Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model.

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6-24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.

Effect of feeding different types of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis).

ß-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of ß-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without ß-glucan; the second and the third group were fed with 2 g kg-1 of ß-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of ß-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with ß-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both ß-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with ß-glucans, especially with ß-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with ß-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing ß-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with ß-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg-1 diet can significantly improve their growth performance and immunity.

Role of sphingosine kinase and sphingosine-1-phosphate receptor in the liver pathology of mice infected with Plasmodium berghei ANKA.

Decreased serum sphingosine 1-phosphate (S1P) has been reported in severe malaria patients, but the expression of receptors and enzymes associated with S1P has not been investigated in the liver of malaria patients. Therefore, this study aimed to investigate the expression of sphingosine kinase (SphK) and S1P receptors (S1PRs) in the liver of malaria-infected mice. C57BL/6 male mice were divided into a control group (n = 10) and a Plasmodium berghei (PbA)-infected group (n = 10). Mice in the malaria group were intraperitoneally injected with 1×106 P. berghei ANKA-infected red blood cells, whereas control mice were intraperitoneally injected with normal saline. Liver tissues were collected on Day 13 of the experiment to evaluate histopathological changes by hematoxylin and eosin staining and to investigate SphK and S1PR expression by immunohistochemistry and real-time PCR. Histological examination of liver tissues from the PbA-infected group revealed sinusoidal dilatation, hemozoin deposition, portal tract inflammation and apoptotic hepatocytes, which were absent in the control group. Immunohistochemical staining showed significant increases in the expression of SphK1 and SphK2 and significant decreases in the expression of S1PR1, S1PR2, and S1PR3 in the endothelium, hepatocytes, and Kupffer cells in liver tissue from the PbA-infected group compared with the control group. Real-time PCR analysis showed the upregulation of SphK1 and the downregulation of S1PR1, S1PR2, and S1PR3 in the liver in the PbA-infected group compared with the control group. In conclusion, this study demonstrates for the first time that SphK1 mRNA expression is upregulated and that S1PR1, S1PR2, and S1PR3 expression is decreased in the liver tissue of PbA-infected mice. Our findings suggest that the decreased levels of S1PR1, S1PR2, and S1PR3 might play an important role in liver injury during malaria infection.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Viral Capsid Change upon Encapsulation of Double-Stranded DNA into an Infectious Hypodermal and Hematopoietic Necrosis Virus-like Particle.

In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30-40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA-capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the "tripod" structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel's opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

Protective Effect of an Anti-HMGB-1 Neutralizing Antibody on Hemozoin-Induced Alveolar Epithelial Cell in a Model of Malaria Associated ALI/ARDS.

BACKGROUND: We aimed to determine whether neutralizing high mobility group box-1 (HMGB-1) prevents the release of HMGB-1 and proinflammatory cytokines on hemozoin (Hz)-induced alveolar epithelial cell in a model of malaria associated ALI/ARDS. METHODS: This study was conducted in the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand in 2020. Human pulmonary alveolar epithelial cells (HPAEpiCs) were exposed to medium alone or 20 µM Hz for 24 h and incubated with different concentrations (1, 5, and 10 µg/ml) of anti-HMGB-1 monoclonal antibody (mAb) for various times (0, 4, 12, 24, and 48 h). The levels of HMGB-1, TNF-α and IFN-γ in the supernatants were measured by ELISA. The mRNA expression of RAGE, TLR-2 and TLR-4 were analyzed by real-time PCR. RESULTS: The HPAEpiCs treated with 10 µg/ml anti-HMGB-1 mAb showed a significant reduction in HMGB-1 release into the supernatant compared with those treated with 1 and 5 µg/ml anti-HMGB-1 mAb. The levels of TNF-α and IFN-γ were significantly decreased in the supernatant of HPAEpiCs treated with 1, 5, and 10 µg/ml anti-HMGB-1 mAb for 4, 12, 24, and 48 h compared with those stimulated with Hz alone. The mRNA expression levels of RAGE, TLR-2, and TLR-4 were significantly decreased after 24 h of anti-HMGB-1 antibody treatment at all concentrations. CONCLUSION: An anti-HMGB-1 antibody could be an effective agent for inhibiting the release of HMGB-1, TNF-α and IFN-γ. Furthermore, a neutralizing anti-HMGB-1 antibody could be applicable for the treatment of malaria-associated ALI/ARDS.

Chimeric virus-like particles (VLPs) designed from shrimp nodavirus (MrNV) capsid protein specifically target EGFR-positive human colorectal cancer cells.

Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Antioxidant and antimalarial properties of Sophora exigua Craib. root extract in Plasmodium berghei-infected mice.

