Research On TLRs And Immune-related Genes Expression Profiles Of Common Carp Infected With Spring Viremia Of Carp Virus

Toll-like receptors(TLRs) is a class of pattern recognition receptors(PRRs) in host cells, which could recognize pathogen associated molecular patterns(PAMPs) of virus and play an important role in the immune response to antiviral infection. After recognizing viral PAMPs, TLRs induce expression of nuclear factor-κB(NF-κB) and interferon regulatory factors(IRFs) through myeloid differentiation factor 88(My D88) dependent or TIR domain-containing adaptor-inducing interferon-β(TRIF) dependent signaling pathway, finally initiate innate and adaptive immunoresponse against virus invasion. At least 20 TLRs have been identified in teleosts, and most of these TLRs can find corresponding orthologs in mammals. But studies have shown that the functional characteristics of fish TLRs differ from those of mammalian.Spring viremia of carp virus(SVCV) is a member of the virus family Rhabdoviridae and the aetiological agent of spring viraemia of carp(SVC), which is an acute haemorrhagic and contagious viraemia in several carp species and of some other cyprinid and ictalurid fish species. SVC causes heavy economic losses with its widespread and high mortality, and has become the biggest threat of cyprinid species culture. Cyprinid species are major freshwater aquaculture species in China, accounting for over 20% of the freshwater aquaculture production. Common carp varieties are the principal host for SVCV and are considered to be most susceptible to SVCV infection, however, the current knowledge about the innate immune response of common carp against SVCV is still very limited.Common carp was employed in this study, the effect of common carp TLRs overexpression on the expression of down-stream interferon associated immune factors m RNA in epithelioma papulosum cyprini(EPC) cells were executed firstly. Secondly, post SVCV infection, we examined the expression profile of some immune-related genes including TLRs in anterior kidney at different time points. These studies of common carp not only give us an insight into the molecular mechanisms of host defense against SVCV, but also help us to develop immune prophylaxis to prevent SVC.Section Ⅰ: Research on antiviral-related TLRs of common carp We cloned the TLR2, TLR3, TLR7, TLR9 and TLR22 genes of common carp(Cc TLRs) by using reverse transcription-polymerase chain reaction(RT-PCR) and ligated them into an expression vector p EGFP-N1. Then five species plasmids of p EGFP-N1-Cc TLR2, p EGFP-N1-Cc TLR3, p EGFP-N1-Cc TLR7, p EGFP-N1-Cc TLR9, and p EGFP-N1 were transfected into EPC cells, respectively. For groups of p EGFP-N1-Cc TLR2, p EGFP-N1-Cc TLR9 and p EGFP-N1, the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h post-transfection were determined by real-time PCR; for groups of p EGFP-N1-Cc TLR3 and p EGFP-N1-Cc TLR7, only expression of IRF3 and IRF7 m RNA at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h post-transfection were determined by real-time PCR. Besides, the infection experiment with SVCV was performed in EPC cells transfected with p EGFP-N1-Cc TLR2. The main results of this thesis were as follows: The expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA were obviously upregulated in EPC cells transfected with p EGFP-N1-Cc TLR2; at 48 h post-transfection, the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA in EPC cells were respectively upregulated 6.3 times, 16.5 times, 15.0 times, 13.0 times, 7.6 times and 92.4 times of that in EPC cells untransfected, and the differences were statistically significant(P<0.01); at 48 h and 72 h post-transfection, the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA in EPC cells transfected with p EGFP-N1-Cc TLR2 were respectively 2 times and 2.2 times, 1.5 times and 2.4 times, 3.9 times and 4.7 times, 3.4 times and 6.7 times, 5.4 times and 3.7 times, 6.6 times and 15.6 times of that in EPC cells transfected with p EGFP-N1, and the differences were statistically significant(P<0.01); the quantity of SVCV in EPC cells transfected with p EGFP-N1 were respectively 1.1 times, 2.4 times, 3.8 times, 11.7 times and 11.4 times of that in EPC cells transfected with p EGFP-N1-Cc TLR2 on 6 h, 12 h, 24 h, 48 h and 72 h post-infection, and the differences were statistically significant(P<0.01). These results indicated that overexpression of a Cc TLR2 gene in EPC cells could upregulate the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA and inhibit the reproduction of SVCV, which suggested that TLR2 of fish could activate the signaling cascade of IRF3 or/and IRF7, leading to secretion of I-IFN to induce the expression of IFN-induced genes such as ISG15、Mx1、PKR and Viperin for anti-virus effect. The expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA were upregulated in EPC cells transfected with p EGFP-N1-Cc TLR9. At 72 h post-transfection, the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA in EPC cells were respectively upregulated 6.9 times, 39.5 times, 65.7 times, 5.0 times, 5.6 times and 156.2 times of that in EPC cells untransfected, and the differences were statistically significant(P<0.01). At 48 h and 72 h post-transfection, the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA in EPC cells transfected with p EGFP-N1-Cc TLR9 were respectively 1.3 times and 2.5 times, 2.8 times and 3.1 times,5.9 times and 10.3 times, 3.0 times