Pathogenesis of Singapore grouper iridovirus (SGIV) and its immune control strategies
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摘要:
虹彩病毒(Iridovirus)是目前海水和淡水养殖鱼类最严重的病毒性病原之一，已从100多种鱼类中分离鉴定出该病毒。石斑鱼虹彩病毒(Singapore grouper iridovirus, SGIV)是从新加坡养殖的患病石斑鱼中分离鉴定的高致病性虹彩病毒，是虹彩病毒科Iridoviridae蛙病毒属Iridovirus 1种新的病毒。本文从以下几个方面对SGIV的研究进行综述：SGIV的形态、超微结构及其在石斑鱼细胞中的复制和装配过程；SGIV病毒基因组、转录组、囊膜蛋白质组及miRNAs的解析；SGIV感染宿主靶标组织的鉴定；病毒侵染的入侵方式、运动轨迹和胞内运输的实时追踪；SGIV感染宿主引起类凋亡的死亡机制及介导的信号通路的揭示；多种宿主免疫抗病基因对病毒感染的调节作用；多种SGIV的检测技术，包括基于抗体的流式细胞技术、微流控芯片检测技术、环介导等温扩增技术及核酸适配体检测方法等；SGIV的灭活疫苗、亚单位疫苗和DNA疫苗的研制等。以期为深入阐明SGIV感染致病机理奠定坚实的理论基础，为发展抗病毒对策提供技术支撑。
Abstract:Iridovirus is one of the most serious viral pathogens in marine and freshwater cultured fish. Up to now, iridoviruses have been isolated and identified from more than 100 fish species worldwide. Singapore grouper iridovirus (SGIV), a novel species of Ranavirus, was isolated from diseased grouper in Singapore. On the basis of establishing a virus-sensitive cell infection model, morphology, ultrastructure, replication and biochemical characterization of SGIV in grouper host cells were studied by electron microscopy and biochemical analysis. The molecular biological characterizations of SGIV, including viral genome, transcriptome, envelope proteome and viral microRNAs, were systematically analyzed by Omics analysis. The interactions between SGIV and host were investigated from many aspects, including identifying the target tissues of SGIV infection, tracking the single virus entry and transport, non-apoptosis cell death induced by SGIV infection, functions of host immune related genes in virus infection. Meanwhile, a variety of SGIV detection technologies have been developed, including antibody-based flow cytometry, microfluidic chip detection technology platform system, loop-mediated isothermal amplification (LAMP) and nucleic acid aptamer detection method. In addition, SGIV inactivated vaccine, subunit vaccine and DNA vaccine were developed. The results provide a theoretical basis for better understanding of the pathogenic mechanism of SGIV infection, and offer technical supports for the prevention and control of SGIV.
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真菌引起的植物病害对农业生产和食品保藏造成了严重的危害，如香蕉炭疽病使香蕉果实变黑、腐烂[1]，小麦赤霉病引起小麦植株出现苗枯、茎腐、秆腐和穗腐等症状[2]。化学农药是目前防治植物真菌病害的主要手段，但也造成病原菌抗药性增强、农药残留等问题[3]。微生物天然产物因具有易降解、毒性低等特点，是化学农药替代品的重点研究对象。已知的昆虫种类达90万种，预测可能有200~3 000万种[4]，而几乎所有昆虫都有共生菌。昆虫共生菌分布于昆虫体内，大部分是酵母类、真菌类和细菌类共生菌[5]。相对于植物共生菌，目前对昆虫共生菌代谢产物的研究远远不足[6]。小菜蛾Plutella xylostella属鳞翅目菜蛾科，1年可传17代，在我国南方尤为泛滥，主要危害甘蓝、青花菜、白菜、油菜、萝卜等十字花科植物[7]。小菜蛾以蔬菜为食，因此会接触到植物病原菌，其共生菌对植物病原菌可能有选择性抑制作用。在前期对小菜蛾共生真菌的研究中，发现分离自肠道的棒曲霉Aspergillus clavatus XCE02菌株在大米固体培养基上培养时，其代谢产物乙醇提取物为 1 mg/mL时对植物病原菌小麦赤霉菌Fusarium graminearum和香蕉炭疽菌Colletotrichum musae显示了较好的抗菌活性，抑菌圈直径分别约为15.61和14.35 mm。本文研究棒曲霉菌株XCE02代谢产物的分离、鉴定及抗菌活性，以期寻找到相应的抗菌先导化合物。
1. 材料与方法
1.1 材料
AV 600核磁共振波谱仪(瑞士BrukerBiospin AG)；MDS SCIEX APCI 2000液质联用仪；Orbitrap高分辨质谱仪(德国Thermo Fisher Scientific Inc.)；SEPA-300旋光仪；GF254和柱层析硅胶为青岛海洋化工厂生产；φ为99%的多菌灵购于上海迈瑞尔化学技术有限公司；试验所用其他试剂均为分析纯。
棒曲霉XCE02分离自小菜蛾肠道[8]。植物病原菌小麦赤霉菌和香蕉炭疽菌引种自华南农业大学农学院，以上菌株均保藏于华南农业大学材料与能源学院。
1.2 菌种鉴定及发酵
将XCE02菌株划线接种于马铃薯蔗糖琼脂(PSA)培养基，26 ℃恒温黑暗条件下培养10 d后，观察其生长情况和菌落形态，并进行初步鉴定。