BACKGROUND: Sophora exigua Craib. is commonly used in Thailand to reduce fever and increase postpartum breast milk production in women who have hypogalactia. However, there has been no report on the antioxidant and antimalarial properties of this plant. This study aimed to investigate the antioxidant and antimalarial activities of S. exigua root extract and to evaluate its acute toxicity in mice to confirm its safety. METHODS: The in vitro antioxidant activities were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide radical, and hydroxyl radical scavenging assays. The in vivo antioxidant activities were determined by detecting the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the livers of malaria-infected mice. The in vivo antimalarial activity was determined by Peters' 4-day suppressive test in mice infected with Plasmodium berghei ANKA and orally administered S. exigua root aqueous and ethanolic extracts at different doses (200, 400, and 600 mg/kg body weight). In addition, the acute oral toxicity of the plant extracts was assessed in mice at a dose of 2000 mg/kg body weight. RESULTS: The ethanolic extract of S. exigua root exhibited inhibition of DPPH radicals, superoxide anions, and hydroxyl radicals, with half maximal inhibitory concentration (IC50) values of 24.63 ± 1.78, 129.78 ± 0.65, and 30.58 ± 1.19 µg/ml, respectively. Similarly, research on the in vivo antioxidant activity indicated that the ethanolic extract of S. exigua root exerted a stronger effect than the aqueous extract. The aqueous extract at doses of 200, 400, and 600 mg/kg had stronger antimalarial activity than the ethanolic extract. The aqueous extract at 600 mg/kg exhibited 60.46% suppression of parasitemia. Increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) and blood urea nitrogen (BUN) were detected in the mice treated with 2000 mg/kg ethanolic extract, which was related to the results of histopathological analysis of liver tissue, showing ballooning degeneration of hepatocytes, diffuse hepatic hemorrhage, and infiltration of inflammatory cells. CONCLUSIONS: This study demonstrated that the ethanolic S. exigua root extract possessed antioxidant properties, and the aqueous extract also had antimalarial activity. Therefore, this plant is an alternative source of new antioxidant and antimalarial agents.

High mobility group box-1 (HMGB-1) and its receptors in the pathogenesis of malaria-associated acute lung injury/acute respiratory distress syndrome in a mouse model.

The DNA-binding protein high mobility group box-1 (HMGB-1) mediates proinflammatory cytokines that contribute to acute lung injury (ALI). Although ALI is a frequent complication of malaria infection, the contribution of HMGB-1 and its receptors to the pathogenesis of malaria-associated ALI/acute respiratory distress syndrome (MA-ALI/ARDS) has not been investigated in a mouse model. Here, the malaria-infected mice were divided into two groups according to lung injury score: the ALI/ARDS and non-ALI/ARDS groups. The expression of HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) in lung tissues was investigated by using immunohistochemical staining and real-time polymerase chain reaction (PCR). Additionally, HMGB-1 and proinflammatory cytokine (TNF-α, IFN-γ, IL-1 and IL-6) levels in plasma and lung tissues were quantified by using enzyme-linked immunosorbent assays. Cellular expression of both HMGB-1 and its receptors (RAGE, TLR-2 and TLR-4) was significantly increased in the lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups. The levels of HMGB-1, TNF-α, IFN-γ, IL-1 and IL-6 were significantly increased in both plasma and lung tissues of the ALI/ARDS group compared with those in the non-ALI/ARDS and control groups, which were similar to the results obtained by real-time PCR. Increased mRNA expression of RAGE, TLR-2 and TLR-4 was found in the lung tissues of the ALI/ARDS group. Furthermore, the plasma HMGB-1 level was positively correlated with TLR-4 mRNA expression in the ALI/ARDS group. HMGB-1 levels were significantly increased in plasma and lung tissues of MA-ALI/ARDS mice and were related to the upregulated expression of HMGB-1 and proinflammatory cytokines. In conclusion, this study demonstrates that HMGB-1 is an important mediator of MA-ALI/ARDS pathogenesis and may represent a target for therapeutic malaria interventions with ALI/ARDS.

Association between ALFPm3 single nucleotide polymorphism and white spot syndrome virus resistance in black tiger shrimp Penaeus monodon.

Here single nucleotide polymorphisms (SNPs) were associated with white spot syndrome virus (WSSV) resistance in black tiger shrimp Penaeus monodon. SNPs were identified by single-strand conformation polymorphism (SSCP) screening and DNA sequencing of shrimp sampled from 3 families (100 shrimp per family) challenged with WSSV. Shrimp that died over the 14 d challenge trial were designated susceptible, with those remaining alive on Day 14 designated resistant. To compare SNPs, 10 samples from the susceptible and resistant groups, each comprising DNA pooled from 3 shrimp, were amplified by polymerase chain reaction (PCR) using primers to 12 selected genes and screened by SSCP. SNPs were only identified in the anti-lipopolysaccharide factor 3 (ALFPm3) gene product. Analysis of complete ALFPm3 gene sequences confirmed the existence of 3 SNPs (g.934C>G, g.1186A>G, and g.1898C>G) that were polymorphic between the susceptible and resistant groups. Further analyses using specific tetra-primer amplification refractory mutation system PCR primer sets associated these 3 SNPS, and particularly the g.1186A>G SNP, with WSSV resistance. This SNP thus has potential for use as a DNA marker to select for WSSV resistance in P. monodon breeding programs.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections.

