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.

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.

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.

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.

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.

Minicistron from a gene of prawn white spot bacilliform virus(WSBV)and its expression

By the sequencing and analysis of genomic DNA and cDNA of WSBV, it was found that there existed a minicistron in the leading sequence of p204 gene. The minicistron utilized rare codons of genes from prawn and WSBV. The p204 gene could be expressed in Escherichia coli whether a miniciston exited in its leading sequence or not, but the amounts of the expressed products of them were different. The minicistron might be related to the expression of p204 gene.

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.

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.

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.

SEM Comparison of Penetration in Artificial White Spots Lesion between an Infiltrant Resin and Two Adhesive Systems

White spot infiltration emerged as an alternative of non-invasive treatment to halt progression of the lesion, through the use of low viscosity resins that would permeate the porous enamel and form a physical barrier that would prevent the acid diffusion produced by micro-organisms. Purpose: To compare penetration levels in artificial white spot lesions, of infiltrant resin ICON&trade;and 2 conventional adhesives systems, XP-Bond&trade;and Single Bond 2&trade;. Methodology: White spot lesions (ICDAS code 2) were caused in 75 premolars or third molars were extracted in good conditions, by immersion in a 0.1 M lactic acid solution (pH 4.5) at 37℃ for 8 weeks. They were divided randomly into 3 groups of 25 samples and applied the following resins, Group A: ICON&trade;, B: XP-Bond&trade;and C: Single Bond 2&trade;. Subsequently, the enamel was removed with hydrochloric acid to expose resin saturated area and the samples were metalized with Au-Pd for SEM observation. The resin tags lengths were measured on microphotographs through software, and the values were analyzed with the statistics ANOVA and Scheffé post-test. Results: There were significant differences (p &trade;(82.7 μm ± 26.8 μm) compared to adhesive systems XP-Bond&trade;(58.5 μm ± 29.3 μm) and Single Bond 2&trade;(44.8 μm ± 32.5 μm). We found no significant differences between the two adhesive systems (p > 0.05). Conclusion: Under the conditions tested, the penetration of infiltrant ICON was significantly higher than the adhesive systems;however, it removes the surface layer of the enamel.

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.

Hemolymph cells apoptosis in imported shrimp Litopenaeus vannamei from Hawaii to Iran,exposed to white spot virus

Objective:To show hemolymph apoptosis in imported shrimp Litopenaeus vannamei from Hawaii to Iran.Methods:One hundred and eighty shrimps[(7.98±0.54)g] which were collcted from a research shrimp farm located in Heleh site in north of Bushehr Province were distributed equally to 6 glass aquariums(50 cm×50 cm×60 cm)as group A in triplicate(imported batch in 2011,without crossing with other generations)with well clean aerated sea water(100 L per aquarium),salinity of 40‰and temperature of 29℃.Shrimps of group B(produced by crossing the adults of imported batches in 2009 up to 2011)were distributed also among 6 aquariums with the same conditions.Both shrimp groups were injected with concentration of LD_(50)=1×10^(5.4)white spot virus.Results:The results showed that in group A,the mortality began approximately 24 h after exposure and reached 100%after 36 h but no mortality was occurred up to 15 d in shrimps of group B.The slide evaluation of hemolymph of group B showed an increasing trend of apoptosis occurrence in all three types of hemolymph cells,hyalinocytes,semi-granulocytes and granulocytes from 24 h to 72 h in contrary to group A that not any apoptosis was observed during the course of the study(15 d).Conclusions:It is concluded that crossing among the speific pathogen free generations could induce the increasing immunity level through apoptosis to protect them against white spot disease.

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.

Four kinds of ENU-induced white spot mice and chromosome locations of the mutant genes

