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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 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 O. niloticus (GIFT strain) 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 was kindly gifted 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 in 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 of the 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 a new light on the pathogenesis of TiLV infection and will pave a new way on the development of effective prevention and control strategy against the epidemic of TiLV in O. niloticus.
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    [33] Reda R M, Mahmoud R, Selim K M, et al. Effects of dietary acidifiers on growth, hematology, immune response and disease resistance of Nile tilapia, Oreochromis niloticus[J]. Fish & Shellfish Immunology, 2016, 50: 255-262.
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Cloning, expression, antibody preparation and tissue distribution of tilapia lake virus nucleoprotein

    Corresponding author: Lijuan ZHAO, 406856929@qq.com
    Corresponding author: Li LIN, linli@zhku.edu.cn
  • Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangzhou, Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Zhongkai University of Agriculture and Engineering, Guangdong    510225, China

Abstract: Oreochromis spp. are important cultured fish species worldwide. 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 O. niloticus (GIFT strain) 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 was kindly gifted 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 in 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 of the 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 a new light on the pathogenesis of TiLV infection and will pave a new way on the development of effective prevention and control strategy against the epidemic of TiLV in O. niloticus.

  • 罗非鱼(Oreochromis spp.)具有食性广、生长快、繁殖力和抗病力强等优点,是联合国粮农组织向全世界推广的重要养殖鱼类[1-2],是动物性蛋白质的重要来源,为发展中国家及全球粮食安全作出了重大贡献[3-6]。然而,近年来以色列、厄瓜多尔、埃及、泰国等国家先后暴发了罗湖病毒(tilapia lake virus,TiLV)病,导致大批野生和养殖罗非鱼死亡,造成严重的经济损失[5, 7-11]。罗湖病毒病又称为罗非鱼合胞体肝炎(syncytical hepatitis of tilapia),其主要症状包括嗜睡、厌食和离群表面游泳,鳃苍白、眼球突出、体色变黑和鳞片脱落[7-8]。TiLV感染最常见的病变包括脑炎和肝炎[12],肝脏组织坏死并出现合胞体[13]。TiLV主要危害罗非鱼仔稚鱼和幼鱼,幼体较成鱼更易感染TiLV[13-16]

    Bacharach等[3]对在以色列分离的Til-4-2011毒株进行了研究,发现TiLV为分节段单链负链RNA病毒,属于正黏病毒科(Orthomyxoviridae)的新成员。TiLV由10个基因组片段组成,总病毒基因组长度约为10 323 bp[3, 8, 17],其中基因组片段1编码的蛋白与C型流感病毒的聚合酶PB1具有弱同源性,其他9个片段编码蛋白还没有解读[17]

    TiLV已经对世界范围内的罗非鱼养殖带来巨大威胁,目前,该病毒已在3大洲的12个国家中被发现[7, 18-19]。TiLV被世界动物卫生组织(OIE)列为烈性传染病原[8, 14, 20],需加强对该病毒的检疫和防范。2017年我国罗非鱼产量达170多万t,是罗非鱼最大生产国[21-22]。目前我国大陆还没有关于TiLV的正式报道。尼罗罗非鱼吉富品系(Oreochromis niloticus,GIFT strain)是我国重要的罗非鱼养殖品种,但是其对TiLV的敏感性还未见报道。关于TiLV的研究主要集中在检测、基因组测定、诊断和组织病理学等方面[18, 23-25],对TiLV的感染目前尚无有效防控措施。本实验克隆并分析了罗湖病毒第6片段基因的cDNA序列。根据序列和进化树分析结果,认为该片段编码的蛋白和正黏病毒科其他成员的核蛋白(nucleoprotein, NP)一致。制备了TiLV-NP蛋白的多克隆抗体,采用苏木精-伊红染色(H.E)、蛋白质印迹(Western Blot)、免疫组化和实时荧光定量PCR(qRT-PCR)等多种技术对TiLV感染尼罗罗非鱼吉富品系进行研究,为防控罗湖病毒在我国传播具有重要意义。

1.   材料与方法
  • 实验动物和病毒 尼罗罗非鱼吉富品系幼鱼购自广东罗非鱼良种场。尼罗罗非鱼吉富品系幼鱼暂养于室内半径为0.4 m,深0.6 m的封闭循环水系统,温度维持在(25±1)°C。罗湖病毒(TiLV)由德国弗里德里希洛弗勒研究所Sven Bergmann博士赠送。新西兰大白兔(Oryctolagus cuniculus)购买于广东省医学实验动物中心。

