(‐)‐Epicatechin gallate,a metabolite of (‐)‐epigallocatechin gallate in grass carp,exhibits antiviral activity in vitro against grass carp reovirus

(‐)‐Epigallocatechin gallate (EGCG), a catechin found in green tea, has been demonstrated to exhibit activity against grass carp reovirus (GCRV). In the current study, we found that EGCG is partially transformed in vivo into (‐)‐epicatechin gallate (ECG), which differs from EGCG only by the absence of a hydroxyl group, and exhibits similar pharmacokinetic behaviour to that of EGCG. ECG is also a major catechin in green tea, but little information on its antiviral activity is available. Therefore, we assessed whether ECG affects GCRV in vitro. We incubated grass carp (Ctenopharyngodon idellus) kidney (CIK) cells with ECG and GCRV‐JX01 at different concentrations, and typical cytopathogenic effect (CPE) values were observed for 5 and 10 µg/ml ECG. However, the CPE in 20 µg/ml ECG treatment group was low; no significant CPE was observed for 40 µg/ml ECG treatment; and a high ECG concentration (80 µg/ml) led to stress response in the CIK cells. Western blot results also revealed that ECG suppresses GCRV replication in CIK cells. Thus, the data indicate that ECG, as well as EGCG, exhibits potential as an antiviral agent for aquaculture.     

In vivo antiviral efficacy of moroxydine hydrochloride against grass carp reovirus and the drug safety assessment

Our previous studies have verified that moroxydine hydrochloride (Mor) could inhibit replication of grass carp reovirus (GCRV) and suppress apoptosis of Ctenopharyngodon idella kidney cells, but be lack of information whether exists on antiviral activity in vivo. The paper was undertaken to explore the antiviral response of Mor against GCRV in grass carp and investigate the safety of drug for aquatic organisms. The results showed that injection treatment of Mor could more effectively inhibit GCRV replication than immersion administration. All the RNA systheses of vp3 and vp6 on day 7 in head kidney, gill, hepatopancreas and dorsal muscle in the Mor injection group were lesser than 0.07‐fold than that of in control group. And the GCRV‐inducing grass carp mortality was effectively controlled within 7 days post Mor injection therapy. Additionally, the reduction of superoxide dismutase activity, total antioxidant capacity and catalase activity in serum was effectively controlled by Mor. Moreover, drug safety assessment results showed that 500 mg/L of Mor was safe to C. idella, Bacillus subtilis, Chlorella vulgaris and Tetrahymena thermophila, which was far higher than the therapeutic concentration. The present study proved Mor as harmless formulations or products had potential value in the control of GCRV in aquaculture, with the advantage of super in vivo antiviral activity and environmental safety.     

EGCG: Potential application as a protective agent against grass carp reovirus in aquaculture

Grass carp reovirus (GCRV) is the primary cause of grass carp haemorrhagic disease. The major catechin in green tea, (–)‐epigallocatechin‐3‐gallate (EGCG), has been found to have anti‐GCRV activity in the C. idellus kidney cell line (CIK). The aim of this study was to test the potential application of EGCG as an anti‐GCRV agent in aquaculture. Here, we demonstrate that various concentrations (99%, 50% and 35%) of EGCG could inhibit GCRV infectivity. EGCG (50%) + GCRV treatment significantly reduced the number of dead fish at 1‐, 2‐, 3‐, 4 ‐and 5‐day post‐challenge compared with the negative control (GCRV challenge without EGCG treatment). The safety of EGCG compound products on cell survival was studied using four fish cell lines; we did not detect a significant change in cell viability within 24 hours of EGCG incubation. We also evaluated toxicity and concentrations of malondialdehyde (MDA), glutathione (GSH) and lysozyme (LZM) in the grass carp, and the results showed that even a high dose of EGCG did not induce toxicity. Following EGCG compound injection, the concentration of MDA decreased and the concentration of GSH and LZM increased compared with the control groups. We also detected EGCG concentration in grass carp plasma and kidney using HPLC with electrochemical detection after intraperitoneal injection at a dose of 150 mg/kg. The concentration of EGCG in the plasma and kidney reached the highest levels (20 μg/ml and 1.5 μg/ml) about 12 hr after injection and then decreased. Overall, EGCG is a safe, effective product that could inhibit GCRV infection and improve immunoactivity in aquaculture.     

