In order to study the infection characteristics of TiLV in the cultured tilapia species and susceptible cells, GIFT Oreochromis niloticus and E-11 cells were chosen as models. For the present study, first of all, the whole nucleotide sequences of the fourth genome segment of TiLV from the experimental infected GIFT O. niloticus were determined. The length of the cDNA of the fourth genome segment was 1 250 bp containing an open reading frame of 1 065 bp, encoding a protein with 354 amino acids. The sequences and phylogenetic tree analysis showed that the fourth genome segment encoded TiLV Hemagglutinin-esterase-fusion (HEF) protein. Subsequently, GST fusion HEF was expressed in Escherichia coli and purified, and it was used to immunize New Zealand white rabbits according to the conventional method to prepare rabbit anti-HEF polyclonal antibody. The results showed that the antiserum titer obtained by ELISA was higher than 1∶51 200, and the serum could specifically recognize the HEF protein from the spleen of TiLV infected GIFT O. niloticus. Through artificial infection experiments, it was found that TiLV infected juvenile GIFT O. niloticus severely and caused surface ulceration, systemic bleeding and ocular lens opacity. Furthermore, hematoxylin and eosin (HE) stain showed the syncytium in liver, hemosiderin and vacuolar degeneration in spleen, necrosis in head kidney lymphocytes, protein precipitation and glomerulus necrosis in trunk kidney. Western blot and immunohistochemistry results showed that the virus was distributed in all the tissues with the higher abundance in the spleen, head kidney and gill than that in the liver, trunk kidney and brain tissues. Through indirect immunohistochemistry assay, it was found that HEF protein was mainly distributed in the cytoplasm in E-11 cells infected with TiLV. Our results demonstrate that TiLV infection could cause disease by targeting liver, spleen, kidney, gill and brain tissues of GIFT O. niloticus.