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

Chimeric MrNV-GE11-VLPs serve as a nano-container to deliver Doxorubicin into cancer cells

We have reported that virus-like particle from shrimp virus, MrNV-VLP, effectively encapsulates and delivers plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. Additionally, modifying VLP with GE-11 peptide extension on the surface (so called, E-MrNV-GE11-VLP) allows them to interact specifically with the EGFR-positive SW480 cancer cells. This work extrapolated the use of E-MrNV-GE11-VLP to encapsulate and deliver doxorubicin (DOX) towards SW480 cells. The results showed that DOX was passively loaded into VLPs in a molar ratio of >200 DOX/VLP equivalent to a loading efficiency of 3%. Specific targeting of E-MrNV-GE11-VLP + DOX and its anti-cancer effect towards SW480 was more pronounced than that of N-MrNV-VLP + DOX, suggesting an interaction and internalization of E-MrNV-GE11-VLP through surface EGFR. This claim was also supported by a lower DOX delivery into MCF7 than SW480 cells. Finally, the cell cytotoxicity assay showed that E-MrNV-GE11-VLP + DOX significantly decreased cell viability in SW480 cells more than that by N-MrNV-VLP + DOX (P<0.05), while its cytotoxicity effect on MFC7 cells was much lower than on SW480 cells. This study provides insights into how to develop target-specific drug delivery for carrying therapeutic agents towards specific tumor cells

Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation. &nbsp;</p

Cellular targets and pathways of yellow head virus infection in lymphoid organ of Penaeus monodon as studied by transmission electron microscopy

Negative-stained intact yellow head virus (YHV) was an enveloped bacilliform particle measuring 40-50 x 175-210 nmwith spike-like projections measuring 7-9 nm. The space between projections was 4-7 nm. YHV nucleocapsid was rod-shaped,measuring 35-40 x 160-200 nm, and the RNA genome had 40-50 turns in a helical structure. YHV infected both stromal matrixcells and haemocytes in the lymphoid tubule wall. The patterns of localisation of viral particles were similar in both cells. Thefully enveloped viral particles were detected at the cell membrane, endosome, rough endoplasmic reticulum, Golgi complexand secretory vesicles, and virions were exocytosed at the cell membrane. In the case of severe infection, unenveloped viralparticles could be detected in the cytoplasm, and they might be released by general breakdown and lysis of the highly infectedcells

Seasonal Changes in Upper Thermal Tolerances of Freshwater Thai Fishes

Seasonal change inferred to climate change inevitably influences Critical thermal maximum (CTmax) of riverine fishes. In this study, we investigated CTmax as thermal tolerance for four common riverine fishes, i.e., Danio regina, Channa gachua, Rasbora caudimaculata and Mystacoleucus chilopterus, in the Kwae Noi river system in western Thailand. The acute thermal tolerance was lower in the wet season (mean river temperature ∼25 °C) and higher in the dry season (mean river temperature ∼23 °C) with medians of wet season-CTmax for those four fishes of 35.3 ± 0.4, 36.2 ± 0.5, 37.3 ± 0.5 and 37.5 ± 0.6 °C, respectively, and high values of dry season-CTmax of 37.4 ± 0.5, 38.3 ± 0.5, 38.7 ± 0.7 and 39.1 ± 0.5 °C, respectively. The variations of CTmax for all of the four species in this study, throughout the wet and dry seasons, attribute to their seasonal plasticity in response to the dynamics of thermal stress. Under climate variability and climate change with increasing the higher temperatures of air and river, and altering the habitat, R. caudimaculata and M. chilopterus had higher capacities to tolerate the acute heat stress across wet and dry seasons

Bilateral eyestalk-ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5–7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13–15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease

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