草鱼呼肠孤病毒VP6蛋白在毕赤酵母中表达的初步研究

根据GeneBank中草鱼呼肠孤病毒(grass carp reovirus,GCRV)104株VP6蛋白的全基因序列(HM234682)设计一对特异引物,以提取的GCRV-104核酸为模板,采用RT-PCR技术扩增VP6蛋白编码基因,并将其克隆到含强启动子AOXⅠ的毕赤酵母(Pichia pastoris)表达载体pPICZB上。重组酵母质粒pPICZB-VP6经SacⅠ线性化,电击转化到毕赤酵母KM71菌株中,Zeocin抗性平板上筛选高拷贝转化子。阳性转化子在30℃,0.5%(V/V)的甲醇添加量的条件下,连续诱导3 d,表达产物经SDS-PAGE和Western-Blotting检测,结果表明成功实现了GCRV-104 VP6蛋白在毕赤酵母中的胞内表达,重组VP6蛋白相对分子质量约46 000,与天然VP6蛋白相对分子质量接近,并且可与鼠抗草鱼GCRV-104 VP6蛋白的多克隆抗体特异性结合。     

草鱼细胞蛋白酶亚基β7与草鱼呼肠孤病毒非结构蛋白NS12相互作用

为研究草鱼I型呼肠孤病毒(grass carp reovirus, GCRV)非结构蛋白NS12的功能,实验利用酵母双杂交实验、GST融合蛋白沉降技术(GST pull-down)和对GCRV感染过表达宿主蛋白酶体亚基β7(proteasome subunit beta type 7, PSMB7)的草鱼卵巢细胞(grass carp ovarian cell,GCO)中ns12转录水平的表达量进行实时荧光定量PCR检测,研究PSMB7和NS12的相互作用。酵母双杂交实验结果表明,PSMB7与GCRV编码的膜相关的非结构蛋白NS12也存在着潜在相互作用。GST-pull-down检测结果证实PSMB7与GCRV编码的膜相关的非结构蛋白NS12存在相互作用;PSMB7过表达能够上调ns12在病毒感染过程中的转录水平表达量;免疫印迹验证NS12对于蛋白降解并不敏感。本实验室先前研究证实PSMB7在病毒感染过程中表达恒定。综上所述,这些结果揭示GCRV NS12与PSMB7存在分子间相互作用,但并没有作为PSMB7的底物。表明其可能竞争性地阻碍蛋白酶体复合物的形成。病毒蛋白干扰蛋白酶体复合物胞内PSMB7的积累可能是其一种针对蛋白酶体介导的先天免疫的免疫逃逸策略。     

草鱼呼肠孤病毒VP6蛋白基因植物表达载体的构建

以据草鱼呼肠孤病毒(GCRV)VP6蛋白的全基因序列(GeneBank AF403394)为模板,采用RT-PCR构建了草鱼呼肠孤病毒VP6蛋白基因植物表达载体的构建。结果显示:实验构建的pCR 2.1-VP6重组质粒含有NcoⅠ和BglⅡ酶切位点;pCR 2.1-VP6重组质粒经PCR扩增和测序显示含有1.3 Kbp的草鱼呼肠孤病毒VP6基因读码框片段。将目的片段VP6酶切、克隆到携带有绿色荧光蛋白(GFP)基因的植物表达载体pCAMBIA1302中,再经酶切、PCR扩增和序列测定显示,pCAMBIA1302-VP6含有1.3 Kbp的草鱼呼肠孤病毒VP6基因片段,说明已插入植物表达载体pCAMBIA1302绿色荧光蛋白基因前,成功构建了融合表达草鱼呼肠孤病毒VP6蛋白和绿色荧光蛋白的植物表达载体pCAMBIA1302-VP6。     

