The Infections Of Tilapia Lake Virus In Nile Tilapia (Oreochromis Niloticus,Gift Strain) And E-11 Cell

In the past ten years,Tilapia lake virus（TiLV）has been endemic in the cultured and wild tilapia in many countries such as Israel,Ecuador,Egypt,Thailand and India,posing a serious threat to the tilapia aquaculture industry.But,there are still no effective measures to prevent and control TiLV infection.In order to study the infection characteristics of the tilapia lake virus in the main domestic genus tilapia（Oreochromis niloticus,GIFT strain）and the striped scorpion cell line SSN-1 clone E-11.The main findings are as follows:1.Preparation and titer detection of TiLV-HEF polyclonal antibody.The full-length cDNA of the fourth segment genome of the tilapia lake virus was obtained from the spleen of the tilapia infected with TiLV.The full-length cDNA of the TiLV was 1250 bp,and the open reading frame length was 1065 bp,encoding a 354 amino acid HEF protein with a predicted molecular weight of 38.30 kDa,5’non-coding region is 48 bp,and 3’ non-coding region is 137 bp.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 is obviously higher higher than1:204,800,and the purified antibody titer can also reach 1:51,200,the serum could specifically recognize the HEF protein from the spleen of TiLV infected tilapia.2.Distribution of TiLV in tilapia（Oreochromis niloticus,GIFT strain）tissues.Through artificial infection experiments,it was found that TiLV infected juvenile tilapia severely and causes surface ulceration,systemic bleeding and ocular lens opacity.Furthermore,hematoxylin and eosin（HE）stain showed liver steatosis and syncytium,hemosiderin and vacuolar degeneration in spleen,necrosis in head kidney lymphocytes,protein precipitation and glomerulus necrosis in the kidney.Western blot and immunohistochemistry results showed that the virus was distributed in liver,spleen,kidney,gill and brain tissues with the higher abundance in the spleen,head kidney and gill than that in the liver,kidney and brain tissues.TiLV infection could cause disease by targeting liver,spleen,kidney,gill and brain tissue of tilapia.3.Characterization of TiLV-infected E-11 cells.E-11 cells showed significant CPE phenomenon in 7-9 days after TiLV infection,and the diseased cells gradually aggregated and plaques appeared.The virus titer was determined to be 2.7x10⁵TCID₅₀/l mL.Through indirect immunofluorescence assay,it was found that HEF protein mainly distributed in the cytoplasm in E-11 cells infected with TiLV.TiLV infection can also cause abnormal expression of IL-8,TNF-αand MHC-I of immune factors.4.The influenza virus neuraminidase inhibitor zanamivir has no inhibitory effect on TiLV.When the concentration of zanamivir was 1 nmol/L,2 nmol/L,4 nmol/L,there was no effect on the viability of E-11 cells,while at a concentration of 8 nmol/L,the normal growth of E-11 cells was inhibited.However,it was found that zanamivir can not inhibit TiLV by PCR,and can not be used to prevent TiLV infection and treatment.Our research results have increased the understanding of TiLV,which is of great significance for further understanding the pathogenesis of TiLV and preventing and controlling the spread of TiLV in China.