    主要试剂 十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶试剂盒购自北京鼎国昌盛生物技术有限责任公司;RNAiso Plus、限制性内切酶均购自宝生物工程(大连)有限公司;质粒小量快速提取试剂盒购自北京艾德莱生物科技有限公司;大肠杆菌(Escherichia coli)Trans5α感受态细胞、BL21(DE3)感受态细胞购自北京全式金生物技术有限公司;ECL显色液、考马斯亮蓝R-250购自北京索莱宝科技有限公司;IPTG、溴酚蓝、氨苄、硝酸纤维素膜购自Biosharp公司;HiScript II qRT SuperMix反转录试剂盒购自南京诺唯赞生物科技有限公司;弗氏完全佐剂、弗氏不完全佐剂购自Sigma公司;分析纯试剂均购自国药集团化学试剂有限公司。脱脂奶粉购自美国BD公司;小鼠(Mus musculus)抗GST-tag一抗,HRP标记山羊(Capra aegagrus hircus)抗小鼠二抗购自北京博奥森生物技术有限公司;RIPA裂解液、TMB显色液、Bradford蛋白浓度测定试剂盒购自上海碧云天生物技术有限公司;通用型组织固定液购自武汉塞维尔生物科技有限公司。

  • 参照预实验数据,对每尾实验鱼腹腔注射200 μL TiLV(病毒浓度为1×106 细胞/mL),对照组注射等量灭菌的PBS缓冲液。感染一定时间后,采集肝脏、脾脏、头肾、体肾、脑和鳃组织,一部分样品用通用型组织固定液固定24 h,送谷歌生物科技有限公司进行石蜡切片和H.E染色。另一部样品置于装有1 mL Trizol的离心管中,随即匀浆。按照Trizol Reagent说明书提取样品总RNA,电泳检测总RNA的完整性,并用分光光度计检测其浓度,根据OD260/OD280判断RNA的纯度。将高质量的总RNA保存于−80 °C备用。取1 μg总RNA,经DNase消化去除DNA污染,并通过Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland)反转录合成cDNA。

  • 罗湖病毒第6片段基因组cDNA由病毒感染的肝脏组织提取的RNA进行克隆获得。通过Clone Manager软件对TiLV第6片段基因cDNA序列进行分析,确定其开放阅读框(ORF);采用DNAMAN 7.0软件对氨基酸序列进行分析,用在线软件BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行核苷酸和氨基酸序列相似性分析,使用在线软件Protparam(http://web.expasy.org/protparam/)在线预测氨基酸序列的理化特性;采用TMHMM法分析蛋白质的跨膜区(http://www.cbs.dtu.dk/services/TMHMM/);信号肽预测运用SignalP (http://www.cbs.dtu.dk/services/SignalP);采用MEGA 7.0中Neighbor-Joining(NJ)法构建系统进化树。

  • 将TiLV-NP基因克隆到pGEX-4T-1质粒上,内切酶分别是EcoR I和Xho I。将重组质粒转化到大肠杆菌BL21(DE3),菌株37 °C摇床培养3 h左右,当菌液浓度OD600 达到0.5~0.8时,使用终浓度为1 mmol/L的IPTG在37 °C条件下诱导6 h,收集菌体后用PBS缓冲液重悬,使用超声波细胞破碎仪破碎菌体,在10 000 r/min 4 °C条件下离心10 min,分离上清液和沉淀,随后进行聚丙烯酰胺凝胶电泳(12%,SDS-PAGE)检测。根据崔振波等[26]报道的方法对包涵体蛋白进行纯化与回收,首先使用20 mL buffer A(50 mmol/L Tris-HCl,5 mmol/L EDTA,pH 8.0)将包涵体沉淀充分混匀悬起,10 000 r/min 4 °C离心20 min,弃上清液,重复1次。加入20 mL buffer B(50 mmol/L Tris-HCl,5 mmol/L EDTA,2 mol脲,pH 8.0)将沉淀充分混匀悬起,10 000 r/min 4 °C离心20 min,弃上清液,重复1次。加入20 mL buffer C(0.1 mol Tris-HCl,10 mmol/L DTT,8 mol脲,pH 8.0)将沉淀充分混匀悬起,置于37 °C恒温摇床上以200 r/min振荡1 h。10 000 r/min 4 °C离心10 min,保留上清液,弃沉淀。最后将上清液装入透析袋中,置于50倍上清液体积的透析液(0.1 mol Tris-HCl,5 mmol/L EDTA,5 mmol/L Cysteins,pH8.0)中,4 °C透析16 h。换用新透析液4 °C透析16 h,10 000 r/min 4 °C离心10 min,保留上清液。将上清液通过0.22 µm滤膜除菌后分装成1 mL冻存于−80 °C备用。将纯化后的蛋白用PBS缓冲液按比例稀释,加入5×loading buffer沸水煮沸15 min,12% SDS-PAGE上样,电泳结束后进行转膜,封闭液 (含有5%脱脂奶粉的TBST溶液) 室温封闭3 h;小鼠抗GST一抗 (1∶1 000) 室温孵育2 h;TBST缓冲液洗涤5次,每次5 min;HRP标记山羊抗小鼠二抗IgG (1∶10 000)孵育45 min后,TBST缓冲液洗涤5次,每次5 min;ECL显色1 min,ChemiScope 6000检测蛋白条带。

