在脊椎动物中viperin已被证明是一种能抵御大多数DNA和RNA病毒的抗病毒蛋白质.本文克隆及鉴定了青鱼viperin.青鱼viperin (bc Viperin) 的c DNA由1 828个核苷酸组成, 编码360个氨基酸.预测的bc Viperin蛋白包含N端低保守的两性α-螺旋结构域、中间自由基S-腺苷甲硫氨酸 (S-adenosylmethionine, SAM) 结构域及高保守C端结构域.bc Viperin m RNA在青鱼心脏、肝脏、脾脏、肾脏、肠、肌肉、皮肤和鳃等组织中均有表达.在草鱼呼肠孤病毒 (grass carp reovirus, GCRV) 和鲤春病毒血症病毒 (spring viremia of carp virus, SVCV) 感染后, bc Viperin m RNA在上述组织中的表达水平提高.在青鱼尾鳍 (Mylopharyngodon piceus fin, MPF) 细胞中, bc Viperin m RNA表达水平同样会在GCRV和SVCV感染后发生上升.蛋白质免疫印迹表明bc Viperin蛋白的相对分子质量约为42 k D;He La和EPC细胞中的免疫荧光染色实验表明bc Viperin是细胞质蛋白.编码bc Viperin的质粒在EPC细胞中过表达后能够显著提高细胞抵御SVCV和GCRV的抗病毒能力.以上研究表明bc Viperin是青鱼天然免疫过程中重要的抗病毒蛋白质.

在哺乳动物细胞系中转基因的效率相对较高而在鱼类细胞系中相对较低, 为了获得鱼类高效转基因细胞系, 我们从模式鱼类青鳉(Oryzias latipes)中分离和构建了肌肉细胞系OLM.该细胞系类似于纤维细胞, 在 28℃ DMEM和10％的胚胎牛血清中培养了超过88代.通过染色体分析, 它属于两倍体并含有48条染色体.在脂质体介导下, 报告基因的转染效率可以达到40％, 在测试的其他鱼类细胞中转染效率最高.此外还构建了稳定转染转基因或者非编码RNA的细胞系.OLM细胞对常见的病毒SVCV、GCRV、SGIV等不敏感.综上所述, 研究在青鳉中建立了一种能高效转染的肌肉细胞系, 它适用于鱼类瞬时和稳定的转基因研究.

糖蛋白(Glycoprotein,G)作为鲤春病毒血症病毒(Spring Virernia of Carp Virus,SVCV)主要的抗原蛋白,已成为现阶段SVCV病毒检测、抗体制备以及疫苗研制的热点.为了对其进行酵母表面展示,研究以SVCV-shlj1分离株基因组为模板,通过RT-PCR技术,体外扩增获得SVCV表面糖蛋白的基因开放阅读框(1530 bp)片段,将其克隆至酵母表面展示载体pYD1,构建重组质粒pYD1-G.利用电转化方法将重组质粒pYD1-G导入酿酒酵母EBY100感受态细胞,经YNB选择培养基筛选和菌液PCR的鉴定,挑选出阳性转化子(命名为EBY100-pYD1-G),对其进行2％半乳糖诱导.利用细胞免疫荧光和流式细胞仪检测G蛋白的酵母表面展示情况.细胞免疫荧光结果显示,诱导后的酵母细胞EBY100-pYD1-G能产生特异性红色荧光,且随着诱导时间的增加,红色荧光的酵母细胞所占比例不断增加,各组之间差异显著(P<0.05).流式细胞仪检测结果显示,酵母细胞的荧光强度与诱导时间呈正比,其中诱导48h与72h的酵母细胞荧光强度不存在显著差异,基本趋于稳定不变的状态.因此,选取诱导48h为酵母表面展示的最佳诱导时间.上述研究结果表明SVCV的G蛋白已经成功展示于酿酒酵母细胞表面,研究为鲤春病毒血症酵母口服疫苗的研发奠定了前期基础.

