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

Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus

Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A2 found in M. nigrocinctus venoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Antivenoms for the treatment of snakebite envenomings: The road ahead

The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders.Universidad de Costa Rica//UCR/Costa RicaInternational Foundation for Science//IFS/SueciaCiencia y Tecnología para el Desarrollo//CYTED/EspañaConsejo Superior de Investigaciones Científicas//CRUSA-CSIC/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

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