Antiviral response of adult zebrafish (Danio rerio) during tilapia lake virus (TiLV) infection

Highlights

• Adult zebrafish are susceptible to TiLV infection by intraperitoneal injection.

• TiLV replicates in multiple organs of zebrafish and induces sickness behavior.

• TiLV induces a high type I IFN response in zebrafish.

• TiLV induces inflammation and adaptive immune response in zebrafish.

Abstract

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1β, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses.

Introduction

The essential immune mechanisms, receptors and pathways for a control of pathogen infections are often well conserved in vertebrates. Zebrafish (Danio rerio) represents a relevant model for studying core immune mechanisms activated by viruses. Several viral infections associated with mass mortality of farmed fish species have been studied using zebrafish as a model (reviewed in Refs. [1,2]). In this case, zebrafish might be used to identify the underlying mechanisms associated with the fish’s immune response to selected viruses, which are causing severe economic losses in aquaculture. This could lead to the development of new drugs and/or therapeutic strategies. Moreover, zebrafish is also used as a model to study the host-pathogen interactions and immune response against human viruses [[3], [4], [5], [6]].

Recently, a novel orthomyxo-like virus associated with disease outbreaks and massive mortality of wild and farmed tilapia fish has been isolated in Israel [7]. This virus, named tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae, order: Articulavirales) [8], has a linear, negative-sense single-strand RNA genome with 10 segments and about 10.323 kb in total length [7,9]. The 14 functional open reading frames (ORFs) were predicted from the 10 gene segments of TiLV [10]. The predicted protein sequence of the first segment has a weak homology to the PB1 subunit of the RNA polymerase of influenza C virus, while the other segments have no homology to any other sequences of known viruses [9]. Interestingly, the conserved sequences at the 5’ and 3’ noncoding regions of the segments shows an organization similar to that described for influenza viruses [9]. TiLV virions are enveloped of a round or oval shape and 60–80 nm in diameter with a central variable electron-dense core [7,11].

TiLV is an important risk for the fast-growing tilapia aquaculture worldwide. Tilapia species comprise the second most important freshwater fish cultured worldwide with a global production of 4.95 million metric tons (estimated value of $10.3 billion) in 2016 (see http://www.fao.org/fishery/statistics/global-aquaculture-production/en). Currently, TiLV has spread in Asia, Africa, South and North America, and caused many disease outbreaks in the main tilapia producing countries [12,13]. TiLV affects several species of cultured tilapia including the most cultured Nile tilapia (Oreochromis niloticus) [11], as well as red tilapia (Oreochromis spp.) [11,14], Mozambique tilapia (O. mossambicus) [15], and hybrids: O. niloticus x O. aureus (also known as gray tilapia) [7,14], and O. niloticus x O. mossambicus [16]. Moreover, a range of wild tilapines are positive for TiLV in Israel, Malaysia, Peru and in Lake Victoria (Tanzania and Uganda) [12]. The outbreaks associated with TiLV infections in farms and natural conditions occur mostly during hot seasons (at a water temperature ranging from 22 °C to 32 °C) leading to varying mortalities between 20 and 90% [7,12,[17], [18], [19]]. However, the total infection rate is presumably much higher as there are also reports of subclinical infections based on the detection of TiLV nucleic acid [20,21]. In experimental trials, TiLV infections of tilapia are associated with high mortality of up to 70–90% [7,11,15]. Three different routes of TiLV infection by intraperitoneal (i.p.) injection [7,11,14,15], intragastric exposure [22] and cohabitation [7,23] have been described for tilapia in laboratory conditions.

There is very little information on immune response of tilapia against TiLV. To the best of our knowledge, there is only one recent study showing an up-regulation of the expression of two pro-inflammatory cytokines il-1β and tnf-α during TiLV infection in red and gray tilapia [14]. However, the sparse number of well characterized sequences of cytokines and other antiviral response genes in tilapia (e.g. interferon type I pathway), limits our understanding of the host response of tilapia to TiLV infection [14]. Therefore, the development and use of a suitable infection model is important to study TiLV induced antiviral immune responses, disease pathogenesis and to develop therapeutic or prophylactic strategies such as vaccines.

The present study was devoted to evaluate the possible use of zebrafish as an animal model to study immune response during TiLV infection. Our results demonstrated that adult zebrafish are susceptible to TiLV and high levels of viral nucleic acids could be detected in all tested organs of i.p. infected fish. Taking advantage of the access to fully sequenced zebrafish genome with well characterized genes encoding molecules involved in the antiviral response, we studied the expression of immune-related genes in spleen and kidney of adult zebrafish during TiLV infection.