The progression of viral infections is notoriously difficult to follow in whole organisms. The small, transparent zebrafish larva constitutes a valuable system to study how pathogens spread. We describe here the course of infection of zebrafish early larvae with a heat-adapted variant of the Infectious Hematopoietic Necrosis Virus (IHNV), a rhabdovirus that represents an important threat to the salmonid culture industry. When incubated at 24°C, a permissive temperature for virus replication, larvae infected by intravenous injection died within three to four days. Macroscopic signs of infection followed a highly predictable course, with a slowdown then arrest of blood flow despite continuing heartbeat, followed by a loss of reactivity to touch and ultimately by death. Using whole-mount in situ hybridization, patterns of infection were imaged in whole larvae. The first infected cells were detectable as early as 6 hours post infection, and a steady increase in infected cell number and staining intensity occurred with time. Venous endothelium appeared as a primary target of infection, as could be confirmed in fli1:GFP transgenic larvae by live imaging and immunohistochemistry. Disruption of the first vessels took place before arrest of blood circulation, and hemorrhages could be observed in various places. Our data suggest that infection spread from the damaged vessels to underlying tissue. By shifting infected fish to a temperature of 28°C that is non-permissive for viral propagation, it was possible to establish when virus-generated damage became irreversible. This stage was reached many hours before any detectable induction of the host response. Zebrafish larvae infected with IHNV constitute a vertebrate model of an hemorrhagic viral disease. This tractable system will allow the in vivo dissection of host-virus interactions at the whole organism scale, a feature unrivalled by other vertebrate models.
The zebrafish larva is uniquely amenable to imaging among vertebrate models because of its small size, transparency, and ease of anesthesia, making it a useful model to understand host-pathogen interactions. We have performed the first detailed analysis of a viral infection in zebrafish. Infection of zebrafish larvae with a salmonid rhabdovirus adapted to growth at the appropriate temperatures resulted in a predictable succession of pathological signs before death. Detection of infected cells in whole larvae revealed that blood vessels were a major target of the virus, providing an explanation to hemorrhages and subsequent loss of blood flow observed in infected larvae. Destruction of vascular cells caused by the viral infection was readily observed in transgenic larvae with fluorescent endothelium. We could identify the critical moments of the infection with simple temperature shift experiments. This work provides a basis to dissect the role of host factors in controlling the propagation of viral infections.