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.

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.

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.

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.

Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus （wssv） and its use as a biosensor

White spot syndrome virus （WSSV） is a major cause of high mortality in cultured shrimp all over the world. VP26 is one of the structural proteins of WSSV that is assumed to assist in recognizing its host and assists the viral nucleocapsid to move toward the nucleus of the host cell. The objective of this work was to produce a polyclonal antibody against VP26 and use it as a biosensor. The recombinant VP26 protein （rVP26） was produced in E. coli （BL21）, purified and used for immunizing rabbits to obtain a polyclonal antibody. Western blot analysis confirmed that the antiserum had a specific immunoreac- tivity to the VP26 of WSSV. This VP26 antiserum was immobilized onto a gold electrode for use as the sensing surface to detect WSSV under a flow injection system. The impedance change in the presence of VP26 was monitored in real time. The sensitivity linear range of 160 160000 of the biosensor was in the copies of WSSV, indicating that it is good and sensitive for analysis of WSSV. The specificity of the biosensor was supported by the observation that no impedance change was detected even at high concentrations when using Yellow Head Virus （YHV）. This biosensor may be applied to monitor the amount of WSSV in water during shrimp cultivation.

Advances in the study of tegument protein VP26 in white spot syndrome virus

White spot syndrome virus(WSSV)has become one of the most widespread causes of mortality in commercial shrimp farming due to its broad host range,rapid spread and high lethality.The tegument protein VP26,which is loosely connected to nucleocapsid and envelope,is one of the major proteins of WSSV and has an important role in the initial stages of viral infection.Recent research has emphasized the vp26 gene,the structure of the VP26 protein,and VP26 binding proteins.Such knowledge is required to develop VP26 immune adjuvant to control WSSV.This paper reviews the related research of VP26 to provide reference for the prevention and treatment of WSSV.

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

我们为 WSSV 开发了敏感、快速的侧面流动的免疫分析(LFIA ) ，用象指示物的胶体的黄金。熔化蛋白质， VP (19+28 ) ，在 E 被表示。coli，净化并且过去常准备 polyclonal 抗体。净化的 anti-VP (19+28 ) IgG 与胶体的黄金被结合。Unconjugated anti-VP (19+28 ) IgG 和山羊反兔子 IgG 在硝化纤维素膜上被使不能调动。在汇编以后，虾样品的三个组(在每个组的 5 个单个动物) 被测试它包括了健康、垂死、死了的虾。为每个组，三不同纸巾(身体蜜汁，鳃和 hepatopancreas ) 同时被测试。在平行，所有样品也为比较用 PCR 被分析。从测试的 45 件样品，当 15 作为否定被分类时， 30 作为积极被检测。LFIA 的结果相关，那些由 PCR 分析获得了，显示这二个察觉方法有在在这初步的研究测试的样品的有限数字的一样的功效。关键词侧面流动的免疫分析 - 白点症候群病毒(WSSV )- VP19 - VP28 CLC 数字 S945.

Molecular characteristics of three thymosin-repeat proteins from Marsupenaeus japonicus and their responses to WSSV infection

β-thymosins, a family of highly conserved peptides, play a vital role in wound-healing, angiogenesis,antimicrobial process and antiviral immunity. Three novel β-thymosin-repeat proteins, named mjthm4, mjthm3 and mjthm2, were cloned from Marsupenaeus japonicus using expressed sequence tags（EST） from suppression subtractive hybridization. The mjthm4, mjthm3 and mjthm2 c DNAs possessed open reading frames that encoded166, 128 and 90 amino acid residue polypeptides and contained four, three and two β-thymosin actin binding modules, respectively. Blast analysis demonstrated that mjthm4, mjthm3 and mjthm2 shared high homology with known invertebrate multi-repeat β-thymosins. These proteins are ubiquitously expressed in all of the examined tissues, and the transcriptional levels were highest in the intestine. Further investigation revealed that mjthm4,mjthm3 and mjthm2 were remarkably up-regulated 6 h after WSSV infection. Moreover, while mjthm4 transcriptional levels displayed no changes, mjthm3 and mjthm2 levels decreased in the virus-resistant shrimps.The results indicate that mjthm4, mjthm3 and mjthm2 are novel multi-repeat β-thymosin homologues, have a close relationship with WSSV infection, and might contribute to a better understanding of host defense and/or virus invasion interactions in shrimps.

Molecular characterization and expression analysis of a novel dual-CRD C-type lectin in kuruma shrimp(Marsupenaeus japonicus)

C-type lectins are among the most significant pattern recognition receptors（PRRs） found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn（termed Mj Lectin） from kuruma shrimp Marsupenaeus japonicus was identified. The full-length c DNA of Mj Lectin was 1 245 bp with a 1 011 bp open reading frame（ORF） that encoded a polypeptide of 336 amino acid residues. Mj Lectin consisted of two tandemly arrayed carbohydrate-recognition domains（CRDs）, unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2＋-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN（Leu134-Pro135-Asn136） in CRD1 and a traditional EPN（Glu299-Pro300-Asn301） in CRD2, respectively. Mj Lectin showed a specific expression pattern in both tissue and cellular levels, for its m RNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus（WSSV） challenge（3.6×108 virions/μL）, the expression of Mj Lectin in the hepatopancreas was up-regulated significantly at 48 h（P〈0.01） compared with the control group. These results suggested that Mj Lectin might be involved in the innate immune defense against WSSV infection.

