Prokaryotic expression of a hub protein of white spot syndrome virus with vaccine potential

Envelope proteins of white spot syndrome virus (WSSV) play an important role in viral entry as well as in triggering host defences. To date, some main envelope proteins such as VP28, VP24 and VP19 have been expressed heterologously and proved effective in WSSV prevention. However, VP62, an envelope protein with hub function as well as better antigenicity, has not been focused on. In an attempt to prepare this protein for rapid purification and further functional analysis, N‐terminus‐truncated VP62 was expressed in Escherichia coli using two common fusion tags, including hexahistidine (his6) and solubility‐enhancing tag thioredoxin (Trx). The results showed that the truncated VP62 fused with C‐terminal His‐tag could not be expressed in either E. coli BL21(Plyss) or Arctic Express, but it could be expressed in the form of inclusion bodies in Arctic Express with N‐terminal tag. After refolding and His‐tag affinity purification, the protein with purity over 90% was obtained. This study laid the foundation for evaluation of its vaccine potential as well as further application in WSSV prevention.     

Molecular docking and simulation studies of 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 against VP26 and VP28 proteins of white spot syndrome virus

White spot syndrome virus (WSSV), an aquatic virus infecting shrimps and other crustaceans, is widely distributed in Asian subcontinents including India. The infection has led to a serious economic loss in shrimp farming. The WSSV genome is approximately 300 kb and codes for several proteins mediating the infection. The envelope proteins VP26 and VP28 play a major role in infection process and also in the interaction with the host cells. A comprehensive study on the viral proteins leading to the development of safe and potent antiviral therapeutic is of adverse need. The novel synthesized compound 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 is proved to have potent antiviral activity against WSSV. The compound antiviral activity is validated in freshwater crabs (Paratelphusa hydrodomous). An in silico molecular docking and simulation analysis of the envelope proteins VP26 and VP28 with the ligand 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 are carried out. The docking analysis reveals that the polar amino acids in the pore region of the envelope proteins were involved in the ligand binding. The influence of the ligand binding on the proteins is validated by the molecular dynamics and simulation study. These in silico approaches together demonstrate the ligand's efficiency in preventing the trimers from exhibiting their physiological function.     

对虾白斑综合征病毒囊膜蛋白VP110在中国明对虾鳃细胞中结合蛋白的鉴定与特性

为了鉴定对虾白斑病综合征病毒(WSSV)囊膜蛋白VP110在中国明对虾(Fenneropenaeus chinensis)鳃细胞中的结合蛋白, 运用pET-32(a)+载体构建了1段含RGD模体的截短VP110原核重组表达质粒, 转化大肠杆菌诱导表达后获得分子量为41 kD的截短重组VP110蛋白(rVP110)。以rVP110作为诱饵蛋白, 运用pull-down实验结合蛋白质谱分析鉴定rVP110结合蛋白, 结果显示, 中国明对虾鳃细胞中的肌动蛋白和精氨酸激酶(arginine kinase,AK)与rVP110具有结合作用。利用PCR扩增中国明对虾AK编码基因, 将其与表达载体pGEX-4T-1连接后转化大肠杆菌诱导表达获得重组AK蛋白(rAK), 通过pull-down实验进一步证实rAK可与rVP110发生结合。克氏原螯虾(Procambarus clarkia)体内中和实验结果显示, rAK对WSSV感染克氏原螯虾具有一定的中和作用, 能延缓螯虾的死亡进程。另外, 中国明对虾在人工感染WSSV后, 荧光定量PCR检测结果显示, AK基因表达水平显著上调, 18 h时达到峰值, 然后下降至正常水平; 酶底物法检测结果同样显示, 鳃细胞中AK酶活性在感染WSSV后发生显著上调。本研究旨在为深入了解WSSV囊膜蛋白VP110在WSSV感染宿主过程中的作用提供基础依据。

     

白斑综合征病毒囊膜蛋白VP19及VP28的研究进展

自二十世纪90年代,白斑综合征病毒(WSSV)就因其暴发范围广、致死率高得到了广泛的关注。研究主要集中在确定该病毒蛋白的结构及功能,以及利用其囊膜蛋白制备亚单位疫苗、研发DNA疫苗等来提高对虾抵抗白斑综合征病毒的能力,尽管免疫防治目前在实验室阶段已取得了显著的保护效果,但因其给药方式局限以及成本较高等因素一直没有应用于实际生产中。VP19和VP28是白斑综合征病毒主要的囊膜蛋白,在WSSV感染对虾的过程中起着非常重要的作用。本文从WSSV的基因组学、VP19和VP28的蛋白质结构及其在免疫防治中的应用等方面概述了VP19和VP28的研究进展,包括蛋白亚单位疫苗、DNA疫苗、RNA疫苗以及相关抗体的研究。在总结了不同类型疫苗的保护效果后发现,VP19和VP28的双价疫苗的保护率较高,为今后制定有效的WSSV控制方法提供了参考。     