参考文献[9]的方法PCR扩增真菌核糖体ITS基因区段鉴定真菌。用CTAB法提取菌株总DNA[10]，用rDNA-ITS序列通用引物ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)与ITS4(5′-TCCTCCGCTTATTGATATGC-3′)[11]进行PCR反应。反应体系与文献[12]相同，具体为：Taq Plus PCR MasterMix [天根生化(北京)科技有限公司] 25 μL，上游和下游引物各2 μL，模板DNA(10 ng) 1 μL，dd H2O 20 μL，总体积50 μL。反应程序为：95 ℃预变性5 min；95 ℃变性30 s，55 ℃退火30 s，72 ℃延伸35 s，共30个循环；72 ℃延伸10 min。PCR产物送至广州天一辉远基因科技有限公司进行测序，已测序的核苷酸序列在NCBI网站进行BLAST相似性比对。
发酵培养：在1 000 mL锥形瓶中装入100 g籼米、100 mL H2O，于121 ℃高温高压灭菌30 min后接种菌株XCE02，28 ℃条件下静置培养30 d，共培养100瓶。
1.3 发酵产物的提取与分离
发酵产物用95%(φ)乙醇溶液浸泡3次，减压浓缩除去水后用乙酸乙酯萃取3次，减压浓缩，经硅胶柱层析，以石油醚−乙酸乙酯(体积比为100∶0~0∶100)溶液、乙酸乙酯−甲醇(体积比为100∶0~80∶20)溶液系统梯度洗脱，得到10种化合物。
1.4 结构鉴定
通过分析化合物的氢谱(1H NMR)、碳谱(13C NMR)、电喷雾质谱(ESIMS)、电喷雾高分辨质谱(HRESIMS)等试验数据与文献或与标准品对照等方法，鉴定10种化合物的结构。
1.5 抗菌活性试验及数据处理
采用滤纸片扩散法[12-13]测试结构较为新颖的化合物对香蕉炭疽菌、小麦赤霉菌的抗菌活性，28 ℃条件下香蕉炭疽菌和小麦赤霉菌分别培养48和24 h，以多菌灵和溶解样品的溶剂(φ为5%的二甲基亚砜，DMSO)分别作为阳性对照和空白对照。鉴于所得样品数量较少，配制的溶液浓度较低，多菌灵和待测样品均为250 μg/mL，用十字交叉法量取抑菌圈直径，每种样品，同一植物病原菌在每个培养皿上测试2组抑菌圈直径，重复3次，共得到6组抑菌圈数据，最终结果以6组抑菌圈数据的平均值±标准误表示。用SPSS 17.0统计软件进行单因素方差(One-way ANOVA)和相关性分析。
2. 结果与分析
2.1 菌株鉴定
菌株在PSA培养基上培养10 d后菌落表面呈蓝灰绿色，基层有较多分生孢子。菌落边缘有一薄层白色菌丝紧贴基质生长，背面暗黄色。孢子穗圆筒形，小梗单层，孢子梗宽为7.5~15.0 μm；顶囊棍棒形；分生孢子椭圆形，(2.5~3.5) μm × (2.8~3.9) μm，表面光滑，壁稍厚序列，符合曲霉属棒曲霉特征[14]。进一步用PCR扩增真菌核糖体ITS序列的方法验证菌株的种属，DNA测序结果与NCBI BLAST数据库中编号为KY765893.1的棒曲霉序列相似性为100%，与编号为AY373847.1、NR121482.1、KF669481.1的棒曲霉序列相似性均为99%。此结果与形态学鉴定结果吻合，表明该菌株属于棒曲霉，该菌株的GenBank登录号为MK828108.1。
2.2 化合物的鉴定及波谱数据
分离鉴定了10种化合物，在V(石油醚)∶V(乙酸乙酯) =85∶15时得到化合物1 (5 mg)；在V(石油醚)∶V(乙酸乙酯)=70∶30时得到化合物2 (2 mg)，在V(石油醚)∶V(乙酸乙酯)=60∶40时得到化合物3(3 mg)，在V(石油醚)∶V(乙酸乙酯) =30∶70得到化合物4 (3.5 mg)和5 (2.5 mg)；在V(石油醚)∶V(乙酸乙酯)=75∶25)时得到化合物6 (10 mg)；在V(乙酸乙酯)∶ V(甲醇)=90∶10时分离得到化合物7 (18 mg)；在V(石油醚)∶V(乙酸乙酯) =65∶35时得到化合物8和9的粗品，再经重结晶得到化合物8 (12 mg)和9 (4 mg)；在V(石油醚)∶V(乙酸乙酯)=45∶55时得到化合物10 (7 mg)。
鉴定化合物1~10分别为gliomasolide A、Sch725674、gliomasolide C、clavatustide A、clavatustide B、20−羟基麦角甾−4,6,8(14),22−四烯−3−酮、黄嘌呤、麦角甾醇、过氧化麦角甾醇和丁二酸。其中化合物1~7的波谱数据如下。
化合物1：无色晶体[V(甲醇)∶V(三氯甲烷)=1∶1]，熔点 89~91 ℃;
$\left[ {\rm{\alpha}} \right]_{\rm{D}}^{25}$ =+30.5 (c 0.15，MeOH)，HRESIMS(m/z): 313.238 4 [M+H]+。1H NMR (600 MHz, CD3OD) δ 6.82 (dd, 15.8, 7.8 Hz, 1H), 5.97 (dd, 15.8, 1.0 Hz, 1H), 4.99 (m, 1H), 4.32 (ddd, 12.0, 7.8, 5.0 Hz, 1H), 3.65 (m, 1H), 1.87 (m, 1H), 1.75 (m, 2H), 1.63 (m, 1H), 1.57 (m, 1H), 1.50 (m, 1H), 1.32~1.43 (m, 13H), 1.23 (m, 2H), 1.14 (m, 1H), 0.90 (t, 7.2 Hz, 3H)。13C NMR (150 MHz, CD3COCD3) δ 168.0, 151.2, 123.3, 77.3, 72.7, 70.9, 36.1, 36.0, 34.3, 32.8, 32.4, 31.3, 29.7, 26.4, 25.9, 24.9, 23.6, 14.3。化合物2：无色晶体。
$\left[ {\rm{\alpha}} \right]_{\rm{D}}^{25}$ =+25.7 (c 0.5，MeOH),HRESIMS(m/z): 329. 233 0[M+H]+。1H NMR (600 MHz, CD3OD) δ 6.87 (dd, 15.8, 6.0, 1H), 6.08 (dd, 15.8, 1.6, 1H), 4.95 (dddd, 9.8, 7.5, 5.0, 2.2, 1H), 4.49 (ddd, 6.0, 3.0, 1.6, 1H), 3.99 (q, 6.5, 1H), 3.85 (ddd, 6.0, 4.7, 3.0, 1H), 1.83 (ddd, 14.7, 6.5, 6.0, 1H), 1.71 (m, 1H), 1.65 (m, 1H), 1.61(m, 1H), 1.58 (m, 1H), 1.54 (m, 1H), 1.30~1.45(m, 11H), 1.19 (m, 2H), 1.16 (m, 1H), 0.90 (t, 6.8, 3H)。13C NMR (150 MHz, CD3OD) δ 168.6, 149.5, 123.3, 77.8, 76.1, 72.9, 69.6, 38.4, 36.9, 36.6, 34.2, 32.9, 29.7, 27.1, 26.5, 25.9, 23.9, 14.6。化合物3：黄色固体。