草鱼成纤维细胞gcngr1基因的表达分析

Ngr1(nogo-66 receptor)是在哺乳类上发现的一种神经元受体,可调节轴突的可塑性并抑制损伤中枢神经系统(CNS)的再生,最新研究还发现它是哺乳动物呼肠孤病毒(mammalian reovirus)的神经受体。草鱼呼肠孤病毒(grass carp reovirus,GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病导致高死亡率,开展GCRV受体相关研究可有助于了解病毒的致病机制。该研究在草鱼吻端成纤维细胞(PSF)中克隆到草鱼ngr1 c DNA序列(下文简称为gcngr1),发现其与哺乳动物呼肠孤病毒神经受体基因Ngr1有相似的结构序列;采用q RT-PCR方法检测该基因在PSF细胞的表达情况,结果显示受草鱼呼肠孤病毒(GCRV-GD108株)感染后gcngr1 m RNA的表达量显著上升,与病毒的增殖趋势基本一致;病毒经病毒抗体孵育后再感染PSF细胞,细胞中病毒的增殖水平下降,gcngr1m RNA的表达量也显著下降。该研究结果提示gcngr1与病毒的感染相关,为进一步分析gcngr1是否为GCRV神经元受体提供依据。     

草鱼jam-a分子在胚胎幼鱼期及受GCRV感染PSF细胞的表达分析

草鱼呼肠孤病毒(Grass carp reovirus, GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病，导致高死亡率。草鱼吻端成纤维细胞(Grass carp snout fibroblast cells, PSF)是GCRV的敏感细胞系。JAM-A (Junctional adhesion molecule A)为免疫球蛋白超家族成员，是多种病毒的细胞受体。本研究在前期克隆到草鱼3种jam-a基因，命名为gcjam-a1，gcjam-a2和gcjam-a3。在获取ORF序列的基础上，利用qRT-PCR分析了3种gcjam-a在草鱼胚胎及幼鱼不同发育时期及PSF细胞中受GCRV(GD108株)感染前后的表达模式。结果显示，检测的13个胚胎及幼鱼发育时期中，gcjam-a1在未受精卵中高表达，在受精卵至出膜前的胚胎表达水平均较低；从出膜后1~3 d表达量开始上升；出膜6~15 d均呈高水平表达。gcjam-a2与gcjam-a3在草鱼胚胎及幼鱼发育各阶段表达水平较低。在无病毒感染的PSF细胞中，gcjam-a只有少量表达。受GCRV-GD108感染后，病毒S7基因在PSF细胞中的拷贝数随时间呈显著上调趋势，gcjam-a的表达量也有不同程度的上调，mRNA上调水平为gcjam-a1>gcjam-a2>gcjam-a3。本研究证实了3种gcjam-a基因在PSF细胞中的表达均与GCRV-GD108感染相关，其中，gcjam-a1的表达水平受GCRV-GD108感染影响最大，同时，它在孵化出膜后表达上升，推测它可能与病毒的感染更相关。gcjam-a1可作为下一步GCRV与宿主互作研究中的候选分子。     

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.     

Antiviral activities of Clinacanthus nutans (Burm.f.) Lindau extract against Cyprinid herpesvirus 3 in koi (Cyprinus carpio koi)

Cyprinid herpesvirus 3 (CyHV‐3) or koi herpesvirus (KHV) is a virulent viral infection in common carp and koi. The disease has caused global epizootic and economic loss in fish aquaculture and in the wild. Clinacanthus nutans (Burm. f.) Lindau is a well‐known medicinal plant used in Thai traditional medicine. Virucidal effects of the plant extract against human herpes simplex virus have been reported. In this study, C. nutans crude extract was tested for antiviral activities against CyHV‐3 in koi carp. Results showed effective antiviral activity against CyHV‐3 pre‐ and post‐infection. The 50% lethal concentration (LC₅₀) of extract was higher than 5 mg/ml. The 50% effective dose (ED₅₀) was 0.99 mg/ml, 0.78 mg/ml, 0.75 mg/ml and 0.71 mg/ml at 1, 2, 3 and 4 hr pre‐infection, respectively. The ED₅₀ from post‐infection tests was 2.05 mg/ml and 2.34 mg/ml at 0 and 24 hr, respectively. These results demonstrated that crude extract expressed antiviral activity against CyHV‐3 and can be applied as a therapeutic agent in common carp and koi aquaculture.     

