| Since 2009, Tilapia lake virus (TiLV) has been endemic and outbreaks in the cultured tilapia in many countries such as Israel, Ecuador, Egypt, Thailand and India, posing a serious threat to the tilapia aquaculture industry. In order to study the infection characteristics of TiLV in the cultured tilapia species and susceptible cells, Nile Tilapia (Oreochromis niloticus，GIFT strain) and E-11 cells were chosen as models. TiLV for present study was kindly gifted by Dr. Sven Bergmann from Institute of Infectology, Friedrich Loffler Institut (FLI). First of all, the whole nucleotide sequences of the fourth genome segment of TiLV from the experimental infected tilapia were determined. The length of the cDNA of the fourth genome segment was 1250 bp containing an open reading frame of 1065 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 1:51200, and the serum could specifically recognize the HEF protein from the spleen of TiLV infected tilapia. Through artificial infection experiments, it was found that TiLV infected juvenile tilapia 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 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 tissue of tilapia, and will shed a new light on the prevention and control of Tilapia Lake virus infection in tilapia.