$\left[ {\rm{\alpha}} \right]_{\rm{D}}^{25}$ =－38.0(c 0.15,MeOH), HRESIMS(m/z): 343.212 4[M−H]－。1H NMR (600 MHz, CD3OD) δ 6.95 (dd, 15.8, 4.8, 1H), 6.18 (dd, 16.0, 1.5, 1H), 5.02 (m, 1H), 4.60 (br. s, 1H), 4.10 (ddd, 7.6, 5.4, 1.5, 1H), 4.00 (dd, 3.5, 3.0, 1H), 3.37 (br s, 1H), 1.77 (m, 1H), 1.68 (m, 1H), 1.61 (m, 1H), 1.55 (m, 1H), 1.33~1.49 (m, 11H), 1.13 (m, 2H), 1.10 (m, 1H), 0.92 (t, 7.2, 3H)。13C NMR (150 MHz, CD3OD) δ 168.5, 147.6, 123.3, 77.9, 77.4, 73.7, 71.2, 71.3, 36.3, 34.7, 32.9, 32.8, 30.4, 27.8, 26.5, 26.4, 23.7, 14.6。化合物4：黄色固体。
$\left[ {\rm{\alpha}} \right]_{\rm{D}}^{25}$ = +22.6(c 0.10, MeOH),ESIMS(m/z): 472.2 [M+H]+, HRESIMS 472.187 2 [M + H]+。1H NMR (600 MHz, CDCl3) δ 10.22 (s, 1H), 8.55 (d, 8.4 Hz, 1H), 8.05 (s, 1H), 7.64 (d, 7.8 Hz, 1H), 7.58 (d, 7.8 Hz, 1H), 7.52 (t, 7.8 Hz, 1H), 7.45 (t, 7.8 Hz, 1H), 7.35~7.38(m, 5H), 7.30(m, 1H), 7.21 (d, 7.8 Hz, 1H), 7.09 (t, 7.8 Hz, 1H), 5.51 (dd, 8.4, 6.6 Hz, 1H), 5.26 (d, 15.0 Hz, 1H), 3.94(m, 1H), 3.43 (dd, 13.8, 8.4 Hz, 1H), 3.29 (d, 1H, 15.0 Hz), 3.27 (m, 1H), 3.25 (m, 1H), 1.02 (t, 7.2 Hz, 3H)。13C NMR (150 MHz, CDCl3) δ 169.6, 167.9, 167.4, 167.4, 138.1, 135.7, 134.5, 132.6, 132.3, 129.7, 129.8, 128.8, 127.8, 127.4, 126.7, 126.3, 125.9, 123.2, 123.1, 122.0, 71.7, 51.4, 43.4, 37.7, 13.9。化合物5：黄色无定形固体。HRESIMS(m/z): 458.172 8 [M+H]+。1H NMR (600 MHz, CDCl3) δ 10.15 (s, 1H), 8.56(s, 1H), 8.50 (d, 8.4 Hz, 1H), 7.64 (d, 7.8 Hz, 1H), 7.56 (d, 7.8 Hz, 1H), 7.44 (t, 7.8 Hz, 1H), 7.41 (t, 7.8 Hz, 1H), 7.32~7.35(m, 5H), 7.31(m, 1H), 7.20 (d, 7.8 Hz, 1H), 7.05 (s, 1H), 5.53 (t, 7.6 Hz, 1H), 5.27 (d, 14.8 Hz, 1H), 3.42 (dd, 13.6, 7.6 Hz, 1H), 3.27 (dd, 13.6, 7.6 Hz, 1H), 3.12 (d, 14.8 Hz, 1H), 3.03 (s, 3H)。13C NMR (150 MHz, CDCl3) δ 169.9, 167.6, 167.5, 167.4, 138.2, 135.6, 135.0, 132.8, 132.4, 129.9, 129.8, 128.9, 127.5, 127.4, 126.7, 126.6, 126.3, 123.4, 123.3, 122.1, 71.7, 54.4, 37.4, 35.9。
化合物6：黄色油状物。HRESIMS(m/z): 409.309 7 [M+H]+。
$\left[ {\rm{\alpha}} \right]_{\rm{D}}^{25}$ = +115.6(c 0.15, MeOH),1H NMR (600 MHz, CDCl3) 6.63 (d, 9.5 Hz, 1H), 6.05 (d, 9.5 Hz, 1H), 5.75 (s, 1H), 5.58 (m, 1H), 5.57 (m, 1H), 2.58 (m, 1H), 2.54 (m, 1H), 2.52 (m, 1H), 2.47 (m, 1H), 2.23 (m, 1H), 2.02 (m, 1H), 1.96 (m, 1H), 1.89 (m, 1H), 1.87 (m, 1H), 1.77 (m, 1H), 1.73 (m, 1H), 1.64 (m, 1H), 1.61 (m, 1H), 1.53 (m, 1H), 1.49 (m, 1H), 1.38 (m, 1H), 1.29 (s, 3H), 1.06 (s, 3H), 0.98 (s, 3H), 0.96 (d, 6.8 Hz, 3H), 0.88 (d, 6.8 Hz, 3H), 0.86 (d, 6.8 Hz, 3H)。13CNMR (150 MHz, CDCl3) δ 199.7, 164.5, 155.6, 136.7, 133.9, 130.6, 124.8, 124.4, 123.1, 74.8, 59.1, 44.4, 44.3, 42.7, 36.8, 36.1, 34.3, 34.2, 33.2, 30.7, 18.9, 24.9, 22.5, 20.6, 20.2, 19.9, 16.8, 17.3。化合物7：白色粉末。HRESIMS(m/z): 153.041 6[M+H]+。1H NMR (600 MHz, DMSO-d6) δ 13.32 (s, 1H), 11.53 (s, 1H), 10.84 (s, 1H), 7.92 (s, 1H)。13C NMR (150 MHz, DMSO-d6) δ 155.5, 151.4, 149.1, 140.6, 106.6。
麦角甾醇(化合物8)、过氧化麦角甾醇(化合物9)和丁二酸(化合物10)为真菌中常见化合物，数据略。
2.3 化合物的结构鉴定
化合物113C NMR δ168.0表明存在1个酯羰基，δ 151.2, 123.3结合1H NMR δ 6.82 (dd, 15.8, 7.8 Hz, 1H), 5.97 (dd, 15.8, 1.0 Hz, 1H)的低场化学位移提示有1个和羰基共轭的双键，13C NMR δ 77.3, 72.7, 70.9结合1H NMR δ 4.99, 4.32, 3.65的信号表明存在3个连氧的—CH基团，提示除了酯羰基外，可能有2个连—CH的羟基。1H NMR δ 0.90 (t, 7.0, 3H)和中高场区重叠的质子信号及碳谱(δ 36.3, 32.9, 26.4, 23.7, 14.6)表明化合物的分子中存在链状结构单元。根据高分辨质谱313.238 4 [M+H]+的分子离子峰，结合碳谱、氢谱及以上分析推导出化合物的分子式为C18H32O4，除去羰基和双键的不饱和度，表明分子存在1个单环，说明化合物属于大环内酯结构类型。查阅文献[15]对照波谱数据、比旋光度等，化合物1鉴定为gliomasolide A，结构见图1。