Antiviral activity of exopolysaccharides from Arthrospira platensis against koi herpesvirus

Although koi herpesvirus (KHV) has a history of causing severe economic losses in common carp and koi farms, there are still no treatments available on the market. Thus, the aim of this study was to test exopolysaccharides (EPS) for its antiviral activity against KHV, by monitoring inhibition and cytotoxic effects in common carp brain cells. These substances can be easily extracted from extracellular algae supernatant and were identified as groups of sulphated polysaccharides. In order to reach this aim, Arthrospira platensis, which is well known for its antiviral activity of intra‐ and extracellular compounds towards mammalian herpesviruses, was investigated as standard organism and compared to commercial antiviral drug, ganciclovir, which inhibits the viral DNA polymerization. The antiviral activity of polysaccharides of A. platensis against KHV was confirmed in vitro using qualitative assessment of KHV life cycle genes, and it was found by RT‐PCR that EPS, applied at a concentration of >18 μg mL^?1 and a multiplicity of infection (MOI) of 0.45 of KHV, suppressed the viral replication in common carp brain (CCB) cells even after 22 days post‐infection, entirely. Further, this study presents first data indicating an enormous potential using polysaccharides as an additive for aquacultures to lower or hinder the spread of the KHV and koi herpesvirus disease (KHVD) in future.     

草鱼呼肠孤病毒Ⅱ型自然发病与人工注射感染草鱼的病理症状和病毒分布研究

为了探究自然发病和人工注射感染草鱼呼肠孤病毒Ⅱ型(GCRV-Ⅱ型)草鱼的临床症状、病理特征和病毒分布的区别，实验采用临床剖检、组织病理学观察、分子生物学检测、实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)等检测方法开展实验，通过对自然发病和人工注射感染草鱼的临床症状进行比较，结果显示，患病草鱼的眼眶、鳃盖、口腔、腹部、鳍条基部、肠道、肌肉出现明显的点状出血，且后者的出血情况比前者更为严重；比较组织病理切片，发现草鱼感染组织出现不同程度的充血和红细胞充盈现象，其中，人工注射感染草鱼的肠道、肌肉和肝胰脏的病变程度更为严重，呈现出较为严重的组织内出血和病变，而自然发病草鱼的鳃和脾脏的病变程度更为严重，鳃呈现出较为严重的充血和炎症增生物，脾脏出现大面积含铁血黄素沉积块病灶。qRT-PCR和Western blot检测结果显示，GCRV在不同的组织中均有分布，头肾在两种感染方式的患病草鱼中的病毒量都比较高，人工注射感染草鱼的肝胰脏、肠道和肌肉的病毒量较高，自然发病草鱼的中肾、鳃和脾脏的病毒量较高。因此，在人工注射感染时，肝胰脏、肠道和肌肉可能是GCRV入侵的主要靶...     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.     

Comparative Study on Physical–Chemical and Biological Characteristics of Grass Carp Reovirus From Different Genotypes

Grass carp reovirus (GCRV) was first isolated in 1983. In China, the virus caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus. Clade analysis of the different GCRV isolates from China indicates there are three distinct branches, which represent genotypes I, II, and III. However, little is known about the physical–chemical and biological characteristics of viruses from these three genotypes. In this study, the morphologic characteristics of JX‐0901 (genotype I), HZ08 (genotype II), and Hubei grass carp disease reovirus (genotype III) were determined using electron microscopy which revealed morphological similarities but viral particles of the isolates arrayed in different ways. A comparison of selected viruses was performed for physical and chemical properties, including stability under different pH conditions, treatment with ether and trypsin, and repeated freeze–thaw cycles. Three isolates showed similar resistance to treatment with ether or trypsin and were stable between pH 3 and pH 10. The viruses exhibited different proliferation curves in the Grass carp swimming bladder cell. No postinfection mortalities or histological lesions were observed in infected rare minnow. Furthermore, cross‐protection assays revealed low cross protection between different genotype viruses. This study contributes to the body of knowledge concerning disease control and vaccine development for grass carp hemorrhagic disease.     

