Interaction between a Peptide and White Spot Syndrome Virus VP28 Envelope Protein

White spot syndrome virus (WSSV) is one of the most important pathogens that endanger the global shrimp aquaculture. Studies have confirmed that in the early stage of infection, VP28, the main envelope protein of WSSV, is used as a viral adhesion protein to bind PcRab7 of Penaeus chinensis, helping virus enter the host cells, resulting in shrimp infection. Hence, inhibition of envelope protein VP28 would be a novel way to deal with the infection. Peptide 2E6 was confirmed to have a high specificity and blocked virus infection. However, the mechanism by which it combines with VP28 is not clear. Clarifying the binding mechanism between peptides and VP28 is of great significance for further optimization and screening of antiviral peptides. In this research, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions aims to clarify the blocking mechanism.

Impact of Vibrio parahaemolyticus and white spot syndrome virus(WSSV)co-infection on survival of penaeid shrimp Litopenaeus vannamei

White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.

Peritrophin-like protein from Litopenaeus vannamei (LvPT) involved in white spot syndrome virus (WSSV) infection in digestive tract challenged with reverse gavage

The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus （WSSV） in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of Litopenaeus vannamei （LvPT） was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of LvPT was predicted with the GenScript OptimumAntigenTM design tool. An anti-LvPT polyclonal antibody was produced and shown to specifically bind a band at -27 kDa, identified as LvPT. The LvPT protein was expressed and its concentration determined. LvPT dsRNA （4 pg per shrimp） was used to inhibit LvPT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection （hpi）. Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after LvPT dsRNA interference than in those of the controls at 48 and 72 hpi. Our results imply that LvPT plays an important role during WSSV infection of the digestive tract.

Construction of white spot syndrome virus (WSSV) whole genome phage display library

A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus （WSSV） genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ～2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 10^5.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ～ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

Proteomic Analyses of the Shrimp White Spot Syndrome Virus

White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.

White spot syndrome virus envelope protein VP124 involved in the virus infection

White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV virions were mixed with three antisera against WSSV envelope proteins （VP39, VP124 and VP187 ）, individually. And then they were injected intramuscularly into crayfish （Procambarus clarkii） to conduct in vivo neutralization assays. The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124, the crayfish mortalities were 100% and 60% on the 8th day postinfection, individually. The virus infection could be delayed or neutralized by antibody against the envelope protein VP124. Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infection. The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish. All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.

White spot syndrome virus inactivation study by using gamma irradiation

The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus （WS SV）. White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 1054/mL. Shrimp post-larvae （1-2 g） were treated with gamma-irradiated （different doses） WSSV （10^o to 10^-4 dilutions） for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14- 15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gamma- irradiated WSSV especially at 14-15 KGy.

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

BALB/c 老鼠与净化的白点症候群病毒(WSSV ) 被使免疫。六根 monoclonal 抗体房间线被 ELISA 选择， VP28 蛋白质在 E 表示了。coli。在 vitro 中立化，实验证明他们中的 4 个能在喇蛄禁止病毒感染。西方污点建议所有这些 monoclonal 抗体对 VP28 的 conformational 结构。monoclonal 抗体 7B4 用胶体的金粒子被标记并且过去常由标记的 immunogold 在病毒信封上定位 VP28。这些 monoclonal 抗体能被用来为 WSSV 感染开发免疫学的诊断方法。关键词怀特点症候群病毒(WSSV )- Mab - 信封 - 本地化 - 中立化 CLC 数字 S945.

Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

凡纳对虾核心育种群生长和抗WSSV性状的遗传参数估计

为开展凡纳对虾(Penaeus vannamei)生长和白斑综合征病毒(white spot syndrome virus,WSSV)抗性复合选育,本研究以凡纳对虾59个核心育种群家系1770尾77~94日龄的个体为实验材料,利用两性状动物模型、公母畜阈值模型,评估在WSSV感染情况下凡纳对虾体长、抗WSSV存活时间和家系WSSV半致死存活率的遗传力和遗传相关。结果显示,凡纳对虾体长、抗WSSV存活时间和家系WSSV半致死存活率遗传力估计值分别为0.17±0.05、0.18±0.05和0.14±0.05,均属于中等遗传力水平,且均与0差异极显著(P<0.01)。凡纳对虾体长与抗WSSV存活时间性状的遗传相关系数为0.15±0.20,与家系WSSV半致死存活率性状的遗传相关系数为0.25±0.22,上述遗传相关与0差异不显著(P>0.05);抗WSSV存活时间性状与家系WSSV半致死存活率性状的遗传相关系数为0.96±0.03,遗传相关与1差异不显著(P>0.05),为高度正相关。结果表明,在该育种群体中,凡纳对虾生长与WSSV抗性可根据育种需要,通过赋值制定综合选择指数,进行多性状复合选育。此外,为优化每代育种的操作过程,可选用家系WSSV半致死存活率作为WSSV抗性的指标性状。本研究为开展凡纳对虾生长和WSSV抗性优良品种的选育提供了基础数据和理论支撑。