化合物2的13C NMR和1H NMR和化合物1非常相似，表明其具有与化合物1相同的碳骨架。化合物2的13C NMR δ 77.8, 76.1, 72.9, 69.6结合1H NMR δ 4.95, 4.49, 3.99, 3.85表明其有4个连氧的—CH，比化合物1多了1个。而且化合物2的质谱显示其相对分子质量比化合物1多16，由此推测化合物2是化合物1取代了1个羟基的衍生物。查阅文献[16]对照波谱数据、比旋光度等，化合物2鉴定为Sch725674，结构见图1。
化合物31H NMR和13C NMR和化合物2相似，表明其也具有和化合物2类似的大环内酯骨架，两者区别在于化合物3有δ 77.9, 77.4, 73.7, 71.2, 71.3 5个连氧的—CH，比化合物2多1个，同时化合物3的质谱显示其相对分子质量比化合物2多16，由此推测化合物3在化合物2基础上多取代了1个羟基。查阅文献[15]对照波谱数据、比旋光度等数据，化合物3鉴定为gliomasolide C，结构见图1。
化合物4 HRESIMS分子离子信号472.187 2 [M + H]+可知其分子式C27H25N3O5，不饱和度为17。13C NMR δ 169.6, 167.9, 167.4, 167.4显示4个碳基碳，16个苯环区碳(δ 122.0~138.1)信号，同时1HNMR显示13个苯环区氢信号，结合耦合常数，表明其分子中存在1个单取代苯环和2个二取代苯环。扣除这些单元后剩余的不饱和度表明化合物4存在环状结构。进一步观察1H NMR发现δ 10.22 (s, 1H), 8.05 (s, 1H)有2个NH信号，13C NMR δ 43.4, 13.9是典型的连接在酰胺氮上的乙基结构单元信号，结合δ71.7的1个连氧碳信号，推测化合物4为2个邻氨基苯甲酸、1个2−羟基苯丙酸以及1个N−乙基甘氨酸组成的环内酰胺类物质。在此基础上查阅文献[17]对照波谱数据、比旋光度等，化合物4鉴定为clavatustide A，分子结构见图1。
化合物5 13C NMR数据和化合物4十分类似，表明具有相同的环状碳架结构。化合物5的1H NMR和化合物4相比少了δ 1.02 (t, 7.2 Hz, 3H)的三重峰甲基信号，多了δ 3.03 (s, 3H)的氮甲基碳信号，同时化合物5的13C NMR比化合物4少了1个高场区的δ 43附近的碳，质谱少了14，相当于CH2的质量单元。这些结果表明二者不同仅在于化合物4连接在酰胺氮上的乙基单元在化合物5中变成了甲基结构。查阅文献[17]对照波谱数据、比旋光度等，化合物5鉴定为clavatustide B，分子结构见图1。
化合物6 1H NMR和13C NMR提示其属于甾醇类，1H NMR δ 0.96 (d, 6.8 Hz, 3H), 0.88 (d, 6.8 Hz, 3H), 0.86 (d, 6.8 Hz, 3H)是连在—CH上的甲基，1.29 (s, 3H), 1.06 (s, 3H), 0.98 (s, 3H)是连在季碳上的甲基，1H NMR δ 6.63 (d, 9.5 Hz, 1H), 6.05 (d, 9.5 Hz, 1H), 5.75 (s, 1H), 5.58 (m, 1H), 5.57 (m, 1H)及13C NMR δ 164.5, 155.6, 136.7, 133.9, 130.6, 124.8, 124.4, 123.1说明有4个双键，13C NMR δ 199.7的信号表明化合物6存在1个α，β不饱和羰基，δ 74.8的连氧碳信号(无对应的1H NMR信号)表明化合物6有1个连季碳的羟基。在此基础上查阅文献，波谱数据和比旋光度与文献[18]中的基本一致，化合物6鉴定为20−羟基麦角甾−4, 6, 8(14), 22−四烯−3−酮，结构见图1。
化合物7 1H NMR显示δ 13.32 (s, 1H), 11.53 (s, 1H), 10.84 (s, 1H)3个NH信号以及7.92 (s, 1H)的双键CH信号，结合13C NMR δ 155.5, 151.4, 149.1, 140.6, 106.6的信号表明化合物7可能是嘌呤类化合物，查阅文献[19]并对照波谱数据等，化合物7鉴定为黄嘌呤。
麦角甾醇(化合物8)、过氧化麦角甾醇(化合物9)和丁二酸(化合物10)的结构通过与实验室标准品进行核磁共振波谱、薄层层析对照确定。
2.4 化合物对植物病原菌的抗菌活性
采用滤纸片扩散法测试了部分化合物对香蕉炭疽菌和小麦赤霉菌的活性。根据文献[12]，抑菌圈直径为[6, 11)、[11, 15)和[15, 20) mm分别表示对该菌株有轻度、中度和高度抗菌作用。由表1可知，在浓度为250 μg/mL时，gliomasolide A、Sch725674和gliomasolide C对香蕉炭疽菌和小麦赤霉菌均显示了高度抗菌活性，且Sch725674、gliomasolide C对小麦赤霉菌的抗菌活性与多菌灵接近；clavatustide A, clavatustide B对香蕉炭疽菌和小麦赤霉菌显示中度抗菌作用，20−羟基麦角甾−4, 6, 8(14), 22−四烯−3−酮对小麦赤霉菌中度抗菌，对香蕉炭疽轻度抗菌。但化合物对2种植物病原真菌抗菌活性均弱于阳性对照多菌灵。
表 1 化合物的抗植物病原菌活性1)Table 1. Antifungal activities of compounds against plant pathogens测试化合物 Tested compound d(抑菌圈 Inhibition zone)/mm 香蕉炭疽菌 Calletotrichum musae 小麦赤霉菌 Fusarium graminearum gliomasolide A 18.19±0.43eE 15.45±0.41eE Sch725674 17.18±0.36dD 17.02±0.35gG gliomasolide C 18.18±0.46eE 16.22±0.54fF clavatustide A 13.75±0.42cC 13.46±0.36cC clavatustide B 13.66±0.24cC 14.47±0.27dD 20−羟基麦角甾−4, 6, 8(14), 22−四烯−3−酮 10.54±0.43bB 12.37±0.53bB 20-hydroxyergosta-4, 6, 8(14), 22-tetraen-3-one 多菌灵 Positive control 26.53±0.20fF 18.57±0.18hH 空白对照 Negative control 0aA 0aA 1)表中数据为平均值±标准误，同列数据后不同小写字母表示组间0.05水平差异显著，不同大写字母表示组间0.01水平差异显著(Duncan’ s法)
1)The data in the table are average values ± standard errors, and different lowercase letters in the same column indicate significant differences among groups at 0.05 level, different capital letters in the same column indicate significant differences among groups at 0.01 level (Duncan’ s method)3. 结论与讨论
