中国对虾杂交优势对自然感染白斑综合征病毒的抗病力分析

中国对虾杂交优势对自然感染白斑综合征病毒的抗病力分析=Analysis of the resistance of heterosis in Fenneropenaeus chinensis to natural infection with white spot syndrome virus ［刊，中］/李素红（中国水产科学研究院黄海水产研究所，青岛 266071)，张天时，孟宪红，孔杰//水产学报，—2007，31(1).-68～75 一个包括42个全同胞家系的中国对虾选育群体，荧光标记后每个家系各取40尾健康个体混养，自然发病并确认感染白斑综合征病毒(white spot-syndrome virus，WSSV)。发病1周后将所有个体取出，统计各家系的存活率，并对活体利用巢式聚合酶链式反应(polymerase chain reaction，PCR)进行WSSV检测。对存活率进行分析，结果显示：4个家系的存活率在60％以上，7个家系的存活率在20％以下；存活率与第一次扩增阳性率、第二次扩增阳性率呈不显著负相关。第一次扩增阳性率与第二次扩增阳性率呈极显著正相关。结合两次扩增的阳性率分析得出：青岛野生群体内自交产生的一个家系和韩国群体养殖家系与乳山野生群体杂交产生的一个家系在抗病力上显著优于其它家系；经独立样本t检验表明，群体间交配组合的存活率差异不显著，因此家系选育可能是进行抗WSSV品种选育的重要途径。多重比较结果发现：韩国群体养殖家系(♂)与乳山野生群体(♀)杂交后代的存活率(56.20%±8.75%) 显著高于韩国群体养殖家系自交后代(22.50%±2.50%) (P<0.05)，说明该杂交后代在抗WSSV方面有一定程度的杂种优势。而韩国群体养殖家系 (♂)和乳山野生群体(♀)的杂交后代所表现的杂种优势(26.95)与其反交后代(3.85)差异显著。图3表5参15 关键词：中国对虾； 巢式PCR； 白斑综合征病毒 E-mail:kongjie@sina.com     

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。     

中国明对虾抗白斑综合症病毒分子标记的筛选

采用随机扩增多态性DNA(RAPD)技术对经过连续4代选育的抗白斑综合症病毒(WSSV)中国明对虾(Fenneropenaeus chinensis)(174)及未经选育的与174来源相同的中国明对虾野生群体(HB)进行分析,以期筛选出与抗病性状相关的分子遗传标记,为进一步的基因定位以及中国对虾的种质资源保护和分子标记辅助育种提供有力的技术支撑和理论依据。共进行220个RAPI)单引物和114对双引物的检测,产生标记数目共计2439个。依据标记在群体中出现的频率和变化规律,共筛选出5个可能与抗病相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析,发现测得片段的序列与数据库中序列的相似性较低,未能找到与所测序列同源性较高的功能基因。这与利用AFIP技术分析的结果一致。由于选育过程施加人工选择,推论这些特异性标记虽不是抗WSSV分子标记,但可能与抗病性状密切相关。     

多重PCR检测对虾白斑综合症病毒和传染性皮下组织坏死病毒

采用对虾白斑综合症病毒和传染性皮下组织坏死病毒的特异引物,在一个PCR反应中同时扩增到大小分别为271 bp和356 bp的病毒特异片段,多重PCR条件优化后可从最少100 fg级病毒DNA模板中定性检测出此两种病毒。     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.     

Optimization of experimental infection protocols for the estimation of genetic parameters of resistance to White Spot Syndrome Virus (WSSV) in Penaeus (Litopenaeus) vannamei