WSSV、IHHNV、EHP和NHPB四重荧光定量PCR检测方法的建立

本研究以对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)的VP664基因、传染性皮下及造血组织坏死病毒(Infectious Subcutaneous and Hematopoietic Necrosis Virus,IHHNV)的NSP2基因、虾肝肠胞虫(Enterocytozoon Hepatopenaei,EHP)的ssr RNA基因以及坏死性肝胰腺炎细菌(Necrotizing Hepatopancreatitis Bacteria,NHPB)的16Sr RNA基因为检测对象,建立了WSSV、IHHNV、EHP和NHPB的四重荧光定量PCR方法,并对该方法的特异性、灵敏性进行了研究。结果表明,建立的四重荧光定量PCR方法特异性良好,对WSSV、IHHNV、EHP和NHPB的最低检测限为4.05 copies·μL^(-1)、6.53 copies·μL^(-1)、5.85 copies·μL^(-1)和6.18 copies·μL^(-1),本实验建立的方法具有较好的应用前景。

白斑综合征病毒(WSSV)灭活制剂对克氏原螯虾抗WSSV的研究

【目的】研究经双乙烯亚胺(BEI)灭活的白斑综合征病毒(WSSV)灭活制剂保护克氏原螯虾Procambarus clarkii抗WSSV感染的效果,以期为白斑综合征的防治提供有效的免疫方法。【方法】应用BEI对WSSV和WSSV超声破碎液进行灭活,通过口服和注射分别对克氏原螯虾进行免疫,再对其进行抗WSSV感染的效果研究。【结果】WSSV在BEI处理24 h可以被完全灭活,通过口服途径用灭活制剂免疫克氏原螯虾7 d后攻毒,克氏原螯虾死亡率显著下降,且口服免疫的效果要好于注射免疫。【结论】经BEI灭活24 h的WSSV灭活制剂对克氏原螯虾是安全的,口服免疫后可以显著降低克氏原螯虾感染白斑综合征病毒的死亡率。图2表3参19。

白斑综合症病毒(WSSV)的宿主调查

用地高辛 (DIG)标记的WSSVDNA探针斑点杂交与原位杂交技术 ,在中国对虾、斑节对虾、南美白对虾、刀额新对虾、脊尾白虾、天津厚蟹、日本大眼蟹体内检测到了WSSV ,它们是WSSV的天然宿主 ;在经人工感染的哈氏美人虾、短脊鼓虾、克氏原螯虾、肉球近方蟹、滕壶体内检测到了WSSV ;在球形侧腕水母、病虾池的桡足类等浮游生物、卤虫无节幼体以及人工浸泡感染卤虫成体体内没有检测到WSSV。经原位杂交检测 ,虾类的甲壳下上皮、胃上皮、附肢、造血组织、鳃等组织器官均可被WSSV侵染 ,其中甲壳下上皮和鳃对WSSV敏感 ;蟹类的甲壳下上皮和鳃对WSSV敏感 ;在中国对虾、南美白对虾、脊尾白虾、注射感染的克氏原螯虾的精巢中 ,精荚的结缔组织细胞和血细胞呈阳性 ,在中国对虾、脊尾白虾以及注射感染的短脊鼓虾的卵巢中 ,结缔组织细胞和滤泡细胞被WSSV感染。

套式PCR检测斑节对虾白斑症病毒(WSSV)

解剖患白斑症斑节对虾的鳃组织 ,制备成不同稀释倍数的模板 ,对其进行白斑症病毒 (WSSV)的 PCR扩增 ,结果显示所建立的套式 PCR的灵敏度大约为一步 PCR的 10 4 倍 ;对感染病毒后不同时期的斑节对虾进行 PCR检测 ,发现感染早期经一步 PCR检测为 WSSV阴性的样品 ,套式 PCR的检测结果为阳性 ;对发病虾塘中的几种甲壳类动物进行 PCR检测 ,发现经一步 PCR检测为阴性的长臂虾、秉氏厚蟹和褶痕相手蟹等宿主 ,套式 PCR的检测结果为阳性。表明所建立的 WSSV套式 PCR检测法较普通的一步