Phenotype-driven is the name for an ap-proach used to study gene functions through mutagenesis, location and cloning of the mutant gene. In this study, 150 male C57BL/6J(B6) mice were treated with ENU and repro-duced a total offspring of 3860. Of these descendants, 210 exhibited mutation phenotypes by screening, and more than 10 of them are hereditable. Four kinds of mutant mice, named Wbct, W-1Bao, W-2Bao, and W-3Bao, showed dominant hereditary white spot mutation with partial albinism on their belly, distal limbs and tail terminal. To map these mutant genes, 39 microsatellites, equally distributed on the mouse genome and with difference between B6 and DBA/2J (D2), were selected to scan the genome after discrimination of the white spots in the F2 mice [(B6×D2)×D2]. It is found that, the log odds score (LODS) between W-1Bao and D5Mit168 is 0.56, and the LODS of W-1Bao and D5Mit352 is 4.47. With the gradual application of microsatellites D5Mit290, D5Mit312, D5Mit308 and D5Mit356 that are close to the mutant gene, and the number of F2 mice going up to 537, the mutant W-1Bao is located between D5Mit356 and D5Mit308 on chro-mosome 5, about 42.19 cM from the centromere. In the same way, W-2Bao and W-3Bao are mapped nearby W-1Bao, and Wbct is located on chromosome 1, about 41.6 cM from the centro-mere. After searching for the mouse genome database (MGD) and performing a one-by-one study of all genes located on chromosome subregion, it is believed that the kit gene is an excellent candidate for the white spot mutations of W-1Bao, W-2Bao and W-3Bao.

视觉评分系统分析透明矫治器和固定矫治器对牙齿光滑面的影响

背景:白斑病变是传统正畸治疗中常见的不良反应。目前有关透光透明矫治器和固定正畸矫治器对正畸治疗患者牙周健康状况、白斑病变发生率、唾液及相关细菌的影响差异仍存在一些争议。目的:采用视觉评分系统对比透明矫治器和固定矫治器对正畸牙齿光滑面的影响。方法:选择2018年1月至2022年12月河北医科大学第二医院口腔正畸科收治的患者80例,采用随机数字表法随机分为对照组(n=40)与观察组(n=40),对照组接受固定矫治器正畸治疗,观察组接受透明矫治器接正畸治疗。治疗前及治疗6,12,24个月后,每位患者选取13、33、16、36牙唇侧或颊侧作为研究对象,采用视觉评分系统评估白斑病变。结果与结论:①治疗前及治疗6,12,24个月后,两组患者牙齿白斑颜色与龋坏严重程度比较差异均无显著性意义(P>0.05);②对于13牙,治疗12个月后的白斑颜色级别与龋坏严重程度均高于治疗前(P<0.05);对于33牙,治疗12个月后的白斑颜色级别与龋坏严重程度均高于治疗前(P<0.05),治疗12个月后的龋坏严重程度高于治疗6个月后(P<0.05);对于16牙,治疗12个月后的白斑颜色级别与龋坏严重程度均高于治疗6个月后(P<0.05);③多水平有序多分类Logistic回归模型分析显示,不同牙位的白斑颜色、严重程度变化与矫治器类型相关性较小,受矫治时间因素影响较大,其中33、13牙初始白斑颜色变化和严重程度在治疗第12,24个月比较有统计学意义;④研究结果尚不能说明固定矫治器和透明矫治器对牙齿光滑面白斑病变的影响有所差异,但可以发现随着正畸治疗时间的延长,牙齿龋坏的风险有所增加。

Homozygous lethality and heterozygous spotting due to a novel missense mutation in the mouse Kit gene

N-ethyl-N-nitrosourea （ENU） mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse （named Kit^W-1 Bao） was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the Kit^W-1 Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers DSMit356 and DSMit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from Kit^W-1 Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp （D） by Val （V） at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the Kit^W-1 Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartie acid residue at position 849 in the Kit gene product

Oral Hygiene in the Presence of Orthodontic Therapy

Orthodontic treatment offers great advantages in improving facial and smile aesthetics, self-confidence and the function of the stomatognathic apparatus. The pursuit of these advantages makes use of orthodontic appliances that could be fixed or removable. However, it’s worth stating that these appliances interfere with tooth brushing, making it more difficult to brush teeth effectively. Orthodontics appliances therefore promote the accumulation of dental plaque, which results in both quantitative and qualitative changes in the oral microbiota, hence, exposing patients to several adverse effects such as White spot lesions, dental caries, periodontal pathologies and halitosis. For this reason, oral assessment of patients before, during and after treatment is necessary as well as oral hygiene instructions and motivation. Orthodontists therefore, should educate patients on oral and periodontal hygiene in order to control dental and periodontal complications. Prescriptions of plaque control materials adapted to each patient are done in order to optimize the final result and minimize unwanted complications.