草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定

为研究Ⅱ型草鱼呼肠孤病毒外纤维蛋白VP56与宿主蛋白的相互作用,实验采用酵母双杂交Gal4系统鉴定与VP56相互作用的草鱼蛋白。首先利用RT-PCR技术从Ⅱ型GCRV感染的草鱼肾细胞中扩增目的基因VP56,构建p GBKT7-VP56诱饵表达载体;在酵母菌株中检测其自激活性后,以VP56为诱饵在草鱼酵母双杂交文库中筛选阳性菌株,并对阳性克隆的序列进行分析。实验也克隆了草鱼JAM-A(Gc JAM-A)基因,并构建p GADT7-Gc JAM-A载体,在酵母中研究草鱼Gc JAM-A与病毒蛋白VP56相互作用的可能性。研究表明构建的诱饵质粒p GBKT7-VP56无自激活作用,可以应用于酵母双杂交筛选;从草鱼肾细胞文库中筛选得到9株阳性克隆,基因测序及序列分析确定其中包含草鱼7个细胞内蛋白和1个细胞外基质蛋白;VP56蛋白与Gc JAM-A蛋白在酵母中不能发生相互作用。本研究初步确定了与Ⅱ型GCRV蛋白VP56存在潜在相互作用的宿主蛋白,为深入探讨VP56蛋白在Ⅱ型GCRV感染宿主过程中的生物学功能奠定了基础。     

Antifungal Activity of Rhein and Aloe‐Emodin from Rheum palmatum on Fish Pathogenic Saprolegnia sp.

Saprolegnia infections cause severe economic losses among freshwater fish farming. In this study, two known compounds, rhein and aloe‐emodin, were isolated from Rheum palmatum, and the in vitro inhibitory activity of both compounds against mycelial growth and spore germination of Saprolegnia was tested. Both rhein and aloe‐emodin were able to decrease Saprolegnia mycelial growth and spore activity in all tested concentrations after exposure for 48 h. Complete inhibition of mycelial growth was observed at 20 mg/L for rhein and at 50 mg/L for aloe‐emodin, while spore germination was 100% prevented at 16 and 40 mg/L for rhein and aloe‐emodin, respectively. Because rhein showed stronger in vitro anti‐Saprolegnia activity, it was further tested in vivo to measure the prevention and treatment efficacy on Saprolegnia infection of grass carp. Its acute activity to grass carp was also evaluated. The results revealed that exposure to rhein at 20 mg/L for 7 d could prevent 93.3% of infections by Saprolegnia in abraded grass carp, while 67.7% of infected fish could be recovered by treatment with rhein. The 48‐h median lethal concentration (48 h‐LC₅₀) to grass carp was 148.5 mg/L, which is about 7.4 times the effective dose indicating the safety for the use of rhein. This study suggests that rhein has promising anti‐Saprolegnia activity and may be an option in preventing and controlling Saprolegnia infection.     

草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立

根据草鱼呼肠孤病毒(grass carp reovirus,GCRV)衣壳蛋白VP6编码基因的序列设计特异性引物,以病毒全基因组RNA为模板,通过对反应条件进行优化,建立了GCRV的逆转录环介导等温扩增(RT-LAMP)检测方法。检测结果表明,本方法可在63℃下1 h内实现靶片段的大量扩增,扩增产物经凝胶电泳呈现梯型条带,反应体系中添加SYBR Green I荧光染料后,绿色阳性结果明显区别于橙色阴性结果。该检测体系针对草鱼呼肠孤病毒的检测灵敏度高,其最低检测限为33 pg,与常规RT-PCR方法相比较,灵敏度高10倍,且与斑点叉尾鮰呼肠孤病毒(CCRV)、鲤春病毒血症病毒(SVCV)、锦鲤疱疹病毒(KHV)、大鲵虹彩病毒(GSIV)等无交叉反应。该方法灵敏度及特异性高,且不需昂贵仪器设备,为快速检测草鱼呼肠孤病毒与诊断草鱼出血病提供了简捷快速的技术手段。