从棒曲霉菌株XCE02的发酵液中共分离鉴定出gliomasolide A、gliomasolide C、20−羟基麦角甾−4,6,8(14),22−四烯−3−酮等10种化合物，结构类型包括有大环内酯、环脂肽、生物碱、甾醇等。滤纸片扩散法抗菌活性测试结果表明，在浓度为250 μg/mL时，化合物gliomasolide A、Sch725674和gliomasolide C对香蕉炭疽菌和小麦赤霉菌显示了高度抗菌活性，推测它们是菌株提取物显示对植物病原菌抗性的重要原因之一，可作为抗菌农药先导化合物开展深入研究。此外clavatustide A、clavatustide B和20−羟基麦角甾−4,6,8(14),22−四烯−3−酮也对香蕉炭疽菌和小麦赤霉菌显示了中度或轻度抗菌活性。据文献报道，gliomasolide A可以抑制人宫颈癌细胞的增殖[15]，Sch725674对酿酒酵母Saccharomyces cerevisiae和白色念珠菌Candida albicans具有强抑制活性[16]，clavatustide A和clavatustide B 具有抑制3种人肝癌细胞HepG2、SMMC-7721和Bel-7402增殖的活性[17]。但鲜见这些化合物对植物病原菌抗菌活性的研究报道，本研究结果丰富了昆虫天然共生真菌源抗菌农药先导化合物库。
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图 1 SGIV病毒的分离、纯化及细胞内的复制增殖[3, 10-11]
a：患病石斑鱼肿大的脾脏；b：正常的石斑鱼脾细胞；c：SGIV感染引起脾细胞明显的细胞病变；d：感染细胞的超微结果观察，VM：病毒装配区，EC：空衣壳，MC：成熟核衣壳，NU：细胞核，标尺1 μm；e~g分别表示SGIV病毒粒子的出芽、释放及重感染邻近细胞，标尺为100 nm
Figure 1. Isolation and purification of SGIV and its replication in cells
a: The enlarged spleen of diseased grouper; b: Mock grouper spleen cells; c: The cytopathic effects induced by SGIV infection; d: Ultrastructural observation of infected cells, VM: viromatrix, EC: empty capsid, MC: matured nucleocapsid, NU: nuclesus, scale bar is 1 μm; e~g indicate budding, release and reinfection of SGIV particles respectively, scale bars are 100 nm
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图 2 SGIV病毒进入介导的内吞方式[32]
a~c：SGIV病毒进入依赖于网格蛋白和巨胞饮，而不依赖于小窝蛋白，标尺为10 μm；d：SGIV进入细胞的模式图
Figure 2. The endocytosis mediated by SGIV entry
a~c: SGIV enters grouper cells via the clathrin-mediated endocytic pathway and micropinocytosis, but not via caveola-dependent endocytosis, scale bars are 10 μm; d: Model entry route of SGIV into GS cells
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图 3 SGIV能显著激活MAPK信号通路中关键的信号分子[34, 36]
a：SGIV能激活ERK的磷酸化；b：SGIV感染对MAPK信号通路的激活情况及MAPK信号通路在病毒感染过程中的作用模式图，SGIV：新加坡石斑鱼虹彩病毒，MAPK：丝裂原活化蛋白激酶，MEK：MAPK激酶，ERK：细胞外调节蛋白激酶，JNK：c-Jun氨基末端激酶，MAPKAPK：MAPK激活蛋白激酶
Figure 3. SGIV infection significantly activates the key molecules in the MAPK signaling pathway
a: SGIV induces phosphorylation of ERK; b: Activation of MAPK signaling pathway induced by SGIV infection and the action of MAPK signaling pathway during virus infection, SGIV: Singapore grouper iridovirus, MAPK: mitogen-activated protein kinase, MEK: MAPK kinase, ERK: extracellular regulated protein kinases, JNK：c-Jun amino-terminal kinase, MAPKAPK: MAPK-activated protein kinase
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[1] GRAY M J, CHINCHAR V G. Ranaviruses: Lethal pathogens of ectothermic vertebrates[M]. SpringerOpen, 2015.
[2] CHINCHAR V G, WALTZEK T B, SUBRAMANIAM K. Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere[J]. Virology, 2017, 511: 259-271. doi: 10.1016/j.virol.2017.06.007
[3] QIN Q W, WU T H, JIA T L, et al. Development and characterization of a new tropical marine fish cell line from grouper, Epinephelus coioides susceptible to iridovirus and nodavirus[J]. J Virol Methods, 2006, 131(1): 58-64. doi: 10.1016/j.jviromet.2005.07.009
[4] HUANG X H, HUANG Y H, SUN J, et al. Characterization of two grouper Epinephelus akaara cell lines: Application to studies of Singapore grouper iridovirus (SGIV) propagation and virus-host interaction[J]. Aquaculture, 2009, 292(3): 172-179.