为进一步了解硬骨鱼类特有的finTRIM (Fish novel tripartite motif,ftr)在斑马鱼(Danio rerio)抗病毒天然免疫中的作用,研究克隆了斑马鱼ftr56基因并分析了其对鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)增殖的抑制作用.根据NCBI中斑马鱼ftr6序列设计引物,采用PCR方法,扩增ftr56 CDS区,连接至真核表达载体pcDNA4.0-His,构建真核表达质粒pcDNA4.0-ftr56-His,进行生物信息学分析.采用实时荧光定量PCR (qRT-PCR)技术检测SVCV感染斑马鱼胚胎成纤维细胞(ZF4)后ftr56 mRNA的变化.系统进化树分析显示,斑马鱼FTR56单独聚为一支.氨基酸多序列比对结果显示,其与黑猩猩、牛、鼠的TRIM56相似度为22％-23％.FTR56二级结构具有1个RING指结构域,1个B-box结构域,1个卷曲螺旋结构域和1个B30.0结构域.qRT-PCR检测结果显示,ftr56在SVCV感染后24h表达量显著上升.在过表达ftr56后,SVCV的G蛋白mRNA水平和蛋白水平在12h和24h相比对照组明显减少,培养基上清中SVCV滴度也明显降低,表明FTR56抑制SVCV的增殖.实验为进一步揭示finTRIM在鱼类病毒性疾病中的免疫调节机制提供参考.

鲤春病毒血症病毒(SVCV)是水生动物病毒中重要的病原体,常引起鲤科鱼类疾病暴发.近些年研究发现,维甲酸诱导基因I样受体家族(RLRs)信号通路在SVCV免疫过程中起到重要的作用.主要功能是在识别病原体相关模式,激活下游信号分子,诱导天然免疫的产生,以及控制病毒的早期复制.当病毒进入机体时会形成病毒-RLRs-IFN互联反馈回路,RLRs相关基因识别SVCV的RNA,最终引起Ⅰ型干扰素(IFN-I)表达量升高,并且RLRs族内成员相互作用增强抗病毒作用.RLRs不仅可以活化天然免疫信号通路,还可增强适应性免疫效应,在控制病毒感染过程中发挥重要作用.介绍RLRs家族,RLRs抗病毒信号调控因子,干扰素诱导的鱼类Mx (myxovirus resistant)蛋白对鲤春病毒血症病毒的抑制作用.

用CRISPR/Cas9技术敲除斑马鱼(Danio Rerio)的trim47,收集TRIM47–/–(trim47基因敲除)和WT(野生型)斑马鱼的大脑和脾脏进行RNA-seq分析,以鉴定差异表达基因(DEGs).总共确定了271个DEGs,经过简要分析,将这些DEGs注释为KEGG途径和GO富集分析,与WT组相比,TRIM47-/-组的脑中DEGs集中在细胞黏附和谷氨酸能突触信号传导途径中.在脾脏中,与野生型组相比,TRIM47-/-组的DEGs在补体和凝血级联信号通路中发生了变化.使用qRT-PCR验证了脑和脾中与补体途径相关的基因,与转录组数据一致.这些结果表明,Trim47在脾脏和大脑中起着重要的生物学作用,尤其是通过补体途径参与先天免疫功能.体内感染实验表明,trim47基因敲除可提高斑马鱼中鲤春病毒血症病毒(SVCV)的感染率.总之,这些发现为TRIM成员在先天免疫中的功能提供了新线索.

为了研究自噬相关基因ATG10(Autophagy related gene 10)在鱼类免疫应答中的功能,研究克隆了大黄鱼(Larimichthys crocea)ATG10基因(LcATG10)的cDNA序列,其开放阅读框全长747个核苷酸,编码248个氨基酸的蛋白,包含1个Autophagy_act_C结构域.系统进化分析显示,LcATG10和其他硬骨鱼类ATG10聚成一支,与金头鲷和石斑鱼ATG10亲缘关系最近.LcATG10在所检测的正常大黄鱼各组织中都有表达.在病毒类似物poly(I:C)刺激后,大黄鱼脾脏和头肾组织中LcATG10的表达水平显著上调,分别在12h和6h达到峰值(9.4和5.9倍).LcATG10在大黄鱼头肾细胞系(LYCK)、原代巨噬细胞、淋巴细胞和粒细胞中也均有表达,在原代粒细胞中的表达量相对较高;在poly(I:C)刺激后,大黄鱼原代头肾粒细胞和LYCK细胞中LcATG10的表达水平显著上调.过表达LcATG10的鲤上皮瘤(Epithelioma papulosum cyprinid,EPC)细胞受鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)感染48h后,细胞病变效应(Cytopathic effects,CPEs)明显低于对照组;细胞培养上清中SVCV病毒滴度为103.55 TCID50/mL,显著少于对照组;SVCV标志基因SVCV-G、SVCV-M和SVCV-P的表达水平也显著低于对照组,分别是对照组的0.022、0.015和0.022倍.这些研究结果表明LcATG10在大黄鱼抗病毒免疫应答中发挥作用,为深入研究自噬在大黄鱼抗病毒免疫中的分子机制奠定了基础.