凡纳滨对虾抗WSSV选育家系的建立及其抗病特性

2002—2007年在人工感染白斑综合征病毒(white spot syndrome virus,WSSV)的基础上进行一代个体选育(G1)后,对凡纳滨对虾连续进行4代家系选育,共建立120个抗WSSV家系,感染实验结果显示,G2～G5选育家系对虾平均成活率分别为5.57%±9.83%,8.66%±11.52%,9.52%±8.84%和13.79%±12.86%;G2～G5选育家系对虾平均成活率的变异系数分别为1.77、1.40、0.97和0.87。根据每个家系对虾的成活情况每个世代可分为敏感、中等抗性和高抗性家系,G2～G5敏感家系在各代选育家系中的比例逐年下降,分别占76.5%、55.2%、51.4%和33.3%,抗病成活率分别为0.44%±1.09%、0.78%±1.70%、2.27%±2.76%和2.44%±3.09%,感染WSSV后2～3 d出现1个急性死亡高峰;中等抗病家系在各代选育家系中的比例逐年上升,分别占0、20.7%、31.1%和38.5%,抗病成活率分别为0、9.08%±1.46%、10.7%±1.41%和11.36%±3.30%,感染WSSV后出现2个死亡高峰,第1死亡高峰值大于第2高峰;高抗家系在各代选育家系中的比例逐年上升(G4除外),分别占23.5%、24.1%、17.1%和28.2%,抗病成活率分别为22.23%±5.21%、22.70%±12.30%、24.45%±6.56%和28.98%±8.09%,感染WSSV后出现2个死亡高峰,第1死亡高峰值小于第2高峰。经连续的定向选育,对虾抗病性状一代比一代强,表现出明显的抗病性能,特别是高抗对虾不仅死亡率低且其死亡高峰推迟2～3 d,延缓了对虾WSSV暴发的时间,但是每代每尾对虾平均产卵量逐年下降。

第四代凡纳滨对虾抗WSSV选育家系的抗病及免疫特性研究

35个第四代凡纳滨对虾抗WSSV选育家系和未选育对虾按每克体重注射103拷贝WSSV病毒量,根据选育家系的抗病性能分3个类群:高抗性类群,成活率达24.45%±6.56%;中抗性类群,成活率为10.70%±1.41%;敏感类群,成活率为2.72%±2.76%,各类群间差异显著(P<0.01)。对分别代表高抗、中抗和敏感类群的12、7和3号家系以及未选育对虾按每克体重注射102、103、104和105拷贝WSSV,高抗性对虾在102、103、104及105感染水平下的存活率分别为100%、23.3%±3.5%、7.8%±1.9%和0%;中抗对虾分别为87.7%±3.9%、12.2%±1.9%、0%和0%;敏感对虾分别为54.4%±3.9%、2.2%±1.9%、0%和0%;未选育对虾分别为51.1%±5.1%、0%、0%和0%。在103拷贝组感染过程中免疫相关因子的变化表明,高抗对虾血液中血细胞数分别比中抗、敏感和未选育对虾提高20.7%(P>0.05),36.7%(P<0.05)和34.4%(P<0.05);PO活力上述三类对虾提高40.0%(P<0.05),76.3%(P<0.05)和63.4%(P<0.05);SOD活力分别比上述三类对虾提高31.1%(P>0.05),58.8%(P<0.05)和32.0%(P>0.05);POD活力分别比上述三类对虾提高29.6%(P>0.05),44.9%(P<0.05)和43.3%(P<0.05);血清蛋白含量分别比分别比上述三类对虾提高31.2%(P>0.05),38.7%(P<0.05)和39.3%(P<0.05),而敏感对虾和未选育对虾之间则无显著差异。结果表明,经四代选育后的高抗对虾免疫性能明显高于其他对虾,表现出良好的抗WSSV性能。

实用WSSV定量检测方法的建立及其应用于脊尾白虾病毒感染规律的研究

为优化当前白斑综合征病毒(WSSV)定量检测方法,实验结合当前WSSV诊断与定量方法应用的实际,试图通过设计两对普通PCR引物来建立一种实用的WSSV绝对定量方法,并利用此方法进一步探明WSSV在脊尾白虾体内的增殖规律及致病性。首先根据WSSV的囊膜蛋白VP28基因序列设计了两对特异引物(F1,R1)和(RF2,RR2),分别用于标准品质粒的构建和扩增子的扩增,以开展SYBR Green染料法定量检测WSSV的研究;随后利用该定量检测方法分析了WSSV在脊尾白虾体内的组织分布及感染规律。研究结果显示:(1)以定量引物RF2和RR2建立的SYBR Green绝对定量检测方法具有敏感度高、特异性强、定量范围广且准确等特点;(2)WSSV能感染脊尾白虾鳃、肝胰腺、肌肉、头胸甲下结缔组织、胃及肠等组织,其中头胸甲下结缔组织病毒含量最高(1.1×107copies/μg),其次是鳃组织(4.1×106copies/μg);正常脊尾白虾感染WSSV,首先经过一个约48 h潜伏期,而后开始大量增殖并造成部分虾死亡,死亡率约为20%。

白斑综合症病毒(WSSV)囊膜蛋白VP28基因的克隆及在蓝藻中表达载体的构建

用蔗糖密度梯度离心法分离纯化白斑综合症病毒(WSSV)。设计并合成引物 ,提取WSSV中国株的基因组DNA作为模板 ,通过PCR ,扩增克隆出VP28基因 ,利用BamHI和EcoRI切点将VP28基因插入克隆载体 pUC19的多克隆位点上 ,得到VP28基因的重组克隆质粒,对其进行双向DNA测序。测序结果表明,该基因含有615个核苷酸 ,与GenBank中已有不同来源的WSSV的序列片段同源性为100 %。利用BamHI切点将VP28的基因插入到穿梭表达载体启动子PpsbA的下游 ,EcoRI酶切鉴定 ,得到正向连接的可在蓝藻表达的重组穿梭表达载体 ,命名为 pRL VP28。