except for 2 carriers that were sampled 278 dpc. b) Venn diagram of probes affected by IHNV status, poly(I:C)-injection and interaction of both factors. Numbers in parentheses indicate numbers of probes retained after a fold change (FC) cut-off of ≥ 1.5. One asterisk (*) indicates that all interaction probes were removed from main effect lists to analyze separately. Two asterisks (**) refer to the number of probes used as input for k-means clustering shown in Fig. 3. c) Venn diagram of fold change-filtered up- and down-regulated probes affected by IHNV status that were separated into survivor- and carrier-relevant probes

Fig. 3

Cluster analysis of probes affected by interaction of poly(I:C)-injection and IHNV status. Based on expression levels of individual probes, clusters I to IV were generated containing 18, 45, 41 and 58 probes, respectively. Fold changes of uniquely annotated probes from cluster I are shown for poly(I:C)-injected survivors (S-pIC) and carriers (C-pIC) relative to naïve-injected fish (N-pIC) (fish group abbreviations as defined in Fig. 2). Colors refer to ranges of fold changes: yellow 1.5 to 2.5; orange 2.6 to 3.5; brown 3.6 to 9.2. Genes marked with an asterisk (*) have been described as virus responsive genes (VRG) in Krasnov et al. [54]