Biodistribution and Toxicity Studies of PRINT Hydrogel Nanoparticles in Mosquito Larvae and Cells.

Mosquito-borne diseases continue to remain major threats to human and animal health and impediments to socioeconomic development. Increasing mosquito resistance to chemical insecticides is a great public health concern, and new strategies/technologies are necessary to develop the next-generation of vector control tools. We propose to develop a novel method for mosquito control that employs nanoparticles (NPs) as a platform for delivery of mosquitocidal dsRNA molecules to silence mosquito genes and cause vector lethality. Identifying optimal NP chemistry and morphology is imperative for efficient mosquitocide delivery. Toward this end, fluorescently labeled polyethylene glycol NPs of specific sizes, shapes (80 nm x 320 nm, 80 nm x 5000 nm, 200 nm x 200 nm, and 1000 nm x 1000 nm) and charges (negative and positive) were fabricated by Particle Replication in Non-Wetting Templates (PRINT) technology. Biodistribution, persistence, and toxicity of PRINT NPs were evaluated in vitro in mosquito cell culture and in vivo in Anopheles gambiae larvae following parenteral and oral challenge. Following parenteral challenge, the biodistribution of the positively and negatively charged NPs of each size and shape was similar; intense fluorescence was observed in thoracic and abdominal regions of the larval body. Positively charged NPs were more associated with the gastric caeca in the gastrointestinal tract. Negatively charged NPs persisted through metamorphosis and were observed in head, body and ovaries of adults. Following oral challenge, NPs were detected in the larval mid- and hindgut. Positively charged NPs were more efficiently internalized in vitro than negatively charged NPs. Positively charged NPs trafficked to the cytosol, but negatively charged NPs co-localized with lysosomes. Following in vitro and in vivo challenge, none of the NPs tested induced any cytotoxic effects.

Biodistribution and trafficking of hydrogel nanoparticles in adult mosquitoes.

BACKGROUND: Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract. CONCLUSIONS/SIGNIFICANCE: Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.

Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito.

BACKGROUND: Aedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock. RESULTS: Herein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways. CONCLUSION: These results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

A large scale laboratory cage trial of Aedes densonucleosis virus (AeDNV).

Aedes aegypti (L.) (Diptera: Culicidae) the primary vector of dengue viruses (DENV1-4), oviposit in and around human dwellings, including sites difficult to locate, making control of this mosquito challenging. We explored the efficacy and sustainability of Aedes Densonucleosis Virus (AeDNV) as a biocontrol agent for Ae. aegypti in and among oviposition sites in large laboratory cages (> 92 m3) as a prelude to field trials. Select cages were seeded with AeDNV in a single oviposition site (OPS) with unseeded OPSs established at varied distances. Quantitative real-time polymerase chain reaction was used to track dispersal and accumulation of AeDNV among OPSs. All eggs were collected weekly from each cage and counted. We asked: (1) Is AeDNV dispersed over varying distances and can it accumulate and persist in novel OPSs? (2) Are egg densities reduced in AeDNV treated populations? AeDNV was dispersed to and sustained in novel OPSs. Virus accumulation in OPSs was positively correlated with egg densities and proximity to the initial infection source affected the timing of dispersal and maintenance of viral titers. AeDNV did not significantly reduce Ae. aegypti egg densities. The current study documents that adult female Ae. aegypti oviposition behavior leads to successful viral dispersal from treated to novel containers in large-scale cages; however, the AeDNV titers reached were not sufficient to reduce egg densities.

Evaluation of mosquito densoviruses for controlling Aedes aegypti (Diptera: Culicidae): variation in efficiency due to virus strain and geographic origin of mosquitoes.

Four mosquito densovirus strains were assayed for mortality and infectivity against Aedes aegypti larvae from different geographic regions. The viral titers were quantified by real-time PCR using TaqMan technology. Firstinstar larvae were exposed to the same titer of each densovirus strain for 48 hours. All strains of densoviruses exhibited larvicidal activity and caused more than 80% mortality and infectivity in the three mosquito strains. AalDNV-exposed larvae had the highest mortality rate. The mean time to death of AalDNV-exposed larvae was shorter than other DNVs-exposed larvae. We can conclude that different densovirus strains exhibit some variations in their pathogenicity to different populations of Ae. aegypti mosquitoes. A few mosquitoes from Chachoengsao and Bangkok exposed to AeDNV and AThDNV survived to the adult stage to lay eggs and showed 22% to 50% vertical transmission in the F1 generation. Phylogenetic analysis of four densovirus strains indicated that mosquito densoviruses are separated into two distinct clades.

Purification of densonucleosis virus by tangential flow ultrafiltration.

Purification at commercial scale of viruses and virus vectors for gene therapy applications and viral vaccines is a major separations challenge. Tangential flow ultrafiltration has been developed for protein purification. Here tangential flow ultrafiltration of parvoviruses has been investigated. Because these virus particles are small (18-26 nm), removal of host cell proteins will be challenging. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow some virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the 30, 50, and 100 kDa membranes, indicating possible entrapment of virus particle in the membrane pores. The permeate flux and level of protein rejection is strongly affected by the cell culture growth medium. The results indicate that when developing a new process, it is essential that the cell culture and purification operations be developed in parallel.

Binding Aedes aegypti densonucleosis virus to ion exchange membranes.

Experimental and numerical results for binding Aedes aegypti densonucleosis virus (AeDNV) using anion and cation exchange membranes are presented. AeDNV particles are adsorbed by anion and cation exchange membranes providing the virus particles and membranes are oppositely charged. Q membranes which are strongly basic anion exchangers were the most effective. Dynamic and static capacities for Q membranes were found to be similar. A numerical model is proposed which assumes a log normal pore size distribution. By estimating the required parameters from static binding experiments, the model may be used to calculate the breakthrough curve for virus adsorption.