[5] OUYANG Z L, HUANG X H, HUANG E Y, et al. Establishment and characterization of a new marine fish cell line derived from red-spotted grouper Epinephelus akaara[J]. J Fish Biol, 2010, 77(5): 1083-1095. doi: 10.1111/jfb.2010.77.issue-5
[6] GOGN J, HUANG Y H, HUANG X H, et al. Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)[J]. J Fish Dis, 2011, 34(9): 677-686. doi: 10.1111/jfd.2011.34.issue-9
[7] ZHOU L L, LI P F, LIU J X, et al. Establishment and characterization of a mid-kidney cell line derived from golden pompano Trachinotus ovatus, a new cell model for virus pathogenesis and toxicology studies[J]. In Vitro Cell Dev-An, 2017, 53(4): 320-327. doi: 10.1007/s11626-016-0112-3
[8] LI P F, ZHOU L L, NI S W, et al. Establishment and characterization of a novel cell line from the brain of golden pompano (Trachinotus ovatus)[J]. In Vitro Cell Dev-An, 2016, 52(4): 410-418. doi: 10.1007/s11626-015-9988-6
[9] WEI S N, YU Y P, QIN Q W. Establishment of a new fish cell line from the caudal fin of golden pompano Trachinotus ovatus and its susceptibility to iridovirus[J]. J Fish Biol, 2018, 92(6): 1675-1686. doi: 10.1111/jfb.2018.92.issue-6
[10] QIN Q W, LAM T J, SIN Y M, et al. Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina[J]. J Virol Methods, 2001, 98(1): 17-24. doi: 10.1016/S0166-0934(01)00350-0
[11] QIN Q W, CHANG S F, NGOH-LIM G H, et al. Characterization of a novel ranavirus isolated from grouperEpinephelus tauvina[J]. Dis Aquat Organ, 2003, 53(1): 1-9.
[12] SONG W J, QIN Q W, QIU J, et al. Functional genomics analysis of Singapore grouper iridovirus: Complete sequence determination and proteomic analysis[J]. J Virol, 2004, 78(22): 12576-12590. doi: 10.1128/JVI.78.22.12576-12590.2004
[13] ZHOU S, WAN Q J, HUANG Y H, et al. Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088[J]. Proteomics, 2011, 11(11): 2236-2248. doi: 10.1002/pmic.200900820
[14] HUANG X H, GONG J, HUANG Y H, et al. Characterization of an envelope gene VP19 from Singapore grouper iridovirus[J]. Virol J, 2013, 10: 354. doi: 10.1186/1743-422X-10-354
[15] ZHANG H L, ZHOU S, XIA L Q, et al. Characterization of the VP39 envelope protein from Singapore grouper iridovirus[J]. Can J Microbiol, 2015, 61(12): 924-937. doi: 10.1139/cjm-2015-0118
[16] TENG Y, HOU Z W, GONG J, et al. Whole-genome transcriptional profiles of a novel marine fish iridovirus, Singapore grouper iridovirus (SGIV) in virus-infected grouper spleen cell cultures and in orange-spotted grouper, Epinephuluscoioides[J]. Virology, 2008, 377(1): 39-48. doi: 10.1016/j.virol.2008.04.011
[17] YAN Y, CUI H C, JIANG S, et al. Identification of a novel marine fish virus, Singapore grouper iridovirus-encoded microRNAs expressed in grouper cells by Solexa sequencing[J]. PLoS One, 2011, 6(4): e19148. doi: 10.1371/journal.pone.0019148
[18] XIA L Q, CAO J H, HUANG X H, et al. Characterization of Singapore grouper iridovirus (SGIV) ORF086R, a putative homolog of ICP18 involved in cell growth control and virus replication[J]. Arch Virol, 2009, 154(9): 1409-1416. doi: 10.1007/s00705-009-0457-y
[19] XIA L Q, LIANG H, HUANG Y H, et al. Identification and characterization of Singapore grouper iridovirus (SGIV) ORF162L, an immediate-early gene involved in cell growth control and viral replication[J]. Virus Res, 2010, 147(1): 30-39. doi: 10.1016/j.virusres.2009.09.015
[20] HUANG X H, HUANG Y H, GONG J, et al. Identification and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) homolog from Singapore grouper iridovirus[J]. Biochem Bioph Res Co, 2008, 373(1): 140-145. doi: 10.1016/j.bbrc.2008.06.003
[21] CAI J, HUANG Y H, WEI S N, et al. Characterization of LPS-induced TNFα factor (LITAF) from orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2013, 35(6): 1858-1866. doi: 10.1016/j.fsi.2013.09.023
[22] HUANG X H, HUANG Y H, CAI J, et al. Identification and characterization of a tumor necrosis factor receptor like protein encoded by Singapore grouper iridovirus[J]. Virus Res, 2013, 178(2): 340-348. doi: 10.1016/j.virusres.2013.09.023
[23] YU Y P, HUANG Y H, WEI S N, et al. A tumour necrosis factor receptor-like protein encoded by Singapore grouper iridovirus modulates cell proliferation, apoptosis and viral replication[J]. J Gen Virol, 2016, 97(3): 756-766. doi: 10.1099/jgv.0.000379
[24] YU Y P, HUANG Y H, NI S W, et al. Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor via modulating host inflammation response[J]. Virology, 2017, 511: 280-289. doi: 10.1016/j.virol.2017.06.025
[25] GONG J, HUANG Y H, HUANG X H, et al. Nuclear-export-signal-dependent protein translocation of dUTPase encoded by Singapore grouper iridovirus[J]. Arch Virol, 2010, 155(7): 1069-1076. doi: 10.1007/s00705-010-0684-2
[26] YAN Y, CUI H C, GUO C Y, et al. An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication[J]. J Gen Virol, 2013, 94: 2759-2770. doi: 10.1099/vir.0.056135-0
[27] YAN Y, CUI H C, GUO C Y, et al. Singapore grouper iridovirus-encoded semaphorin homologue (SGIV-sema) contributes to viral replication, cytoskeleton reorganization and inhibition of cellular immune responses[J]. J Gen Virol, 2014, 95: 1144-1155. doi: 10.1099/vir.0.060608-0
[28] YAN Y, GUO C Y, NI S W, et al. Singapore grouper iridovirus (SGIV) encoded SGIV-miR-13 attenuates viral infection via modulating major capsid protein expression[J]. Virus Res, 2015, 205: 45-53. doi: 10.1016/j.virusres.2015.05.010
