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 L itopenaeus vannamei(Lv PT) was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of Lv PT was predicted with the Gen Script Optimum Antigen? design tool. An anti-Lv PT polyclonal antibody was produced and shown to specifically bind a band at ~27 k Da, identified as Lv PT. The Lv PT protein was expressed and its concentration determined. L v PT ds RNA(4 μg per shrimp) was used to inhibit Lv PT 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 L v PT ds RNA interference than in those of the controls at 48 and 72 hpi. Our results imply that Lv PT plays an important role during WSSV infection of the digestive tract.

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 inactivation study by using gamma irradiation

The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus(WSSV). 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 10 5.4 /mL. Shrimp post-larvae(1–2 g) were treated with gamma-irradiated(different doses) WSSV(10 0 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 gammairradiated WSSV especially at 14–15 KGy.

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.

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.

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.

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。

白斑综合征病毒(WSSV)在拟穴青蟹体内增殖的研究

拟穴青蟹(Scylla paramamosain)因肉质鲜美、生长快速而成为我国主要的海水养殖经济蟹类,但是随着养殖规模的不断扩大,病害问题也越发严重。本研究通过流行病学调查和定量PCR跟踪检测的方法,研究了拟穴青蟹对白斑综合征病毒(WSSV)的易感性和WSSV在拟穴青蟹体内的增殖情况。结果表明,拟穴青蟹是WSSV的自然宿主,自然携带率为8.47%。WSSV可通过口服途径感染拟穴青蟹,并在拟穴青蟹体内快速增殖,注射感染5 d后,病毒量达到感染1d时的1.1×109倍,当病毒累积到一定量后即可致死拟穴青蟹。研究表明,拟穴青蟹是WSSV的天然宿主,在虾蟹混养过程中可以通过摄食WSSV感染虾而带毒或发病,并因此成为WSSV传播媒介,从而对虾蟹混养条件下WSSV的防治效果产生重要影响。

凡纳滨对虾和斑节对虾对WSSV敏感性的比较

用6.0×104拷贝、1.2×104拷贝和6.0×103拷贝3种剂量白斑综合症病毒(WSSV)对凡纳滨对虾和斑节对虾进行人工注射感染,比较了两种对虾对WSSV敏感性的差异。结果表明,凡纳滨对虾死亡时间随病毒剂量降低而延长,斑节对虾死亡时间没有明显差异;随病毒剂量的降低,凡纳滨对虾人工注射感染后病毒复制高峰时间显著延长,斑节对虾感染后病毒复制高峰时间相同,WSSV在凡纳滨对虾体内比在斑节对虾体内复制慢。对虾携带WSSV数量最低为3.3×107拷贝.g-1,最高为4.3×108拷贝.g-1。凡纳滨对虾比斑节对虾对WSSV的抵抗性更强。

WSSV人工感染量和饵料对中国明对虾存活时间的影响

为揭示不同白斑综合征病毒(white spot syndrome virus,WSSV)病毒量对于中国明对虾存活时间和存活率的影响,实验设计了逐尾、定量人工感染实验,在确保每尾中国明对虾进食特定量WSSV毒饵后进行观察、分析。结果显示,分别喂食含5.2×108copies、1.0×109copies、2.1×109copies WSSV的毒饵,对虾平均存活时间分别是389.3、323.3和187.3 h,差异极显著(P<0.01);对虾最终累计死亡率都为100%。研究表明,致死量范围内,WSSV的感染量越低,对虾的平均存活时间越长。为了揭示饵料对中国明对虾抗病性能的影响,对已感染WSSV的中国明对虾投喂不同饵料。结果显示,分别喂食活卤虫、鲜蛤肉和配合饲料,对虾平均存活时间分别是281.7、173.9和164.9 h;喂食活卤虫的实验组平均存活时间显著高于喂食配合饲料和鲜蛤肉的实验组(P<0.01);喂食配合饲料和鲜蛤肉的对虾平均存活时间无显著差异(P>0.05);3组累计死亡率都为100%,结果表明,与配合饲料和鲜蛤肉相比,喂食活卤虫更能增强对虾抗WSSV的能力。

常规PCR与巢式PCR法快速检测克氏原螯虾白斑综合征病毒(WSSV)

对传统对虾白斑综合征病毒(WSSV)的PCR检测方法中的病毒模板DNA的提取方法加以改进,建立了一种快速检测克氏原螯虾(Procambarus clarkii)WSSV的PCR方法。本方法将克氏原螯虾肝胰腺组织匀浆液冻融3次进行差速离心后,在病毒沉淀中加入50μL蛋白酶K溶液,室温消化5 min,沸水中煮10 min后,10000 r/min高速离心5 min即可取上清液用于PCR扩增,结果显示:与传统的病毒模板DNA提取方法相比,本方法的PCR结果特异性强,扩增效率高,准确率高,可应用于快速大规模检测克氏原鳌虾中的WSSV。

铜绿微囊藻对WSSV潜伏感染对虾的致死效应

【目的】明确池塘优势蓝藻对潜伏感染白斑综合征病毒(White spot syndrome virus,WSSV)凡纳滨对虾(Litopenaeus vannamei)的致死效应,为完善养殖对虾病害生态防控技术提供理论依据。【方法】分别设阳性对照组(PC)、阴性对照组(NC)、铜绿微囊藻(Microcystis aeruginosa)活藻组(MAL)和铜绿微囊藻死藻组(MAD),加藻组的铜绿微囊藻初始密度均为106cells/mL,每组各设3个平行,试验周期144 h。除NC组对虾不携带WSSV外,其他组的对虾均经WSSV人工注射感染。采用实时荧光定量PCR-TaqMan探针检测对虾样品中的WSSV携带状况,并通过显微计数法统计铜绿微囊藻细胞数量。【结果】NC组对虾在试验结束时(144 h)的累计死亡率为11.1%;PC、MAL和MAD组的对虾累计死亡率随时间推移呈明显升高趋势,至试验结束时的累计死亡率分别为64.4%、82.2%和88.9%,表现为加铜绿微囊藻(MAL和MAD)组的对虾累计死亡率远高于2个对照组(PC和NC)。MAL、MAD和PC组对虾肌肉样品均携带104copies/g以上的WSSV,其中又以MAD组的WSSV携带量最高。在铜绿微囊藻试验组中,对虾死亡率与铜绿微囊藻呈显著相关(P<0.05),与WSSV的相关性则未达显著水平(P>0.05)。【结论】铜绿微囊藻能有效加速WSSV感染对虾的死亡速率,增加对虾累计死亡率,即使是死亡的微囊藻细胞也能促进对虾体内WSSV的增殖。