Two infection protocols, individual oral (IO) and waterborne infection (WB), were evaluated to challenge Penaeus (Litopenaeus) vannamei with White Spot Syndrome Virus (WSSV). Five different batches of full and half-sib families were infected experimentally and tested for growth performance under commercial growth conditions. The genetic variance for WSSV resistance was estimated using a linear sire-dam repeatability model that considers test-day survival as the dependent variable. The heritability estimates using the IO protocol ranged from 0.01 ± 0.00 to 0.02 ± 0.01, whereas the heritability estimates using the WB infection protocol were not significantly different from zero. The genetic correlations measured as the product moment correlation between full-sib family breeding values for resistance to WSSV and harvest body weight in ponds were unfavourable in three of the five batches and favourable in one of them. The dosage of WSSV was better controlled with IO oral infection than with other methods, with all animals being exposed to approximately the same risk of infection at the same time. This should improve the accuracy of the genetic parameters and hence improve the accuracy of the breeding values. It should, however, be noted that once the outbreak was established and the mortalities began, the shape and the magnitude of the slope of the mortality curves showed little difference in the infection pattern between batches irrespective of the dosage and infection protocol, and in most of the cases the cumulative mortality was greater than 80%. The main reason for this is probably the high densities of animals in the tanks needed for the genetic evaluations.     

Protection of Procambarus clarkii against white spot syndrome virus using recombinant subunit injection vaccine expressed in Pichia pastoris

ABSTRACT:   The potentiality of injection vaccine against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. WSSV envelope proteins VP19 and VP28 were expressed in yeast Pichia pastoris GS115. The purified recombinant proteins (2 µg/g of crayfish) were injected intramuscularly, and the same dose injected as a booster shot on fifth day after vaccination. The vaccinated crayfish were divided into two even groups and later challenged orally by WSSV-infected dead crayfish muscle (2 g/individual) on the third and 21st days after the booster shot. The relative percent survival (RPS) in the third-day group was the highest in VP28 (91%), followed by VP19 + VP28 (84%), and VP19 (45%). The RPS for the 21st-day group was the highest in VP28 (78%), followed by VP19 + VP28 (76%), and VP19 (17%). Development of vaccine by using recombinant proteins VP19 and VP28 expressed in yeast is feasible.     

Pathogenicity of white spot syndrome virus on postlarvae and juveniles of Penaeus (Litopenaeus) vannamei

White Spot Syndrome Virus (WSSV) has decimated the shrimp aquaculture around the world. Breeding efforts to generate resistant stocks are necessary but there is a lack of basic information on challenge test strategies focused on genetic selection. Infection routes and developmental stages were evaluated on Penaeus vannamei as a first step in a strategy to select white spot virus (WSSV)-resistant stocks. Mortalities could not be induced before the PL30 stage. The impact of infection by immersion and blended tissue was intermediate on mortalities when compared to the minced tissue treatment on PL30. Blended and minced tissue treatments produced the highest mortalities on PL40 while immersion was intermediate. A general tendency towards higher susceptibility associated with older stages was detected. Additionally, juveniles of 1 g average weight from three local breeding programs were challenged. There were no differences in survival between the programs, although two of them derived their progenies from survivors of strong WSSV events. The implications of these results to the WSSV epidemiological characteristics and breeding programs are discussed.     

人工选育中国对虾两个群体WSSV感染相关免疫与生化因子的变化

通过分析中国对虾人工选育两个群体WSSV感染相关免疫与生化因子的变化，研究选育的两个中国对虾群体对WSSV的敏感性和抵抗力。结果表明，两个群体感染WSSV后，总细胞数（THC）在24h达到最大，随后呈下降趋势；两个群体血淋巴蛋白在感染初期和中期变化不同，但在后期均呈下降趋势；相比而言2＇对虾群体比6＇对虾群体血蛋白含量和THC下降幅度稍慢；2＇对虾群体和6＇对虾群体感染24h后酸性磷酸酶（ACP）稍有下降，随后保持较高的活性，而碱性磷酸酶（AKP）的变化除2＇对虾群体在感染48h有显著增大外，6＇对虾群体变化不明显；过氧化氢酶（CAT）、活性氧（ROS）、超氧化物歧化酶（SOD）与WSSV感染有密切的联系，两个群体抗氧化酶活性随WSSV增殖的变化略有不同；细胞内酚氧化酶（proPO）原变化趋势也表现出差异，斑点杂交结果显示6＇对虾群体比2＇对虾群体阳性反应较早，揭示不同群体间对WSSV感染的敏感性存在一些差异。