Protection of crayfish, Cambarus clarkii, from white spot syndrome virus by polyclonal antibodies against a viral envelope fusion protein

White spot syndrome virus (WSSV) is a large double-stranded DNA virus, causing considerable mortality in penaeid shrimp and other crustaceans. WSSV produces five major structural proteins, including two major envelope proteins, VP28 and VP19. To produce VP28 and VP19 as a single protein for antibody production, DNA sequences encoding both open reading frames were fused together and cloned into pET-22b(+) expression vector. The fusion protein, VP(19+28), was expressed in Escherichia coli, purified using Ni2+ His affinity chromatography and injected into a rabbit. Antiserum collected from the immunized rabbit was tested in vivo for ability to protect crayfish, Cambarus clarkii, from disease caused by WSSV. Fifteen days after challenge with WSSV, treatment with VP(19+28) antiserum gave 100% protection against disease in the ambient temperature range of 15-22 degrees C and 65% protection at a constant temperature of 26 degrees C. These results demonstrated VP(19+28) antiserum is effective in protection of crayfish from WSSV and confirmed that VP19 and VP28 play an important role in WSSV host infection. Targeting both VP19 and VP28 may be effective for the design of both immunotherapeutic medicines and reagents to detect WSSV.     

Virulence status,viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of India

The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non‐virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first‐step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS‐PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV‐infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV‐infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality.     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

WSSV与IHHNV竞争对虾鳃细胞膜受体的研究

本研究对白斑综合征病毒(White Spot Syndrome Virus,WSSV)与传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Haematopoietic Necrosis Virus,IHHNV)能否竞争虾鳃细胞膜上的NM23蛋白受体进行探索。先用蔗糖梯度离心法提纯WSSV的全蛋白,利用病毒覆盖蛋白印迹技术(VOPBA)与对虾鳃细胞膜NM23蛋白作用,将疑似蛋白条带进行LC-MS/MS分析,初步筛选出3种WSSV蛋白,分别为WSSV013、Wsv497和Wsv035,再构建这3种蛋白的原核表达载体,通过VOPBA和免疫共沉淀(co-immunoprecipitation)验证了Wsv497、Wsv035能与NM23蛋白相互作用,WSSV013不能与NM23蛋白相互作用。初步推测WSSV与IHHNV可能竞争对虾鳃细胞膜上的NM23蛋白受体,该结果为今后研究两种病毒竞争细胞膜受体和虾病毒蛋白作用机制提供理论基础。     

Protection of Fenneropenaeus chinensis (Osbeck, 1765) against the white spot syndrome virus using specific chicken egg yolk immunoglobulins by oral delivery

Two kinds of specific chicken egg yolk immunoglobulins (IgYs), IgY‐WSSV and IgY‐VP28, were, respectively, raised against the 2 mM binary ethylenimine (BEI)‐inactivated white spot syndrome virus (WSSV) and a principal envelope protein VP28. The activity of purified specific IgYs was stable under the conditions of 20–70 °C, pH 3.0–10.0 and 0–700 g L^?1 sucrose solution. In the neutralization assay, these high‐affinity IgY antibodies can specifically bind with the virus particles to protect shrimp (Fenneropenaeus chinensis) against WSSV infection. After oral delivery for 20 days, the IgY‐WSSV exerted a higher protection effect (RPS: 71.5%) than IgY‐VP28 (RPS: 63.7%). Moreover, an increase in RPS (79.2%) was found on addition of IgY‐WSSV:VP28 (0.1% IgY‐VP28 plus 0.2% IgY‐WSSV). This may indicate that neutralization of WSSV refers to the multiple‐hit model. By time‐course study of the levels of the specific IgYs in vivo, the data showed that the titre was enhanced to a relatively high level (P/N=8.35±0.45) at 3 days post administration, declined slightly (P/N=7.13±1.01) at 7 days post administration and then remained stable for further investigation. The stable antibody level potentially contributes towards blocking a large number of WSSV particles from entering and infecting on the major tissues at the early and late stages after challenge in shrimp.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

抗WSSV囊膜蛋白（VP28和VP19）单抗的体内中和效果研究

通过酶联免疫吸附法（ELISA）测定不同稀释度对虾白斑综合征病毒（WSSV）与已制备的WSSV囊膜蛋白单克隆抗体结合的OD值。利用克氏原螯虾Cambarus proclarkii动物模型,将不同稀释度病毒与单抗1：1混合孵育2h后,肌肉注射克氏原螯虾（50μl／只）,观察记录螯虾的死亡情况。ELISA结果显示,在1×10^-3病毒稀释度下两种单抗均足量。在螯虾体内中和实验中,当病毒浓度为1×10^-3、1×10^-4、1×10^-5和1×10^-6稀释度时,MAb1D6（VP28）螯虾组最终死亡率分别为100％、90％、16．7％和6．7％,而MAb2E9（VP19）螯虾组最终死亡率分别为100％、100％、100％和93．3％。这表明随病毒浓度的降低,MAb1D6（VP28）的中和效果越明显。而MAb2E9（VP19）并无明显的中和效果。     