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.

Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection.

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

Nanocontainer designed from an infectious hypodermal and hematopoietic necrosis virus (IHHNV) has excellent physical stability and ability to deliver shrimp tissues.

BACKGROUND: A virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied. METHODS: IHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in E. coli. Their physical properties against three strong physical conditions including long term storage (0-30 days) in 4 °C, physical stabilities against broad ranged pH (4-9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection. RESULTS: Upon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8-9) and to a lesser extent in acidic condition (pH 4-6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues. DISCUSSION: There are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field.

Body painting to promote self-active learning of hand anatomy for preclinical medical students.

BACKGROUND: The purpose of this study was to use the body painting method to teach hand anatomy to a group of preclinical medical students. METHODS: Students reviewed hand anatomy using the traditional method and body painting exercise. Feedback and retention of the anatomy-related information were examined by a questionnaire and multiple-choice questions, respectively, immediately and 1 month after the painting exercise. RESULTS: Students agreed that the exercise was advantageous and helped facilitate self-active learning after in-class anatomy lessons. While there was no significant difference in knowledge retention between the control and experimental groups, the students appreciated the exercise in which they applied body paint to the human body to learn anatomy. CONCLUSION: The body painting was an efficient tool for aiding the interactive learning of medical students and increasing the understanding of gross anatomy.

Nodavirus-based biological container for targeted delivery system.

Biological containers such as virus-like particles (VLPs) have gained increasing interest in the fields of gene therapy and vaccine development. Several virus-based materials have been studied, but the toxicity, biodistribution, and immunology of these systems still require extensive investigation. The specific goal of this review is to provide information about nodaviruses, which are causative infectious agents of insects and aquatic animals, but not humans. By understanding the structure and biophysical properties of such viruses, further chemical or genetic modification for novel nanocarriers could be developed. Therefore, their application for therapeutic purposes, particularly in humans, is of great interest.

Encapsulation and delivery of plasmid DNA by virus-like nanoparticles engineered from Macrobrachium rosenbergii nodavirus.

Virus-like particles (VLPs) are potential candidates in developing biological containers for packaging therapeutic or biologically active agents. Here, we expressed Macrobrachium rosenbergii nodavirus (MrNv) capsid protein (encoding amino acids M1-N371 with 6 histidine residuals) in an Escherichia coli BL21(DE3). These easily purified capsid protein self-assembled into VLPs, and disassembly/reassembly could be controlled in a calcium-dependent manner. Physically, MrNv VLPs resisted to digestive enzymes, a property that should be advantageous for protection of active compounds against harsh conditions. We also proved that MrNv VLPs were capable of encapsulating plasmid DNA in the range of 0.035-0.042 mol ratio (DNA/protein) or 2-3 plasmids/VLP (assuming that MrNV VLPs is T=1, i made up of 60 capsid monomers). These VLPs interacted with cultured insect cells and delivered loaded plasmid DNA into the cells as shown by green fluorescent protein (GFP) reporter. With many advantageous properties including self-encapsulation, MrNv VLPs are good candidates for delivery of therapeutic agents.

Chimeric hepatitis E virus-like particle as a carrier for oral-delivery.

Oral delivery with virus-like particles (VLPs) is advantageous because of the inherited entry pathway from their parental viral capsids, which enables VLP to withstand the harsh and enzymatic environment associated with human digestive tract. However, the repeat use of this system is challenged by the self-immunity. In order to overcome this problem, we engineered the recombinant capsid protein of hepatitis E virus by inserting p18 peptide, derived from the V3 loop of HIV-1 gp120, into the antibody-binding site. The chimeric VLP resembled the tertiary and quaternary structures of the wild type VLP and specifically reacted with an HIV-1 antibody against V3 loop. Different from the wild type VLP, the chimeric VLP was vulnerable to trypsin cleavage although it appeared as intact particle, suggesting that the intermolecular forces of attraction between the recombinant capsid proteins are strong enough to maintain the VLP icosahedral arrangement. Importantly, this VLP containing the V3 loop did not react with anti-HEV antibodies, in correspondence to the mutation at its antibody-binding site. Therefore, the insertion of peptides at the surface antigenic site could allow VLPs to escape pre-existing anti-HEV humoral immunity.