At 28 dpc, the cumulative mortality in Mozambique tilapia with low (1 × 10³ TCID₅₀/mL) and high (1 × 10⁵ TCID₅₀/mL) concentration of TiLV was 48.89% and 77.78%, respectively (Figure 1). At 4 dpc, moribund Mozambique tilapia developed clinical signs of TiLV infection, including skin hemorrhage, lethargy, pale skin, abdominal swelling, skin erosion, congestion around the eye, and exophthalmos (Figure 2). Notably, mortality in fish exposed to high TiLV concentration began at 4 dpc, while the mortality in fish exposed to low TiLV concentration did not start until 7 dpc. Mortality in both low TiLV and high TiLV-exposed fish ended at 19 dpc (Figure 1). Intraperitoneal challenges (IP) of red hybrid tilapia with TiLV at 1 × 10⁵ TCID₅₀/mL were conducted to compare clinical signs, pathology, and mortality patterns. Similar to the Mozambique tilapia, the red hybrid tilapia also showed skin hemorrhage, swimming at the bottom of the tank, skin and fin erosion, exophthalmos, and scale protrusion, with the first mortality at 6 dpc. At the end of the experiment, the cumulative mortality of the red hybrid tilapia was 66.67% (Figure 1). No clinical signs and mortality developed in the control Mozambique and red hybrid tilapia.

To further confirm the susceptibility of Mozambique tilapia to TiLV, spleens were collected from low and high TiLV-challenged fish at 3, 6, and 12 dpc. As shown in Table 1, TiLV was detected in 3 out of 5 fish (60%) and 4 out of 5 fish (80%) exposed to the low concentration of TiLV at 3 and 6 dpc, respectively, with the virus concentration ranging from 9.7 × 10–4.8 × 10⁵ copies per μg of total RNA. No TiLV was detected in the fish exposed to the low concentration at 12 dpc. Conversely, all of the fish exposed to high concentration of TiLV had TiLV in their spleen as early as 3 dpc (1.1 × 10⁴–3.5 × 10⁴ copies per μg of total RNA), which remained detectable in all fish until 12 dpc (3.3 × 10⁵–2.3 × 10⁶ copies per μg of total RNA). Throughout the studied period, no TiLV was detected in the unchallenged fish.

Normal hepatocytes with adequate glycogen storage were found in the liver of control fish (Figure 3A,C). No extensive histopathological changes were found in the livers and spleens of Mozambique tilapia that were challenged by the low concentration of TiLV (Figure 3C,D). However, severe histopathological changes could be found in red hybrid tilapia and Mozambique tilapia exposed to high concentration of TiLV at 6 dpc. Depletion of glycogen storage, loss of sinusoid structure, presence of syncytial giant cells containing multiple nuclei, and intracytoplasmic inclusion bodies were observed in the livers of both species (Figure 3A,E). A few melanomacrophage centers (MMCs) and normal lymphocytes surrounded by a sheath of reticular cells were observed in the spleen of the control red hybrid and Mozambique tilapia (Figure 3B,D), while an abundance of MMCs and depletion of red blood cells were observed in the spleens of the fish exposed to the high TiLV concentration (Figure 3B,F). No remarkable change was observed in the gills or intestines of the challenged fish. The localization of TiLV RNA in infected tissues including liver, intestines, heart, and gills was further confirmed by ISH. Intriguingly, positive signals with green coloration were clearly observed in all tested Mozambique tilapia tissues exposed to the high TiLV concentration, while positive signals were noted to a lesser extent in liver and intestines of Mozambique tilapia exposed to the low TiLV concentration. Conversely, no positive hybridization signal was detected in either the infected tissues exposed to a non-irrelevant probe or the uninfected control tissue samples (Figure 4).