YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.     

Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA

RNA interference (RNAi) has been shown to inhibit viral replication in some animals and plants. Whether the RNAi is functional in shrimp remains to be demonstrated. In vitro transcribed dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 were transfected into shrimp primary cell culture and found to inhibit YHV replication. dsRNA targeted to nonstructural genes (protease, polymerase, and helicase) effectively inhibited YHV replication. Those targeted structural genes (gp116 and gp64) were the least effective. These findings are the first evidence that RNAi-mediated gene silencing is operative in shrimp cells. This could be a powerful tool for studying gene function and to develop effective control of viral infection in shrimp.     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病素养(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病素养(基因组约290kDa,双链环状).     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病毒(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病毒(基因组约290kDa,双链环状)。WSSV病毒粒子为卵形杆状,外被囊膜,囊膜在尾部延伸成一长尾。它不仅能感染对虾,还能感染其它淡水及海水甲壳类。养殖对虾被感染后,3—10d内累积死亡率可达100％,给对虾养     

斑点杂交检测对虾白斑综合征病毒青岛株在螯虾体内的动态分布

近年来 ,我国学者对人工养殖对虾暴发性病毒病的病原进行了较为系统的研究[1～ 5] ,本试验应用螯虾这一动物模型[6] ,利用斑点杂交方法 ,研究了白斑综合征病毒 (ＷＳＳＶ ,前称无包埋体对虾病毒Ｎｏｎ -Ｏｃｃｌｕｄｅｄ -ＳｈｒｉｍｐＶｉｒｕｓＮＯＳＶ )青岛株在螯虾体内的动态分布 ,为研究该病毒的传播途径、增殖致病机理提供了参考。1　材料与方法1.1　实验动物克氏原螯虾 (Ｃａｍｂａｒｕｓｐｒｏｃｌａｒｋｉｉ ,以下简称螯虾 ) 40尾 ,购自南京某农贸市场 ,实验室饲养一周以上 ,健康存活。1.2　种毒处理及接种白斑综合征病毒青岛株 (…     

对虾白斑综合症病毒囊膜蛋白VP28的表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp28的序列,设计并合成引物,PCR扩增得到vp28基因,成功构建重组表达载体pET22b-vp28并转化大肠杆菌BL21(DE3)。基因工程菌株37℃IPTG诱导,表达产物经Western-blot和SDS-PAGE检测显示有与预期大小32kDa相符合的目的蛋白。用Ni2 -柱纯化的目的蛋白分别直接注射螯虾和包被饲料投喂螯虾,实验结果表明vp28在大肠杆菌中的表达产物有显著提高虾体抗WSSV感染力的作用,而且注射效果更好。     

对虾白斑综合症病毒囊膜蛋白VP19的融合表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp19的序列,设计并合成引物,PCR扩增得到vp19基因并克隆到pGEM‐T载体中,经过BamHⅠ/HindⅢ酶切、连接并将vp19插入到pET32b表达载体中。用重组质粒pET32b-vp19转化大肠杆菌Origam(iDE3)pLysS,在IPTG诱导下,融合蛋白Trx-VP19以可溶性的形式得到表达,经SDS-PAGE和Western-blot检测显示其分子量与预期的大小相符合。目的蛋白经Ni2 柱纯化并定量后分别直接注射鳌虾和包被饲料投喂鳌虾。实验结果表明注射Trx-VP19可以提高鳌虾个体抗WSSV感染力的作用。     

对虾白斑综合征病毒中国地方株变异区基因的比较

对虾白斑综合征病毒(White spot syndromevirus,WSSV)是对虾养殖的主要病原之一,它是目前发现的基因组最大的动物病毒,为环状双链DNA病毒[1,2],全基因组序列分析结果显示,对虾白斑综合征病毒和其他杆状病毒相差甚远,最新病毒分类报告已将该病毒划归新建立的线头病毒科(Nima-viridae)白斑病毒属(Whispovirus)[3,4]。目前Gen-Bank公布有3个版本的WSSV全序列[1,2],其基因组大小的测定结果相差较大。不同的WSSV毒株可能在形态结构、理化性质上无法区分,但病毒基因组限制酶切片段长度多态性(RFLP)可以将之区分开来,Marks等[6,7]通过计…     

Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae

We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microalgae (Isochrysis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japonicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after 1 and 3 days, were negative after 10days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae.     

Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shr...     

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

根据GenBank上WSSV囊膜蛋白基因vp19和vp28的序列,设计并合成两对引物,PCR扩增得到vp19和vp28两基因,大小分别为370bp和630bp。通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b( )中,构建出重组表达载体pET-vp(19 28)并转化大肠杆菌BL21(DE3)。基因工程菌株35℃IPTG诱导,表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带。用Ni^2 -柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对WSSV的中和效率达到了100％。     

Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp

Sulfated galactans (SG) were isolated from the red seaweed Gracilaria fisheri (G. fisheri). Chemical analysis revealed SG contains sulfate (12.7%) and total carbohydrate (42.2%) with an estimated molecular mass of 100 kDa. Structure analysis by NMR and FT-IR spectroscopy revealed that SG is a complex structure with a linear backbone of alternating 3-linked β-d-galactopyranose and 4-linked 3,6-anhydrogalactose units with partial 6-O-methylate-β-d-galactopyranose and with sulfation occurring on C4 of d-galactopyranose and C6 of l-galactopyranose units. SG treatment enhanced immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase in shrimp Penaeus monodon. Shrimp fed with Artemia salina enriched with SG (100 and 200 μg ml⁻¹) and inoculated with white spot syndrome virus (WSSV) showed a significantly lower mortality rate and lower viral VP 28 amplification and expression than control. The results suggest that SG from G. fisheri exhibits immune stimulatory and antiviral activities that could protect P. monodon from WSSV infection.     

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

根据GenBank上WSSV囊膜蛋白基因vp19和vp28的序列,设计并合成两对引物,PCR扩增得到vp19和vp28两基因,大小分别为370bp和630bp.通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b(+)中,构建出重组表达载体pET-vp(19+28)并转化大肠杆菌BL21(DE3).基因工程菌株35℃IPTG诱导,表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带.用Ni2+-柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对WSSV的中和效率达到了100%.     

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immun-ological diagnosis methods for WSSV infection.     

Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP （19＋28） Fusion Protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using colloidal gold as an indicator. The fusion protein, VP (19 28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19 28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19 28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group) of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 were classified as negative. The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.     

Development of lateral-flow immunoassay for WSSV with polyclonal antibodies raised against recombinant VP (19+28) fusion protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV,using colloidal gold as an indicator.The fusion protein,VP (19+28),was expressed in E.coli,purified and used to prepare polyclonal antibodies.The purified anti-VP (19+28) IgG were conjugated with colloidal gold.Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes.After assembly,three groups (5 individual animals in each group) of shrimp samples were tested which included healthy,moribund and dead shrimps.For each group,three different tissues (body juices,gills and hepatopancreas) were tested at the same time.In parallel,all the samples were also analyzed using PCR for comparison.Out of 45 samples tested,30 were detected as positive while 15 were classified as negative.The results of LFIA correlate with those obtained by the PCR analysis,indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.     

对虾白斑综合征病毒囊膜蛋白VP53B原核表达及抗血清的制备

【目的】研究对虾白斑综合征病毒(White spot syndrome virus,WSSV)囊膜蛋白sVP53B克隆、表达、纯化及抗血清制备。【方法】根据WSSV囊膜蛋白基因序列,设计引物,PCR扩增出功能序列(Svp53B),构建到pET-16b载体后,转化至大肠杆菌Rosetta 2诱导表达,用SDS-PAGE、Western blotting检测优化表达。表达产物采用Ni-NTA琼脂糖磁珠进行纯化、割胶回收融合蛋白,以纯化的Svp53B-his为抗原,免疫兔子获得多克隆抗体,通过间接ELISA检测抗体的效价。【结果】构建重组质粒pET-16b-Svp53B,在大肠杆菌Rosetta 2中以1 mmol/L IPTG诱导表达量最高,主要以包涵体形式表达。纯化包涵体蛋白免疫兔子,获得多克隆血清,效价达到1:150 000。【结论】原核表达并纯化得到高纯度的WSSV囊膜蛋白sVP53B,制备的兔源多克隆血清亲和力高、特异性好,这对后期进一步研究VP53B与经口侵染相关功能奠定了基础。