and 1.9 times, 2.3 times and 2.6 times, 6.5 times and 11.9 times of that in EPC cells transfected with p EGFP-N1, and the differences were statistically significant(P<0.01). The results indicated that overexpression of a Cc TLR9 gene in EPC cells could upregulate the expression of IRF3, IRF7, ISG15, Mx1, PKR and Viperin m RNA, and the upregulation of Viperin, ISG15 and IRF7 were more obvious and PKR was least among them; and these suggested that TLR9 of fish could activate the signaling cascade of IRF7 to induce the secretion of I-IFN, which could induce the expression of IFN- stimulated genes such as ISG15、Mx1、PKR and Viperin for anti-virus effect. The expression of IRF3 and IRF7 m RNA were upregulated in EPC cells transfected with p EGFP-N1-Cc TLR3 and p EGFP-N1-Cc TLR7, but had not significant differences with that in EPC cells transfected with p EGFP-N1(P?0.05). The results indicated that overexpression of a Cc TLR3 or Cc TLR7 gene in EPC cells could not upregulate the expression of IRF3 and IRF7. Unfortunately, we did not succeed to clone TLR22 gene from common carp.Section Ⅱ: Research on expression profiles of TLRs and immune-related genes of common carp infected with SVCV In the present study, a less lethal concentration(to achieve a lower rate of mortality) of SVCV was injected in common carp to have a better understanding of the response of innate immune genes very early in infection as well as during the recovery. Furthermore, the m RNA expression profiles of genes in individual fish encoding for proteins with various functions during the TLRs pathway, from pattern recognition receptors to antiviral genes, were examined. This includes(1) the toll-like receptors: TLR2, TLR3 and TLR7;(2) the interferon regulatory factors: IRF3 and IRF7;(3) the interferon genes: IFNa1, IFNa2 and IFNa1S;(4) the interferon-stimulated genes: ISG15, Mx1, Vig1, PKR, ADAR;(5)the pro-inflammatory cytokines genes: IL1β, IL6 and IL10. The main results of this thesis were as follows: Viral copies were found immediately upon infection from 3 hpi and were considerably increased at 6 and 12 hpi to reach maximum at 3 dpi. At 5 dpi, the viral copies were markedly reduced; but at 10 dpi, samples still had a high viral load. In all sampling groups, variation in viral copies between the fish was quite high.Observing viral copies in the infected fish, it can be noted that most of the sample groups contain one fish(33%) with high level of viral copies that is similar to mortality rate. Hence we can assume that the fish carrying high viral load succumb to the infection at a later stage of infection. To identify the potential role of immune-related genes in anti-virus immune responses in fish, we monitored the expression change of some genes in anterior kidney of common carp post-SVCV infection.(1) TLR2, TLR3 and TLR7 showed a significant upregulation of transcript level at 3 hpi and were increased to reach a maximum at 3 dpi, 12 hpi and 1 dpi, respectively; then gradually decreased, at 10 dpi, expression of these three TLRs were still at a low level upregulation.(2) IRF3 and IRF7 showed a significant upregulated of transcript level at 3 hpi and were increased to reach a maximum at 1 dpi and 3 dpi, respectively; then gradually decreased, but kept upregulation throughout the experimental period; the upregulation of IRF7 were more obvious than that of IRF3, and still at a high level upregulation at 10 dpi.(3) Among the interferons, IFNa1 and IFNa2 were significantly upregulated at 3 hpi and were increased to reach a maximum at 3 dpi, however, IFNa1 S was underexpressed or expressed at a very low level. IFNa1 and IFNa1 S became similar to the control at 5 dpi and 7 dpi, but IFNa2 kept upregulation with a high transcript level.(4) ADAR, ISG15, Mx1, PKR and Vig1 were all strongly upregulated at 12 hpi; ADAR, ISG15 and PKR were significantly increased to reach a maximum at 1 dpi, thereafter, expression was gradually reduced at 3 dpi, and expressed higher than control throughout the experimental period; Mx1 and Vig1 were expressed more strongly at 1 to 3 dpi and reach a maximum, then was reduced, but still comparatively quite high, at 5 dpi.(5)IL1β, IL6 and IL10 was initially expressed at a lower level at 3 and 6 hpi, but were upregulated at 12 hpi and 1 dpi; then IL6 and IL10 were reduced(lower than the control) on 5 dpi, but IL1β expressed higher than control throughout the experimental period. These results above showed that expression of all immune-related genes in this study were upregulated with viral detection and generally consistent with the amount of viral copies in the fish.In conclusion, virus becomes active in a very short period of time and increases at an exponential rate in common carp post SVCV infection. TLR2, TLR3 and TLR7 are immediately expressed at a relatively high level upon viral entry to initiate immune signal transduction, stimulating the expression of IRFs, I-IFNs and IL at an early stage of infection; and the IFN-induced genes such as ADAR, ISG15、Mx1、PKR and Vig1 were expressed strongly to play an anti-virus effect during the middle and late period of infection. These suggested that SVCV(rhabdoviruses) could upregulate its innate immune factors to counter the viral activities mainly mediated by I-IFNs in common carp, which was initiated by recognition of virus by carp TLR2, TLR3 and TLR7, and activated the innate immune responses through a IRFs/I-IFNs /ISGs signaling pathway.