[29] GUO C Y, YAN Y, CUI H C, et al. miR-homoHSV of Singapore grouper iridovirus (SGIV) inhibits expression of the SGIV pro-apoptotic factor LITAF and attenuates cell death[J]. PLoS One, 2013, 8(12): e83027. doi: 10.1371/journal.pone.0083027
[30] HUANG C H, ZUANG X, GIN K Y, et al. In situ hybridization of a marine fish virus, Singapore grouper iridovirus with a nucleic acid probe of major capsid protein[J]. J Virol Methods, 2004, 117(2): 123-128. doi: 10.1016/j.jviromet.2004.01.002
[31] FU J, HUANG Y H, CAI J, et al. Identification and characterization of Rab7 from orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2014, 36(1): 19-26. doi: 10.1016/j.fsi.2013.10.002
[32] WANG S W, HUANG X H, HUANG Y H, et al. Entry of a novel marine DNA virus, Singapore grouper iridovirus, into host cells occurs via clathrin-mediated endocytosis and macropinocytosis in a pH-dependent manner[J]. J Virol, 2014, 88(22): 13047-13063. doi: 10.1128/JVI.01744-14
[33] PAN Y, WANG S W, SHAN Y, et al. Ultrafast tracking of a single live virion during the invagination of a cell membrane[J]. Small, 2015, 11(23): 2782-2788. doi: 10.1002/smll.201403491
[34] HUANG X H, HUANG Y H, OUYANG Z L, et al. Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling[J]. Apoptosis, 2011, 16(8): 831-845. doi: 10.1007/s10495-011-0616-y
[35] HUANG Y H, HUANG X H, YAN Y, et al. Transcriptome analysis of orange-spotted grouper (Epinephelus coioides) spleen in response to Singapore grouper iridovirus[J]. BMC Genomics, 2011, 12: 556. doi: 10.1186/1471-2164-12-556
[36] HUANG X H, HUANG Y H, OUYANG Z L, et al. Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells[J]. J Gen Virol, 2011, 92: 1292-1301. doi: 10.1099/vir.0.029173-0
[37] CAI J, HUANG Y H, WEI S N, et al. Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection[J]. Fish Shellfish Immunol, 2011, 31(6): 1129-1136. doi: 10.1016/j.fsi.2011.10.004
[38] WEI S N, HUANG Y H, HUANG X H, et al. Characterization of c-Jun from orange-spotted grouper, Epinephelus coioides involved in SGIV infection[J]. Fish Shellfish Immunol, 2015, 43(1): 230-240. doi: 10.1016/j.fsi.2014.12.033
[39] GUO M L, WEI J G, ZHOU Y S, et al. MKK7 confers different activities to viral infection of Singapore grouper iridovirus (SGIV) and nervous necrosis virus (NNV) in grouper[J]. Fish Shellfish Immunol, 2016, 57: 419-427. doi: 10.1016/j.fsi.2016.09.002
[40] GUO M L, WEI J G, ZHOU Y S, et al. c-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis[J]. Dev Comp Immunol, 2016, 65: 169-181. doi: 10.1016/j.dci.2016.06.009
[41] GUO M L, WEI J G, ZHOU Y S, et al. Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2016, 49: 355-363. doi: 10.1016/j.fsi.2015.12.001
[42] GUO M L, WEI J G, HUANG X H, et al. JNK1 derived from orange-spotted grouper, Epinephelus coioides, involving in the evasion and infection of Singapore grouper iridovirus (SGIV)[J]. Front Microbiol, 2016, 7: 121.
[43] BANKS L, PIM D, THOMAS M. Viruses and the 26S proteasome: Hacking into destruction[J]. Trends BiochemSci, 2003, 28(8): 452-459. doi: 10.1016/S0968-0004(03)00141-5
[44] RANDOW F, LEHNER P J. Viral avoidance and exploitation of the ubiquitin system[J]. Nat Cell Biol, 2009, 11(5): 527-534. doi: 10.1038/ncb0509-527
[45] HUANG X H, WEI S N, NI S W, et al. Ubiquitin-proteasome system is required for efficient replication of Singapore grouper iridovirus[J]. Front Microbiol, 2018, 9: 2798. doi: 10.3389/fmicb.2018.02798
[46] HUANG Y H, YU Y P, YANG Y, et al. Fish TRIM8 exerts antiviral roles through regulation of the proinflammatory factors and interferon signaling[J]. Fish Shellfish Immunol, 2016, 54: 435-444. doi: 10.1016/j.fsi.2016.04.138
[47] YANG Y, HUANG Y H, YU Y P, et al. RING domain is essential for the antiviral activity of TRIM25 from orange spotted grouper[J]. Fish Shellfish Immunol, 2016, 55: 304-314. doi: 10.1016/j.fsi.2016.06.005
[48] YU Y P, HUANG X H, LIU J X, et al. Fish TRIM32 functions as a critical antiviral molecule against iridovirus and nodavirus[J]. Fish Shellfish Immunol, 2017, 60: 33-43. doi: 10.1016/j.fsi.2016.11.036
[49] YANG Y, HUANG Y H, YU Y P, et al. Negative regulation of the innate antiviral immune response by TRIM62 from orange spotted grouper[J]. Fish Shellfish Immunol, 2016, 57: 68-78. doi: 10.1016/j.fsi.2016.08.035
[50] LV S Y, ZHAN Y, ZHENG J Y, et al. Negative regulation of the interferon response by finTRIM82 in the orange spotted grouper[J]. Fish Shellfish Immunol, 2019, 88: 391-402. doi: 10.1016/j.fsi.2019.03.004
[51] HUANG Y H, ZHANG J C, LIU J X, et al. Fish TRIM35 negatively regulates the interferon signaling pathway in response to grouper nodavirus infection[J]. Fish Shellfish Immunol, 2017, 69: 142-152. doi: 10.1016/j.fsi.2017.08.019
[52] YU Y P, HUANG X H, ZHANG J C, et al. Fish TRIM16L exerts negative regulation on antiviral immune response against grouper iridoviruses[J]. Fish Shellfish Immunol, 2016, 59: 256-267. doi: 10.1016/j.fsi.2016.10.044
[53] HUANG Y H, YANG M, YU Y P, et al. Grouper TRIM13 exerts negative regulation of antiviral immune response against nodavirus[J]. Fish Shellfish Immunol, 2016, 55: 106-115. doi: 10.1016/j.fsi.2016.05.029
[54] QIN Q W, GIN K Y, LEE L Y, et al. Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture[J]. J Virol Methods, 2005, 125(1): 49-54. doi: 10.1016/j.jviromet.2004.12.005
[55] LIU W T, ZHU L, QIN Q W, et al. Microfluidic device as a new platform for immunofluorescent detection of viruses[J]. Lab Chip, 2005, 5(11): 1327-1330. doi: 10.1039/b509086e
[56] MAO X L, ZHOU S, XU D, et al. Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification[J]. J ApplMicrobiol, 2008, 105(2): 389-397.
