Cloning, expression, antibody preparation and tissue distribution of tilapia lake virus nucleoprotein

Recently, tilapia lake virus (TiLV) has been epidemic in many countries and posed a serious threat to Oreochromis spp. aquaculture industry. China has contributed the most amount of cultured Oreochromis spp. in the world. Up to date, there is no report of the TiLV epidemic in Oreochromis spp. in the mainland of China. However, since GIFT O. niloticus is one of the most cultured Oreochromis spp. species in the mainland, therefore it is necessary to characterize the features of the GIFT strain infected with TiLV. Taking the advantage of the TiLV which was kindly given as a gift by Dr. Sven Bergmann from Institute of Infectology, Friedrich Loffler Institute, 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 O. niloticus were determined. The length of the cDNA 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 ku. There is 5′ end non-coding region of 19 bp and 3′end non-coding region of 972 bp. The sequences and phylogenetic tree analysis showed that the sixth genomic segment encoded TiLV nucleoprotein (NP). Subsequently, 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 1:51 200, and the antibody could specifically recognize the NP protein from the tissues of O. niloticus infected with TiLV. Hematoxylin-eosin staining (H.E) was performed on different tissues of O. niloticus. 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 O. niloticus infected with TiLV. The results showed that the highest amount of NP protein was expressed in the liver, followed by the brain, trunk kidney and head kidney. In order to elucidate the immune responses of O. niloticus to the TiLV infection, real-time quantitative PCR (qRT-PCR) was used to measure the mRNA expressions of TNF-α and TGF-β 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 infection), the expression of both TNF-α and TGF-β was significantly inhibited by the viral infection, indicating that TiLV might inhibit these cytokines so as to facilitate its early replication in the host. The current study will shed new light on the pathogenesis of TiLV infection and will pave a new way for the development of effective prevention and control strategy against the epidemic of TiLV in O. niloticus.