  • 将纯化的重组NP蛋白免疫新西兰大白兔。免疫前,提取少量免疫前的血清作为空白对照血清。每次免疫蛋白剂量为0.5 mg,第一次免疫为蛋白抗原与等体积弗氏完全佐剂混匀乳化,第二次免疫和第三次免疫为蛋白抗原与等体积弗氏不完全佐剂混匀乳化。采用皮下多点注射法对新西兰大白兔注射免疫原。免疫后第44天心脏取血,收集血清,分装后−80 °C保存。采用酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)检测TiLV-NP多克隆抗体的效价。取感染TiLV患病尼罗罗非鱼肝脏组织0.1 g剪成细小的碎片,加入1 mL RIPA裂解液(包括1 mmol/L PMSF,10 mmol/L氟化钠,1 mmol/L原矾酸钠),用玻璃研磨棒于冰上研磨,直至充分裂解,4 °C下12 000 r/min离心20 min,取上清液。用Bradford蛋白浓度测定试剂盒测定其浓度,上样总蛋白量为30 μg。后续SDS-PAGE电泳、转膜、封闭、抗体孵育、显色等实验步骤按上述“TiLV-NP重组质粒构建、融合蛋白表达及其检测”描述的方法,检测多克隆抗体的特异性。

  • 随机挑选健康的体质量为(30±5) g尼罗罗非鱼幼鱼分为2组,每组30尾,感染组每尾实验鱼腹腔注射200 μLTiLV(病毒浓度1×106 细胞/mL),对照组注射等量灭菌的PBS缓冲液。每日观察实验尼罗罗非鱼发病情况。感染在封闭循环水系统内进行,对于实验用水进行严格消毒杀灭病毒后才对外排放。采集病鱼肝脏、脾脏、头肾、体肾、鳃、脑等组织,用TRizol法提取总RNA。按照HiScript II qRT SuperMix反转录试剂盒说明书对RNA进行反转录获得cDNA。PCR引物根据Tattiyapong等[27],Nested ext-2 (5′-TTGCTCTGAGCAAGAGTACC-3′);Nested ext-1(5′-TATGCAGTACTTTCCCTG-CC-3′),扩增TiLV-S3的片段(491 bp)。PCR反应条件:95 °C预变性2 min;95 °C变性30 s,56 °C退火30 s,72 °C延伸0 s,35个循环;72 °C终延伸7 min,16 °C中保持30 min。1.5%琼脂糖胶中电泳检测扩增的DNA片段。

  • 取3尾人工感染发病尼罗罗非鱼的肝脏、脾脏、头肾、体肾、鳃和脑组织各0.1 g,剪成碎片后混合,加入1 mL RIPA裂解液,用玻璃研磨棒于冰上研磨,直至充分裂解,4 °C 12 000 r/min离心20 min,取上清液。用Bradford蛋白浓度测定试剂盒测定蛋白浓度,上样量为30 μg,以β-actin作为内参蛋白。后续SDS-PAGE电泳、转膜、封闭、抗体孵育、显色等实验步骤按“TiLV-NP重组质粒构建、融合蛋白表达及其检测”描述方法,检测NP蛋白在罗非鱼组织的表达。

