The Function Of Thioredoxin And Oncoprotein In WSSV Infection And Metabolite Changing After Infected By WSSV In Litopenaeus Vannamei

White spot syndrome virus(WSSV) is a serious infectious pathogen of cultured shrimp,appeared in Southeast Asia at the beginning of the 1990 s, often leading to large scale mortalities resulting in diminished shrimp production and severe huge economic losses in the past decades to farmers worldwide. The virus has a broad host range and is reported to infect a wide range of aquatic animals that includes marine and brackish water crustaceans, penaeids, crabs, freshwater prawns and crayfish, aquatic arthropods and planktons[1-4].Part 1:Thioredoxin(TRX), a major intracellular antioxidant, has a wide range of biological functions. It was up-regulated and targeted by WSSV. However, the relevant of TRX with WSSV infection and signaling pathway remains largely unknown. Sequence analysis indicated that TRX might interact with the WSSV030(VP362) and WSSV454(TK-TMK) of WSSV. In this study, TRX, VP362 and TK-TMK were expressed and the interaction of TRX between VP362 or TK-TMK was investigated. Furthermore, how TRX affect the process of WSSV infection and the gene transcription of IKK, Lv P38 and STAT in the hemocytes and hepatopancreas was explored. Far-western blot and ELISA results showed that TRX interacted with VP362 and TK-TMK. The m RNA expression of IKK, Lv P38 and STAT were significantly affected by the over-expression of TRX of Litopeneaus vannamei. The experiment of in vivo neutralization indicated that TRX introduced the transcription expression of VP28 and increased the viral copy numbers in the early stage of WSSV infection and it may accumulate the death of shrimps infected by WSSV.Part 2:As is known to us, Nucleoside diphosphate kinase(NDK), which sequences are the same parallel to oncoprotein(OP) of human, can lead to nucleoside triphosphates for DNA replication and to be regulated by viral infection in Litopenaeus vannamei. This paper describes some methods to identify what the relationship between NDK and WSSV. He NDK was synthesized by Prokaryotic expression firstly. From the results of Western-blot, Far-western blot and ELISA, we confirm interaction existed in themselves. The copy numbers of WSSV and m RNA levels of IE1 and VP28 were significantly increased in shrimps at 72 h after WSSV-infected, which were injected with recombinant NDK. After synthesizing the ds RNA-OP and confirming the efficacy of OP silencing, cumulative mortality was recorded of the experiment of WSSV-infected shrimps which were injected with NDK and ds RNA-OP. Make a comparison between these results we demonstrate that the silence of NDK delayed the death of shrimps obviously. These findings indicate that NDK have an important role in influencing thereplication of WSSV in shrimps. Meanwhile it might be acted as a new therapeutic strategy for WSSV infection in shrimp.Part 3:Here, we comprehensively analyzed the metabolic change of hepatopancreas from Litopenaeus vannamei which were infected with white spot syndrome virus(WSSV) artificially by intramuscular injection of the virus inoculum by 1H nuclear magnetic resonance(NMR). In this study, hepatopancreas samples were obtained from WSSV-infected shrimps(N=5) and control groups injected PBS solution(N=5). Through the NOESYPR1 D spectrum combined with multi-variate pattern recognition analysis, including principal component analysis(PCA)and orthogonal partial least-squares discriminant analysis(OPLS-DA) models, we can further distinguish WSSV-infected groups from healthy subjects. It was capable of diagnosing WSSV by using the OPLS-DA model, and the model has much higher sensitivity and specificity rate.From the analysis about WSSV hepatopancreas samples, concentrations of acetate 、 lipid(-CH2-CH=CH-)、lysine、tyrosine、N-acetyl glycoprotein were lower,opposite to α-Glucose andβ-Glucose in comparison with the control group. In the study, these observations offered novel insights into the results indicating that it could be used by NMR-based metabolomic methods,because the sufficient sensitivity and specificity to distinguish WSSV-infected shrimps from healthy controls it had. So we can act the method as a potential tool in order to develop a clinically useful diagnostic analysis about the WSSV in aquaculture, and could also contribute to a further understanding of disease mechanisms at a functional level.Part 4 : In order to understand the gene expression associated with the oxidative and signaling pathway, quantitative real-time PCR was performed to analysis the changes of m RNA expression of thioredoxin(TRx), Litopenaeus vannamei p38(Lv P38), catalase(CAT),peroxidase(POD) gene in tissues of lymphoid and gills to white spot syndrome virus infection at different time( 0, 6, 12, 24, 48 and 72 h). Shrimps were divided into two groups(WSSV-injected group and PBS-injected group). After injection, 3 shrimp from the WSSV-injected and PBS-injected group were taken separately at 0, 6, 12, 24, 48 and 72 h post-injection. Individual samples of lymphoids and gills were taken each time. After extraction of total RNA, first-strand c DNA was synthesised with kit and quantified by Nondrop 3000. Quantitative real-time PCR was carried in a Rotor Gene 3000. The results showed that the expression levels of TRx、Lv P38、CAT、POD in lymphoids reached the maximum value at 72 h post infection(WSSV-injected group), and there was no significant difference at the period from 6 h to 48 h between WSSV-injected and PBS-injected group. However, the significantly higher expression levels were reached at 12 h post infection in the tissues of gills and decreased with the period of infection in the WSSV-injected group. Otherwise, significant difference of TRx m RNA expression were observed at 48 and 72 h respectively, while CAT m RNA expression showed asignificant difference at 48 h, between WSSV-injected and PBS-injected group. Evidences in our study indicated that TRx、Lv P38、CAT、POD correlated with WSSV infection.