人α——干扰素及其对草鱼出血病毒的干扰作用

病毒的干扰现象最早于1926年由Mekinney在研究植物病毒时发现的，1932年Gildmeister又发现了动物病毒的干扰现象，直到1957年英国的病毒学家A．Isaccs和J．Lindenmann才发现了干扰素。在以后的36年里，这一领域的研究工作，使人们对干扰素的认识有了根本的改观。现在认为干扰素既是病毒干扰现象的介质，又是调节细咆功能的一类重要物质，它已成为病毒学、细胞学、免疫学、肿瘤学、分子生物学等学科共同的新型研究领域。     

常见渔病用药处方指南（三）

一、蟹类病毒性疾病 蟹类病毒性疾病病原主要为白斑病毒、桃拉病毒、呼肠弧病毒、疱疹病毒等。主要症状为：病蟹呈昏迷状、附肢瘫痪、血液混浊、呈白色并充满微细颗粒。该病主要发生于养殖中后期，由于养殖时间长，底质逐渐败坏，养殖密度大，虾类往往于中后期感染病毒病后出现并发症（吃死虾）。     

1种可用于检测WSSV蛋白酶的微量酶活测定技术

以酪蛋白为底物 ,对常规蛋白酶检测方法进行改进并与偶氮酪蛋白法进行比较 ,建立适应病毒微量蛋白酶的快速检测方法。结果表明 ,改进的微量蛋白酶检测方法具有微量、快速、灵敏度高的特点 ,适于病毒微量蛋白酶的检测。蛋白酶的最低检出量可达ng级。     

鲤春病毒血症病毒(SVCV)的研究进展

鲤春病毒血症病毒（SVCV）是一种能够引起鲤科鱼类急性传染性病毒病-鲤春病毒血症（SVC）的病原，一旦暴发会造成巨大的经济损失。该病主要在欧洲的鲤鱼养殖中广泛传播，主要症状为体内出血，腹膜炎及腹水。SVCV具有囊膜，暂时列为弹状病毒科水泡性病毒属。病毒基因组为线性单股不分段的负链RNA，长11019个核苷酸，主要包含5个基因，分别编码核蛋白N、磷蛋白P、基质蛋白M、糖蛋白G和RNA聚合酶L。日前，中国出入境检验检疫检测SVCV的行业标准是逆转录PCR法。本研究从病毒的生物学特征，感染的临床症状及流行特点，病毒基因组及其多肽特征，病毒的分类地位以及检测技术等方面对国内外研究的现状作一综述，以期为研究者提供理论参考。     

动物病毒细胞受体研究及应用

细胞受体是介导病毒入侵的决定因素。本文阐述动物病毒与细菌受体间的关系，一般来说二者之间是一对一，但也有多个病毒共用一个受体或一个病毒有多个受体的情况。病毒与受体的相互作用启动子病毒的生命特环，包括结合和进入的过程；一些寻找病毒受体的直接观察法及分子生物法。介绍了一些已经确定的病毒受体及其在对虾病毒病研究中的应用。     

罗氏沼虾苗种肌肉白浊病诺达病毒的分离和特性研究

本实验在以往罗氏沼虾(Macrobrachium rosenbergii)苗种肌肉白浊病病毒致病性的基础上对病毒特性进行深入研究。病虾除菌过滤液以1：50和1：250浸泡感染可复制出典型的症状，病毒对氯仿抵抗；用PEG法浓缩病毒后，35000r／min超速离心部分纯化病毒，在电镜下可观察到大量直径24nm的球形病毒颗粒，无囊膜，此外还观察到大小为14nln的病毒粒子；采用Trizol试剂对病毒进行了核酸提取，电泳结果发现有4条核酸条带，分别为3．0kb、1．3kb、0．8kb、0．75kb，对RNA核酸酶、S1核酸酶敏感，对DNA酶抵抗．为单链RNA病毒。用罗氏沼虾诺达病毒核酸探针进行分子杂交，探针可与提纯核酸及病虾样品匀浆液反应，Southern杂交表明3．0kb、1．3kb条带分别属于诺达病毒的RNAl和RNA2。对不同的发病样品进行了核酸电泳，4个典型症状的样品均存在诺达病毒条带，2个样品还存在小病毒核酸。上述结果表明，诺达病毒是引起罗氏沼虾苗种肌肉白浊病的主要病原。14nm小病毒与罗氏沼虾肌肉白浊病中的关系有待进一步的研究。     