    将获得的切片进行免疫组化分析。首先将切片依次放入二甲苯Ⅰ20 min,二甲苯Ⅱ 20 min,无水乙醇Ⅰ 10 min,无水乙醇Ⅱ 10 min,95%酒精5 min,90%酒精5 min,80%酒精5 min,70%酒精5 min,蒸馏水清洗2次,每次10 min;随后将组织切片置于盛满EDTA抗原修复缓冲溶液(pH 9.0)的修复盒中于微波炉内进行抗原修复。中低火至沸腾10 min。自然冷却后将玻片置于PBS缓冲液 (pH7.4) 中在脱色摇床上晃动洗涤3次,每次5 min;切片放入3%过氧化氢溶液,室温避光孵育20 min,将玻片置于PBS缓冲液 (pH7.4) 中,在脱色摇床上晃动洗涤3次,每次5 min;向切片滴加用5% NGS稀释好的一抗 (1∶200) 覆盖组织。切片平放于湿盒内4 °C孵育过夜后,置于TBST缓冲液 (pH7.4) 中在脱色摇床上晃动洗涤3次,每次5 min;切片稍甩干后滴加HRP偶联二抗 (山羊抗兔) 覆盖组织,室温孵育50 min,置于TBST (pH7.4) 中在脱色摇床上晃动洗涤3次,每次5 min;切片稍甩干后滴加新鲜配制的DAB显色液,显微镜下控制显色时间,阳性为棕黄色,自来水冲洗切片终止显色;Harris苏木素复染3 min左右,自来水冲洗,1%的盐酸酒精分化数秒,将切片浸入双蒸水中,清洗2次,每次5 min,终止反应。等切片干燥后,用显微镜 (ECLIPSE E100, 尼康) 拍照。

  • 随机选取健康的尼罗罗非鱼分成实验组与对照组各30尾,分别暂养在2个水族箱中。水温为25 °C,持续充氧。暂养1周后,实验组罗非鱼腹腔注射罗湖病毒溶液200 μL TiLV(病毒浓度1×106 细胞/mL),对照组腹腔注射等体积的PBS缓冲液。分别在感染后0、6、24和48 h各随机取3尾实验鱼的脾脏和头肾,分别置于装有1 mL Trizol的1. 5 mL离心管中,随即匀浆,按照上述方法进行总RNA提取、检测纯度并进行反转录,−80 °C保存备用。肿瘤坏死因子(TNF-α)、转化生长因子(TGF-β)基因和β-actin基因引物序列见表1,其中β-actin基因作为内参。实时荧光定量PCR (qRT-PCR) 反应使用AceQ qPCR SYBR® Green Master Mix(Vazyme公司,南京)试剂,反应于Roche LightCycler 480 (Roche, 美国)上进行。每个样品3次重复。试验数据采用2−ΔΔCT 法计算,数据表示方法为平均值±标准差,使用GraphPad Prism 7软件分析统计分析,P<0.05表示差异显著,P<0.01表示差异极显著。

    引物名称
    primer
    序列(5′-3′)
    sequence from 5′ to 3′
    应用
    application
    TNF-α-F CTTCCCATAGACTCTGAGTAGCG qRT-PCR
    TNF-α-R GAGGCCAACAAAATCATCATCCC
    TGF-β-F TGCGGCACCCAATCACACAAC qRT-PCR
    TGF-β-R GTTAGCATAGTAACCCGTTGGC
    β-actin-F GCTACTCCTTCACCACCACAG qRT-PCR
    β-actin-R CGTCAGGCAGCTCGTAACTC

    Table 1.  Primers used in the experiments

2.   结果
  • 罗湖病毒第6片段基因组cDNA全长1 044 bp,ORF为954 bp,编码317个氨基酸(aa);5′非编码区(NCR)为18 bp,3′非编码区(NCR)为72 bp。Protparam在线软件预测ORF编码的蛋白分子量为36.38 ku,等电点为8.86,是亲水酸性蛋白,无跨膜结构和信号肽(图1)。利用MEGA 7.0软件NJ法对该基因和其他5个正黏病毒科病毒的核蛋白基因进行系统进化分析,结果显示,该蛋白与传染性鲑贫血病毒的NP蛋白基因最为接近(图2),可以认定罗湖病毒第6片段基因组编码蛋白为罗湖病毒的NP蛋白。