[57] LI P F, YAN Y, WER S N, et al. Isolation and characterization of a new class of DNA aptamers specific binding to Singapore grouper iridovirus (SGIV) with antiviral activities[J]. Virus Res, 2014, 188: 146-154. doi: 10.1016/j.virusres.2014.04.010
[58] LI P F, WEI S N, ZHOU L L, et al. Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells[J]. J Gen Virol, 2015, 96(11): 3348-3359. doi: 10.1099/jgv.0.000270
[59] LI P F, ZHOU L L, WEI J G, et al. Development and characterization of aptamer-based enzyme-linked apta-sorbent assay for the detection of Singapore grouper iridovirus infection[J]. J Appl Microbiol, 2016, 121(3): 634-643.
[60] ZHANG X B, HUANG C H, QIN Q W. Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon[J]. Antiviral Res, 2004, 61(2): 93-99. doi: 10.1016/j.antiviral.2003.08.019
[61] XU D, WEI J G, CUI H C, et al. Differential profiles of gene expression in grouper Epinepheluscoioides, infected with Singapore grouper iridovirus, revealed by suppression subtractive hybridization and DNA microarray[J]. J Fish Biol, 2010, 77(2): 341-360. doi: 10.1111/jfb.2010.77.issue-2
[62] HUANG Y H, ZHANG J C, OUYANG Z L, et al. Grouper MAVS functions as a crucial antiviral molecule against nervous necrosis virus infection[J]. Fish Shellfish Immunol, 2018, 72: 14-22. doi: 10.1016/j.fsi.2017.10.035
[63] WEI J G, GUO M L, GAO P, et al. Isolation and characterization of tumor necrosis factor receptor-associated factor 6 (TRAF6) from grouper, Epinephelus tauvina[J]. Fish Shellfish Immunol, 2014, 39(1): 61-68. doi: 10.1016/j.fsi.2014.04.022
[64] YU Y P, HUANG Y H, YANG Y, et al. Negative regulation of the antiviral response by grouper LGP2 against fish viruses[J]. Fish Shellfish Immunol, 2016, 56: 358-366. doi: 10.1016/j.fsi.2016.07.015
[65] HUANG Y H, YU Y P, YANG Y, et al. Antiviral function of grouper MDA5 against iridovirus and nodavirus[J]. Fish Shellfish Immunol, 2016, 54: 188-196. doi: 10.1016/j.fsi.2016.04.001
[66] HUANG Y H, OUANG Z L, WANG W, et al. Antiviral role of grouper STING against iridovirus infection[J]. Fish Shellfish Immunol, 2015, 47(1): 157-167. doi: 10.1016/j.fsi.2015.09.014
[67] HU Y, HUANG Y H, LIU J X, et al. TBK1 from orange-spotted grouper exerts antiviral activity against fish viruses and regulates interferon response[J]. Fish Shellfish Immunol, 2018, 73: 92-99. doi: 10.1016/j.fsi.2017.12.010
[68] HUANG Y H, HUANG X H, CAI J, et al. Identification of orange-spotted grouper (Epinephelus coioides) interferon regulatory factor 3 involved in antiviral immune response against fish RNA virus[J]. Fish Shellfish Immunol, 2015, 42(2): 345-352. doi: 10.1016/j.fsi.2014.11.025
[69] CUI H C, YAN Y, WEI J G, et al. Identification and functional characterization of an interferon regulatory factor 7-like (IRF7-like) gene from orange-spotted grouper, Epinephelus coioides[J]. Dev Comp Immunol, 2011, 35(6): 672-684. doi: 10.1016/j.dci.2011.01.021
[70] GAO R, HUANG Y H, HUANG X H, et al. Molecular cloning and characterization of two types of IκBα orthologues in orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2014, 38(1): 101-110. doi: 10.1016/j.fsi.2014.02.019
[71] HUANG Y H, HUANG X H, YANG Y, et al. Involvement of fish signal transducer and activator of transcription 3 (STAT3) in nodavirus infection induced cell death[J]. Fish Shellfish Immunol, 2015, 43(1): 241-248. doi: 10.1016/j.fsi.2014.12.031
[72] ZHANG J C, HUANG X H, NI S W, et al. Grouper STAT1a is involved in antiviral immune response against iridovirus and nodavirus infection[J]. Fish Shellfish Immunol, 2017, 70: 351-360. doi: 10.1016/j.fsi.2017.09.030
[73] WEI J G, XU D, ZHOU J G, et al. Molecular cloning, characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2010, 28(1): 178-186. doi: 10.1016/j.fsi.2009.10.020
[74] JI H S, WEI J G, WEI S N, et al. Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides[J]. J Fish Biol, 2014, 84(2): 436-447. doi: 10.1111/jfb.2014.84.issue-2
[75] CHEN X L, WEI J G, XU M, et al. Molecular cloning and characterization of a galectin-1 homolog in orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2016, 54: 333-341. doi: 10.1016/j.fsi.2016.02.036
[76] ZHOU JG, WEI JG, XU D, et al. Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2011, 30(2): 559-568. doi: 10.1016/j.fsi.2010.11.021
[77] GUO M L, WEI J G, HUANG X H, et al. Antiviral effects of β-defensin derived from orange-spotted grouper (Epinephelus coioides)[J]. Fish Shellfish Immunol, 2012, 32(5): 828-838. doi: 10.1016/j.fsi.2012.02.005
[78] WEI S N, HUANG Y H, CAI J, et al. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides[J]. Fish Shellfish Immunol, 2012, 33(2): 186-196. doi: 10.1016/j.fsi.2012.03.027
[79] WEI S N, HUANG Y H, HUANG X H, et al. Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides[J]. Dev Comp Immunol, 2014, 46(2): 401-412. doi: 10.1016/j.dci.2014.05.006
[80] OUYANG Z L, WANG P R, HUANG X H, et al. Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides[J]. Dev Comp Immunol, 2012, 38(2): 254-261. doi: 10.1016/j.dci.2012.07.004
[81] OUYANG Z L, WANG P R, HUANGY H, et al. Selection and identification of Singapore grouper iridovirus vaccine candidate antigens using bioinformatics and DNA vaccination[J]. Vet Immunol Immunopathol, 2012, 149(1/2): 38-45.
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