草鱼呼肠孤病毒的致病机制及抗病毒新对策

草鱼呼肠孤病毒(Grass carp reovirus,GCRV)引发草鱼病毒性出血病,是危害性最大的草鱼病原体。该病毒基因组由11条双链RNA组成,共编码7种结构蛋白和5种非结构蛋白。报告总结了呼肠孤病毒在基因表达、蛋白质合成、细胞凋亡、细胞融合、细胞膜渗透、干扰素分泌、细胞分裂以及细胞压力小体等方面与宿主细胞相互作用的生物学研究进展;论述了蛋白酶抑制剂、RNA干扰(RNAi)机制、干扰素诱导素、GCRV干扰颗粒及小分子化合物等抗病毒策略的分子作用机制。报告预测基于中草药的免疫增强剂和口服化基因工程抗病毒疫苗将是最容易被市场化的两种绿色抗草鱼出血病药物。     

海洋贝类病毒性疾病研究概述

目前已发现20多种感染贝类的病毒，其中几种已被证实可导致养殖贝类流行性疾病和大量死亡。人工养殖贝类个体密度较高，为病毒传播提供条件，因此贝类病毒研究越来越引起人们的重视。     

刺参一种囊膜病毒的分离及其超微结构观察

利用电镜负染技术检测发病的养殖刺参[Apostichopus japonicus（Selenka）]组织提取液。观察发现，提取液中存在大量病毒样粒子，该病毒粒子近似球形，具有囊膜。囊膜内可见高电子密度的核心结构。完整的病毒粒子直径为80～100nm，囊膜厚6～10nm，核心结构直径35-45nm，呈六边形。应用超薄切片技术对刺参的触手顶部、触手臂、疣足、呼吸树、背肠血管、肠等组织的病毒感染状况进行观察，发现该种病毒粒子大量存在于所检测各组织内。感染细胞超微结构表现为大量细胞器崩解形成空泡结构，并出现“髓袢样”结构等病理变化。根据观测结果，该病毒是一种无包涵体病毒。     

草鱼呼肠孤病毒HZ08株的分离与鉴定

从浙江省湖州地区采集发病草鱼(Ctenopharyngodon idellus)样本中,取症状明显病料的肝、脾、肾组织,经过滤除菌处理后接种草鱼肾脏细胞(CIK).盲传8代,CIK细胞未出现明显细胞病变,但感染细胞固定后经电镜观察发现,细胞质内有大量病毒聚集,病毒无囊膜,近球形,直径约70 nm,形态与已报道的草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)相似.将病毒提纯后分别经DNA酶、RNA酶和绿豆核酸酶消化,证实为双链RNA(dsRNA)病毒.十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,病毒基因组由11个dsRNA组成,呈现水生呼肠孤病毒基因组典型特征.采用RNA水平3味端加接头的方法获得了S6节段的全长序列,测序结果表明,S6由2 030个核苷酸组成,推测其编码一个分子量约为68.4 kD的蛋白.聚类分析结果显示,该病毒为水生呼肠孤病毒,但在氨基酸水平上与草鱼呼肠孤病毒代表株873株的差异较大,同源性为33%,提示该病毒为一株新型的草鱼呼肠孤病毒.本研究旨在为草鱼出血病防治方法的深入研究奠定基础.     

Proteases secreted by strains of Aeromonas salmonicida

Abstract. The proteases secreted by four strains (MT004, 1102, 480 and 480P^-) of Aeromonas salmonicida grown in liquid culture have been studied. Strains MT004, 1102 and 480 all show a similar pattern with two types of proteases produced; one of molecular weight 70 000 which is active against casein and gelatin and one (or more) of lower molecular weight (about 20 000) which is (are) active against gelatin but not casein. Strain 480P^- produces only the latter type of protease(s). The protease of molecular weight 70 000 is classified as a serine-type protease, but further characterization of the features of its active site has not yet proved possible. The results are discussed in terms of the previously published but often contradictory data on the proteases of A. salmonicida.     

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase^®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.     

Protease in sturgeon sperm and the effects of protease inhibitors on sperm motility and velocity

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.     

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca²⁺ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.     