    Figure 1.  The complete nucleotide sequences of the sixth genomic segment of TiLV and the deduced amino acid sequences of the encoding protein

    Figure 2.  Phylogenetic tree of TiLV-NP and nucleoprotein from other orthomyxoviruses

  • 通过双酶切,电泳可清晰观察到2个条带,且大小与理论片段大小相一致(图3-a)。SDS-PAGE蛋白胶分析显示,重组的NP蛋白主要表达在包涵体中(图3-b),通过GST-tag标签抗体检测表达的重组蛋白,结果显示,重组的GST-NP蛋白在62 ku左右有一条明显的条带,且大小和重组NP蛋白预测一致(图3-c)。

    Figure 3.  Cloning and expression of NP gene

  • 对获得的GST-NP多克隆抗体血清进行酶联免疫吸附测定法进行效价测定(图4-a),结果显示,多克隆抗体的效价达1∶51 200。为了进一步验证多克隆抗体对NP蛋白的特异性,实验通过Western Blot检测感染TiLV的尼罗罗非鱼肝脏组织,结果显示,在36 ku左右处可以观察到一条清晰的条带,大小和预测的TiLV-NP相一致(图4-b),表明制备的抗体可以特异性识别病毒表达的NP蛋白。

    Figure 4.  The measure of the titer of GST-NP polyclonal antibody and the detection of NP in viral infected tissue.

  • 病毒感染的尼罗罗非鱼表现出食欲不振、嗜睡、行为异常、游动迟缓、鱼鳞脱落、体色变黑、鳍糜烂、贫血以及晶状体白内障。感染晚期,眼球肿胀,偶尔穿孔,晶状体破裂,内容物凝固(图5-a)。对肝脏组织总RNA进行qRT-PCR检测,结果显示,对照组未出现特异条带,而感染组在491 bp处有一特异性条带,大小和预测一致(图5-b)。

    Figure 5.  The symptoms of O. niloticus artificially infected with TiLV and the detection of viral nuclei acid

  • 组织病理H.E染色结果显示,TilV感染可引起尼罗罗非鱼不同组织的病变:在肝脏中出现肝细胞解离和空泡,部分细胞出现合胞体(图版Ⅰ -2);脾脏中的部分细胞出现空泡、坏死以及含铁血黄素增多(图版Ⅰ -4);在头肾中,部分细胞坏死(图版Ⅰ -6);在体肾中出现细胞坏死(图版Ⅰ -8);鳃丝上皮细胞发生明显解离和脱落,鳃小片黏连以及空泡(图版Ⅰ -10);脑组织细胞肿大,轻微出血,细胞坏死(图版Ⅰ -12)。

    Figure 图版Ⅰ.  H.E staining of the tissues from O. niloticus

  • 通过Western Blot和免疫组织化学方法检测NP蛋白在罗非鱼组织中的分布情况。对尼罗罗非鱼肝脏、脾脏、头肾、体肾、鳃和脑等6个组织的蛋白进行Western Blot,结果显示,在肝脏、头肾、体肾和脑中出现较明显的阳性信号,其中肝脏中阳性信号最明显,脾脏和鳃中信号较弱(图6)。免疫组化结果显示,分别在肝脏(图版Ⅱ-2)、脾脏(图版Ⅱ-4)、头肾(图版Ⅱ-6)、体肾(图版Ⅱ-8)、鳃(图版Ⅱ-10)及脑(图版Ⅱ-12)中检测到不同程度的阳性信号。免疫组织化结果与Western Blot结果基本一致。

    Figure 6.  Expression of NP protein in the tissues of TiLV infected Oreochromis niloticus.

    Figure 图版Ⅱ.  Immunohistochemical assay the expression of NP protein in the tissues from TiLV infected O. niloticus

  • 为了解病毒感染和宿主的免疫应答,实验集中研究病毒感染尼罗罗非鱼0、6、24和48 h后脾脏和头肾2种主要免疫组织,qRT-PCR检测2种重要免疫因子TNF-αTGF-β的表达。在脾脏和头肾组织中的TNF-α检测结果显示,除了在头肾6 h组外,病毒感染的尼罗罗非鱼脾脏和头肾组织中的表达都显著低于对照组(图7-ab);TGF-β在脾脏和头肾组织中的检测结果显示,在病毒感染尼罗罗非鱼6和24 h后,TGF-β在毒感染的尼罗罗非鱼脾脏和头肾组织中的表达都显著低于对照组,但在病毒感染罗非鱼48 h后,TGF-β在病毒感染和对照组罗非鱼脾脏和头肾中表达无显著差异(图7-cd)。研究表明,在感染早期,病毒可以显著抑制宿主这2种免疫因子的表达。

