Cloning,Expression,Antibody Preparation And Tissue Distribution Of Tilapia Lake Virus Nucleoprotein

Oreochromis spp are important cultured fish species worldwide.Recently,tilapia lake virus(TiLV)has been epidemic in many countries and caused great economic loss in the tilapia culture industry.China is the largest tilapia aquacultured country with total amount of more than 1.7 million tons in 2017.Up to date,there is no report of the TiLV epidemic in tilapia in the mainland of China.However,since Oreochromis niloticus(GIFT strain)is one of the most cultured tilapia species in the mainland,therefore it is necessary to characterize the features of the GIFT strain infected with TiLV.In this report,we studied the cloning,expression,antibody preparation and tissue distribution of tilapia lake virus nucleoprotein.The main results are followings:1.We performed the infection of TiLV in the GIFT strain.The whole nucleotide sequences of the sixth genomic segment of TiLV from the experimental infected tilapia were determined.The length of the c DNA of the sixth genomic segment was1 044 bp containing an open reading frame of 954 bp encoding a protein with 317 amino acids with predicted molecular weight of 36.38 k Da.The sequences and phylogenetic tree analysis showed that the sixth genomic segment encoded TiLV nucleoprotein(NP).2.GST fusion NP was expressed in Escherichia coli and purified,and it was used to immunize New Zealand white rabbit(Albus lepus)according to the conventional method to prepare rabbit anti-NP polyclonal antibody.The results showed that the antibody titer obtained by ELISA was higher than 1:25 000,and the antibody could specifically recognize the NP protein from the tissues of tilapia infected with TiLV.3.To observe the distribution of TiLV in various tissues of tilapia,hematoxylin-eosin staining(H&E)was performed on different tissues of tilapia.The results showed that there were apparent pathological changes in the observed tissues,including hepatic necrosis and syncytium;vacuolization,necrosis and increased amount of hemosiderin in the spleen;necrosis and inclusion body in the head kidney;dissociation and shedding of the epithelial cells of the gill filament,small pieces adhered to each other;vacuoles of nerve cells in the brain tissue.Western blot and immunohistochemistry(IHC)were used to detect the expression of the NP protein in different tissues of tilapia infected with TiLV.The results showed that the highest amount of NP protein was expressed in the liver,followed by in the brain,trunk kidney and head kidney.4.In order to elucidate the immune responses of tilapia to the TiLV infection,real-time quantitative PCR(qRT-PCR)was used to measure the m RNA expressions of TNF-? and TGF-? in the spleen and head kidney which are the two major immune tissues of fish.The results showed that during the early period of the infection(12-24 h post of the infection),the expression of both TNF-? and TGF-? was significantly inhibited by the viral infection,indicating that TiLV might inhibit these cytokines so as to facilitate its early replication in the host.TiLV could infect E-11 cells and caused significant cytoplasmic vacuoles and plaque formation at 7 day post of the infection.The NP protein of TILV mainly distributed in the cytoplasm.The current study will pave a new way on the diagnosis and the development of effective prevention and control strategy against the epidemic of TiLV in tilapia.