Prediction of Interaction Sites of PcRab7-VP28

White spot syndrome virus (WSSV) is one of the most important pathogens in the world. Since its outbreak in 1993, the virus has caused huge economic losses. Studies have confirmed that in the early stage of infection, VP28, the main envelope protein of WSSV, as a viral adhesion protein, binds to PcRab7 of Penaeus chinensis to help the virus enter the host cells. Understanding the mechanism of PcRab7-VP28 interaction is of great significance to understand the mechanism of WSSV infection and the development of antiviral drugs. In this research, the interaction interface and interaction sites were predicted by using the methods of molecular simulations. Results showed that VP28 binds to the second β-sheet (L73-D86) of PcRab7, which is consistent with the region detected in previous studies. Furthermore, we speculated the possible interaction sites in PcRab7 are E81, F77 and D76. These results may contribute to a deep understanding of the infection mechanism of WSSV on the host.

Effects of transport stress on immune response, physiological state, and WSSV concentration in the red swamp crayfish Procambarus clarkii

Transport is an essential part of the aquaculture and research of the main freshwater aquaculture crayfish Procambarus clarkii in China.However,transport is often accompanied by a low survival rate.Assessing the physiological state of P.clarkii before and after transport may discover the cause of this high mortality rate.In this study,ice-cold and exposed-to-air transport methods were compared using an array of parameters,including relative expression level of heat shock protein 70(HSP70),content of serum glucose and cortisol,immune parameters(enzyme and immune-related genes),and white spot syndrome virus(WSSV)concentration were investigated to understand the physiological state of P.clarkii before and after transport,as well as the cause of dying crayfish on days 5 and 7 after transport stress.Histological sections of hepatopancreas,gills,and intestines reflected pathological changes.The survival rate of crayfish with ice-cold transport was significantly higher than that with exposed-to-air transport,and mortality peaked at 3-9 days after transport stress.A prolonged response to oxidative stress and short-term immunosuppression was present after transport,and the trend of the WSSV concentration in the hepatopancreas was similar to the mortality rate of P.clarkii.The contents of serum glucose and cortisol,antioxidant enzymes and immune-related indexes,and the concentration of WSSV in hepatopancreas of dying crayfish were significantly higher than those of vibrant crayfish on the 5th and 7th days after transport.The hepatopancreas,intestines,and gills of dying crayfish had varying degrees of damage,and the hepatopancreas and intestines were severely damaged.The results suggested that the death of P.clarkii after transport stress is caused by oxidative stress,the imbalance of reactive oxygen species regulation,and decreased WSSV resistance,which eventually led to irreversible tissue damage.The increase of WSSV in the body of crayfish might be the direct cause of crayfish death.

Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity

Anti-lipopolysaccharide factors(ALFs)exhibit a potent antimicrobial activity against a broad range of bacteria,filamentous fungi,and viruses.In previous reports,seven groups of ALFs(groups A–G)were identified in penaeid shrimp.Among them,group D showed negative net charges and weak antimicrobial activity.Whether this group has antiviral function is not clear.In this study,the ALF sequences of penaeid shrimp were analyzed,and eight groups of ALF family(groups A–H)were identified.The four ALFs including MjALF-C2,MjALF-D1,MjALF-D2,and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli,and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus(WSSV)infection.Results showed that the expression of Vp28(WSSV envelope protein)decreased significantly in the MjALF-D2-injected shrimp only.Therefore,MjALF-D2 was chosen for further study.Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV.The WSSV replication was detected in RNA,genomic DNA,and protein levels using VP28 and Ie1(immediate-early gene of WSSV)as indicators in MjALF-D2-injected shrimp following WSSV infection.Results showed that WSSV replication was significantly inhibited compared with that in the rTRX-or PBS-injected control groups.After knockdown of MjAlf-D2 in shrimp by RNA interference,the WSSV replication increased significantly in the shrimp.All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity,and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV internalization.Methods:In this study,we have cloned PmRab7 and expressed in Escherichia coli,further purified rPmRab7 was used for antibody production,isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein.Moreover,high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR.Results:651 bp amplicon size gene was successfully amplified,ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel.Subcloned(pRSET-B)651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page,furthermore soluble fraction of rPmRab7(26 kDa)was purified by ni-NTA column.AntiPmRab7 antibody was received by Merk Pvt.Ltd.,and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction.Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct=1.0 to Ct=8.5.Conclusions:Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation,other hand WSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.