乳糖诱导对虾白斑病毒囊膜蛋白VP28基因在重组大肠杆菌中的表达

以含对虾白斑综合症病毒（white spot syndrome virus，WSSV）囊膜蛋白VP28编码基因质粒的重组大肠杆菌Escherichia coliB121作为研究对象，研究了乳糖或乳清粉代替IPTG作为诱导剂诱导重组囊膜蛋白VP28的表达。结果表明，乳糖不仅能够作为诱导剂诱导重组大肠杆菌进行外源蛋白的表达，而且能作为碳源促进菌体的生长。通过对诱导条件的优化，乳糖在发酵培养基的添加量为8g·L^-1，发酵时间为12h时可以获得最高的目的蛋白表达量，为97．36mg·L^-1。试验亦使用乳清粉作为发酵培养基的碳源和诱导工程菌表达的诱导剂。结果表明，在发酵培养基中添加乳清粉作为碳源和诱导剂，使其乳糖终浓度为10g·L^-1，发酵时问为13h时可以获得最高的目的蛋白表达量，为86．24mg·L^-1。     

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS‐PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross‐reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody‐based assays.     

对虾白斑综合征病毒囊膜蛋白VP28和VP26的毕赤酵母组成型分泌表达

近年来,重组 VP28和 VP26蛋白作为蛋白亚单位疫苗,在增强对虾抗白斑综合征病毒(WSSV)感染的过程中具有重要作用。本研究根据GenBank中WSSV的基因序列设计引物,以WSSV粗提液为模板进行普通PCR扩增,得到VP28和VP26基因,再用引物悬挂法将EcoRⅠ和XbaⅠ酶切位点分别添加到 VP28和 VP26基因的5￠端和3￠端。目的基因经双酶切后插入到表达载体pGAPZαA,转化TOP10大肠杆菌,经博莱霉素(Zeocin)抗性筛选阳性重组酵母表达载体。AvrⅡ酶切线性化之后,电击转化 X-33毕赤酵母感受态细胞,经 Zeocin 抗性筛选得到阳性重组酵母。SDS-PAGE电泳分析重组酵母表达上清液的目的蛋白,没有检测到VP28和VP26重组蛋白。随后,采用蛋白质银染法,结果显示,与空载pGAPZαA组相比,VP28和VP26表达上清液组有明显的条带,证明VP28和VP26在毕赤酵母中成功表达,蛋白分子量大小约为32 kDa。     

Nucleotide sequence variations of the major structural proteins (VP15, VP19, VP26 and VP28) of white spot syndrome virus (WSSV), a pathogen of cultured Litopenaeus vannamei in Mexico

White spot syndrome virus (WSSV) was first reported in farmed Litopenaeus vannamei stocks in Sinaloa and Sonora, Mexico during 1999 and continues to cause severe shrimp losses. WSSV genes encoding nucleocapsid (VP26 and VP15) and envelope proteins (VP19 and VP28) of a Mexican isolate were cloned in the pMosBlue vector. The nucleotide sequences of these genes were compared with WSSV isolates in GenBank. VP15 is highly conserved, and VP26 showed 99% homology to a Chinese isolate. The VP28 fragment demonstrated 100% homology to the majority of the isolates analysed (UniProt accession no. Q91CB7), differing from two Indian WSSV and one Chinese WSSV isolates by two non-conserved and one conserved replacements, respectively. Because of their highly conserved nature, these three structural proteins are good candidates for the development of antibody-based WSSV diagnostic tools or for the production of recombinant protein vaccines to stimulate the quasi-immune response of shrimp. In contrast, VP19 of the Mexican isolate was distinguishable from almost all isolates tested, including an American strain of WSSV (US98/South Carolina, GenBank accession no. AAP14086). Although homology was found with isolates from Taiwan (GenBank accession no. AAL89341) and India (GenBank accession no. AAW67477), VP19 may have application as a genetic marker.     

Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus

In order to investigate whether protein structure has an effect on protective effect of envelope protein of WSSV, VP28 protein was expressed both in Escherichia coli (pET-VP28) and insect (BmN) cells (BmNPV-VP28). The baculovirus (BmNPV) expression system was used to obtain correctly folded VP28 protein. Procambarus clarkii crayfish were intramuscularly injected with lysates of cells infected with recombinant pET-VP28 and BmNPV-VP28, respectively, and then challenged by intramuscular injection of WSSV to assess the duration of protection. The crayfish injected with BmNPV-VP28 showed generally lower mortality rates when compared to crayfish injected with pET-VP28, resulting in relative percent survivals of 92% and 39%, respectively, when compared to the control groups injected with empty vectors BmNPV and pET-30a. These results showed that VP28 protein produced in BmN cells gave much better protection than VP28 protein produced in E. coli.