为进一步研究程序性细胞死亡6互作蛋白Pdcd6ip(Programmed cell death 6-interacting protein)分子功能,探讨其表达对鲤春病毒血症病毒(Spring viraemia of carp virus,SVCV)增殖的影响,研究通过逆转录-聚合酶链式反应扩增得到两端带有Nhe I和EcoR I酶切位点的pdcd6ip编码片段,并将其克隆至真核表达载体pCI-neo上,构建了真核表达质粒pCI-pdcd6ip.获得的过表达质粒转染至EPC细胞后进行RT-qPCR和western blot检测Pdcd6ip的表达情况,并利用CCK-8法检测其对细胞增殖的影响.细胞转染后24h进行SVCV感染,检测Pdcd6ip过表达对SVCV增殖的影响.结果显示,Pdcd6ip蛋白真核重组质粒能够在EPC细胞中大量表达,转染后72h细胞活性较对照组无显著差异.同时,免疫荧光、RT-qPCR和western blot检测结果均显示,Pdcd6ip蛋白过表达后,SVCV M蛋白mRNA水平和蛋白表达水平较对照组显著下降,表明Pdcd6ip过表达显著抑制了SVCV的增殖.研究为抗SVCV药物的研发提供了新的研究基础和设计思路.

Since the anti-inflammatory effect of hydrogen has been widely known,it was supposed that hydrogen could suppress tissue damage by inhibiting virus-related inflammatory reactions.However,hydrogen is slightly soluble in water,which leads to poor effect of oral hydrogen-rich water therapy.In this study,the nano-bubble hydrogen water(nano-HW)(about 0.7 ppm)was prepared and its therapeutic effect against viral infection was investigated by utilizing spring viraemia of carp virus(SVCV)-infected zebrafish as model.Three-month-old zebrafish were divided into nano-HW treatment-treated group and aquaculture water treated group(control group).The results revealed that the cumulative mortality rate of SVCV-infected zebrafish was reduced by 40％after treatment with nano-bubble hydrogen water,and qRT-PCR results showed that SVCV replication was significantly inhibited.Histopathological examination staining showed that SVCV infection caused tissue damage was greatly alleviated after treatment with nano-bubble hydrogen water.Futhermore,SVCV infection caused reactive oxygen species(ROS)accumulation was significantly reduced upon nano-HW treatment.The level of proinflammatory cytokines IL-1 β,IL-8,and TNF-α was remarkably reduced in the nano-HW-treated group in vivo and in vitro.Taken together,our data demonstrated for the first time that nano-HW could inhibit the inflammatory response caused by viral infection in zebrafish,which suggests that nano-HW can be applied to antiviral research,and provides a novel therapeutic strategy for virus-caused inflammation related disease.

Spring viremia of carp virus(SVCV)is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range.To develop effective agents to control SVCV infection,we selected 16 naturally active small molecules to assess their anti-SVCV activity.Notably,dihydroartemisinin(DHA)(100 μmol/L)and(S,S)-(+)-tetrandrine(TET)(16 μmol/L)exhibited high antiviral effects in epithelioma papulosum cyprinid(EPC)cells,with inhibitory rates of 70.11％and 73.54％,respectively.The possible antiviral mechanisms were determined as follows:1.Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells,suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted;2.Although neither had an effect on viral adhesion,TET(but not DHA)interfered with SVCV entry into host cells(＞80％),suggesting that TET may have an antiviral function in early viral replication.For in vivo study,both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3％,significantly decreased viral load,and modulated the expression of antiviral-related genes,indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection.Overall,our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication,thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.