Murine leukemia virus clearance by flocculation and microfiltration.

Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated. Murine leukemia virus is a retrovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of retrovirus-like particles. Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used. Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate. The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles. The virus particles are 80-130 nm in size. The feed streams were flocculated using a cationic polyelectrolyte. Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes. The level of virus clearance in the permeate was determined. For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells. While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams. Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant. However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes. The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration. Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles. Flocculation of the feed stream leads to an increase in the permeate flux.

Densonucleosis virus purification by ion exchange membranes.

Preparative chromatography is widely used in the downstream purification of biopharmaceutical products. Replacement of resins by membranes as chromatographic supports, overcomes many of the limitations associated with resin-based chromatography such as high-pressure drops, slow processing rates due to pore diffusion and channeling of the feed through the bed. In particular, adsorptive membranes may be ideally suited for virus capture. Virus capture is critical in a number of applications. In gene therapy and vaccine production, large-scale purification of virus vectors is often essential. In the manufacture of biopharmaceuticals, validation of virus clearance is critical. Here results for purification of Aedes aegypti densonucleosis virus (AeDNV) using anion and cation exchange membranes are presented. AeDNV is a non-enveloped, single-stranded mosquito-specific parvovirus. Virus particles are around 20 nm in size. AeDNV could find potential applications in integrated vector-borne disease control programs. In addition, capture of parvovirus for validation of virus clearance in the manufacture of biopharmaceuticals is of commercial importance. By adjusting the pH of the feed stream, AeDNV particles may be adsorbed by both anion and cation exchange membranes. However, strongly basic anion exchange membranes were the most effective in adsorbing AeDNV particles. Adsorption and subsequent elution of AeDNV by anion exchange membranes leads to significant virus concentration. Dynamic and static capacities for anion exchange membranes were similar. Further, a sharp elution curve was obtained suggesting that pore diffusional resistances are insignificant. The adsorption of AeDNV particles by anion exchange membranes may be described by a linear isotherm.

Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Clearance of minute virus of mice by flocculation and microfiltration.

Clearance of minute virus of mice (MVM) from CHO cell suspensions by flocculation and microfiltration has been investigated. MVM is a parvovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of parvoviruses. The feed streams were flocculated using a cationic polyelectrolyte. Virus clearance in excess of 10,000-fold was obtained in the bulk permeate for flocculated feeds streams. However, the level of clearance was only about 10- to 100-fold for unflocculated feed streams. The results suggest that virus clearance involves interactions between the MVM particles, the cationic polyelectrolyte, and the CHO cells present. Validating virus clearance is a major concern in the biotechnology industry. New unit operations are frequently added to the purification train simply to validate virus clearance. However, many of these unit operations are less effective at validating clearance of nonenveloped viruses. Validating clearance of parvoviruses is often particularly problematic as they are nonenveloped and the virus particles are small (18 to 24 nm), making physical removal difficult. The results obtained herein indicate that addition of the cationic polyelectrolyte not only results in significant clearance of MVM but also leads to an increase in permeate flux.

Infection and pathogenicity of the mosquito densoviruses AeDNV, HeDNV, and APeDNV in Aedes aegypti mosquitoes (Diptera: Culicidae).

These studies compared three genetically distinct mosquito densoviruses Aedes aegypti (AeDNV), Hemagogus equinus (HeDNV), and Aedes Peruvian (APeDNV) densoviruses in a laboratory investigation to begin to evaluate their potential as mosquito control agents. A real-time polymerase chain reaction (PCR) assay for quantification of viral genomes and a standardized mosquito infection protocol were developed. Mortality associated with exposure to AeDNV increased in a dose-dependent manner, with the maximum mortality of 75.1% occurring in those organisms exposed to the highest dose of virus. The majority of death occurred as larvae. Similar results were observed with AeDNV produced from ground larvae and AeDNV produced from cell culture. Exposure of mosquitoes to HeDNV and APeDNV resulted in lower mortality, with values peaking at 33.5% for HeDNV and 27.8% for APeDNV. AeDNV-exposed larvae develop at a slower rate than nonexposed and HeDNV- and APeDNV-exposed larvae. Decreased virulence does not reflect a decrease in virus replication. PCR analysis of infectivity rates and titers in adults revealed reproduction of all three viruses, with an average viral titer of approximately 10 logs/mosquito after exposure to the highest dose of each virus. Accumulation of virus in the larval-rearing water was also observed with values approaching 10-11 logs/ml for each virus. These data indicate that there are dramatic differences in the pathogenicity among mosquito densoviruses.

The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain.

Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma-leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.

The midgut epithelium of aquatic arthropods: a critical target organ in environmental toxicology.

The midgut epithelium of aquatic arthropods is emerging as an important and toxicologically relevant organ system for monitoring environmental pollution. The peritrophic matrix of aquatic arthropods, which is secreted by the midgut epithelium cells, is perturbed by copper or cadmium. Molecular biological studies have identified and characterized two midgut genes induced by heavy metals in the midgut epithelium. Many other metal-responsive genes (MRGs) await characterization. One of the MRGs codes for an intestinal mucin, which is critical for protecting the midgut from toxins and pathogens. Another codes for a tubulin gene, which is critical for structure and function of the midgut epithelial cells. Perturbation of expression of either gene could condition aquatic arthropod survivorship. Induction of these MRGs is a more sensitive and rapid indicator of heavy-metal pollution than biological assays. Characterization of genes induced by pollutants could provide mechanistic understanding of fundamental cellular responses to pollutants and insight into determinants of aquatic arthropod population genetic structure and survivorship in nature.