    Figure 7.  Expression of TNF-α and TGF-β in the spleen and the head kidney of O. niloticus during the infection of TiLV

3.   讨论
  • 相对于其他养殖鱼类而言,罗非鱼具有较强的疾病抵抗力。然而最近在野生罗非鱼和养殖罗非鱼群体中均暴发由TiLV感染的病毒病,已对世界罗非鱼的健康养殖造成了巨大威胁[27]。该病毒病显著的特征是高死亡率和发病率,目前尚无有效防控TiLV感染措施。尽管我国大陆还没有TiLV的正式报道,鉴于尼罗罗非鱼吉富品系是我国重要的罗非鱼养殖品种,开展对TiLV的感染特性的研究也具有重要意义。本研究从人工感染TiLV的尼罗罗非鱼吉富品系肝脏组织中克隆和测定了TiLV第6片段基因组,序列分析结果表明该片段编码NP蛋白。NP蛋白参与病毒生活周期的多个阶段,影响病毒的转录和复制等功能[28]。相关研究表明,各型流感病毒NP存在一个保守的抗原决定簇。针对这抗原决定簇制备的抗体可抑制病毒的转录,表明NP蛋白在RNA的转录过程中起着关键作用[29]。然而,关于NP蛋白在TiLV中的研究目前还未见报道。本研究首先制备了NP蛋白多克隆特异性抗体,开展了病毒感染的组织病理和免疫因子的研究,结果发现,尼罗罗非鱼吉富品系对TiLV敏感,病毒感染的罗非鱼表现出食欲不振、嗜睡、鱼鳞脱落、体色变黑、晶状体不透明、肝脏和肾脏出血等,结果与其他报道一致[3, 8]

    通过Western Blot检测,TiLV-NP在肝脏、头肾、体肾和脑组织中均有表达,其中肝脏组织的含量最多,与TiLV的临床症状即肝脏和肾脏出血相符,也与前人报道的结果一致[3, 8, 15-16, 30]。已有研究表明,肝脏和脑是病毒主要的靶器官,感染的肝细胞脱离并已失去其多面体形状[31],肝细胞坏死和淋巴细胞浸润[32]。通过人工感染试验发现,肝脏、脑、头肾和体肾组织发生广泛的组织病理变化,包括肝脏合胞体形成,头肾细胞坏死等。利用免疫组化检测TiLV感染的尼罗罗非鱼组织,发现在肝脏、脾脏、头肾、体肾、鳃和脑中均存在TiLV-NP阳性信号,TiLV-NP在肝脏、体肾、头肾和脑中分布较多,而在鳃和脾脏中分布较少。Dong等[14]采用地高辛标记的DNA探针原位杂交法检测TiLV基因组的第3片段基因在组织中的分布,结果显示,肝脏、肾脏、脑、鳃、脾脏和肌肉结缔组织有阳性信号,且肝脏、肾脏和鳃中信号较强。而本实验使用NP蛋白的抗体,在蛋白水平检测NP蛋白的表达,结果与Dong等[14]人的研究结果略有差异。这有可能是检测方法不同导致结果的差异,此外毒株、采样时间点和鱼种差异等因素也会导致研究结果出现差异。

    TNF-α可激发免疫细胞、促进呼吸暴发和一氧化氮的产生[33]。TGF-β在免疫细胞功能调节,细胞增殖分化和迁移,血管生成、组织重塑和修复中发挥重要调控作用[34-35]。这些细胞因子在病毒感染宿主过程中对促进细胞吞噬、细胞凋亡、淋巴细胞活化和白细胞迁移过程中发挥重要作用[36]。为了解罗非鱼对病原体TiLV的免疫反应,本实验测定主要免疫因子TNF-αTGF-β在脾脏和头肾中的表达。结果发现,在感染早期(12~24 h),TiLV病毒可以显著抑制TNF-αTGF-β在脾脏和头肾中的表达。也许病毒通过抑制宿主这些免疫因子来促进自身在感染早期的复制。TiLV对罗非鱼的免疫因子表达调控的分子机制还需进一步研究。

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