酶法提取烤鳗下脚料水解动物蛋白的研究

以烤鳗下脚料为原料，经蛋白酶水解后，提取水解动物蛋白，使废充物得到充分利用。通过正交实验，确定了木瓜蛋白酶、枯草杆菌蛋白酶、胃蛋白酶的最佳水解条件，选择了木瓜蛋白酶为最佳水解酶。     

Genogrouping of birnaviruses isolated from marine fish: a comparison of VP2/NS junction regions on genome segment A

Yellowtail ascites virus and related strains isolated from marine fish have been shown to be similar to infectious necrosis virus (IPNV) in terms of biological and serological characteristics. This paper explores the relationship of aquatic birnaviruses at the genetic level. The junction region on the genome segment A coding viral capsid protein VP2 and viral protease NS was amplified by PCR in six marine strains. Analysis of nucleotide and the deduced amino acid sequences revealed that the six marine strains have amino acid variations in the possible amino terminus of NS when compared to IPNV. The six marine strains form a new genogroup which is distinguished from three serotypes ofPNV.     

Characterization of protease activity in developing discus Symphysodon aequifasciata larva

The activity of different protease classes was monitored in developing discus (Symphysodon spp.) larvae using a combination of biochemical assays and substrate SDS–PAGE techniques. Results showed the presence of alkaline proteases of serine proteases such as trypsin with a significant increase in activity levels detected beginning 3 days after hatching. Other alkaline proteases such as metallo‐proteases and chymotrypsin, a type of serine protease, were only detected in older larval stages, at around 20–30 days after hatching. Acidic protease activity was very low during the first 20–25 days of development before showing a significant (P < 0.01) rise. This is despite the formation of a stomach observed 10 days after hatching. Based on the development of the protein digestive system observed, the use of microdiets to replace Artemia should be considered 25 days after hatching.     

Proteases and protease inhibitors of possible clinical relevance in COPD of horses

The importance of proteases and protease inhibitors for the pathogenesis of pulmonary emphysema and chronic bronchitis of the horse is described. Endogenous elastases from neutrophil granulocytes and macrophages, which probably provoke emphysema in the human being, are not relevant in horse emphysema. Exogenous elastases from different species of streptomyces may be responsible for emphysema generation in this species. Part of the exogenous elastases are poorly or not inhibited at all by the equine blood protease inhibitors especially by alpha 1-protease inhibitors. A disorder similar to genetic alpha 1-protease inhibitor deficiency in the human being could not be found in the horse. Proteases and protease inhibitors are probably also relevant for the pathogenesis of chronic bronchitis. Neutral proteases from neutrophil granulocytes may be relevant as initiators or amplifiers of an inflammation in the human being and in the horse. Under physiological conditions the proteases are controlled by the secretory protease inhibitor called HUSI-1 in the human being. In contrast, the horse lacks a protease inhibitor proper to secretion in its respiratory ducts. Protease activity, which correlates with the degree of the COPD, was detected in equine inflamed tracheobronchial secretions. This finding is useful in diagnostic evaluation of the individual disease.     

Effect of plant protease inhibitors on digestive proteases in two fish species, Lutjanus argentiventris and L. novemfasciatus

This work provides a comparative study of the inhibitory effect of several plant protein sources on digestive proteases of two snappers: yellow snapper (Lutjanus argentiventris) and dog snapper (Lutjanus novemfasciatus). Seed extracts did not affect gastric proteases whereas they significantly inhibit intestinal proteases. Inhibition of alkaline proteases showed that pancreatic proteases of L. argentiventris were more sensitive to seed protease inhibitors than those of L. novemfasciatus. Legume seeds showed the highest inhibitory capacity on alkaline proteases causing inhibition higher than 50% in total proteolytic activity. Protease inhibition on digestive extracts was assessed using different relative concentration of seed extracts and represented by constructing dose response curves. In order to reduce the inhibitory effect, seed extracts were acid-treated before the inhibition assay. Results showed that acid treatment did not affect the inhibitory capacity of seeds on alkaline proteases in both species. However, when the action of gastric enzymes was simulated on seed extracts, the inhibitory capacity was reduced significantly, mainly in the case of L. novemfasciatus. The responses of fish enzymes to heat-treated seed extracts were also tested. Only higher temperatures were capable of reducing the inhibitory capacity of seed, with the specific response to the snapper species. The use of biochemical assays allows us to quantify the action of inhibitors on total proteolytic activity. In addition, zymograms obtained by substrate-SDS-PAGE provided qualitative information about the number and type of proteases affected by each inhibitor. Each seed extract produces a characteristic profile of inhibition on alkaline protease. The results obtained are important for future formulation of feeds for these snapper species.     

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.