2.8. Experimental Challenge and Sampling Regime

For the experimental challenges, the goldfish nidovirus was propagated in E11 cells and the viral stock titrated, as described above.

Single tanks containing either 30 Atlantic salmon parr or 30 common carp, both reared from ova in the bio-secure stock aquarium areas of the Cefas Weymouth lab, or 30 goldfish, sourced from a local farm subjected to health checks and quarantine, weighing between 10 and 15 g, were exposed to the virus via either intraperitoneal injection (IP), with a viral inoculum of 100 µL at 1.7 × 10⁸ TCID₅₀ mL⁻¹, or by a static bath immersion, consisting in 15 mL of virus stock at 1.7 × 10⁸ TCID₅₀ mL⁻¹ added to 30 L of tank water (5 × 10⁴ TCID₅₀ mL⁻¹ dose) for 4 h, after which the flow rate was restored to 0.5 L min⁻¹. The water temperature was maintained at 18 °C for common carp and goldfish, or at 14 °C for Atlantic salmon. Two control tanks for each species, containing 30 fish in each tank, were either mock IP-injected or mock bath-challenged.

The fish were monitored for 33 days throughout the challenge. Five fish per species and treatment (IP and bath challenge, including respective controls) were sampled at 10 days post-challenge (pc) (for common carp and goldfish) and 12 days (for Atlantic salmon). At 20 days pc, 10 Atlantic salmon and common carp and 8 goldfish were sampled, while all the remaining fish were sampled at 33 days.

For each sampled animal, a pool of brain, kidney, heart and spleen were taken for virology and processed as described above. Gill, kidney, spleen, liver, heart and brain were also fixed in 10% neutral-buffered formalin (NBF) and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (GLUT) for histology and transmission electron microscopy (TEM) examinations, respectively. For all the survivors sampled at 33 days pc, equivalent to 594-degree days (DD) for common carp and goldfish and at 462 DD for Atlantic salmon, blood was drawn from the caudal vein for serology tests.

To determinate the viral load in infected tissues, RNA was extracted from the heart and kidney of selected specimens and analysed using Taqman qPCR, as described above.