Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV’s ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib—an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyx-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells

Detection of Tilapia Lake Virus (TiLV) in Clinical Samples by Culturing and Nested RT-PCR

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia

Tilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5′ and 3′ termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease

Recovery of Streptococcus iniae from Diseased Fish Previously Vaccinated with a Streptococcus Vaccine

Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish