Development of cell culture based peste des petits ruminants (PPR) virus vaccine candidate from Bangladeshi isolates.

This study was conducted to develop a cell culture based PPR virus vaccine candidate using recent Bangladeshi strain of peste des petits ruminant's (PPR) virus. PPR virus was isolated from field outbreaks, confirmed by RT-PCR and used as viral inoculum for serial passaging in Vero cells for adaptation and attenuation. 60th serial passage had completed and RT-PCR and real time RT-PCR were done in every 5 passages for confirmation of PPR virus in tissue culture fluid (TCF). To assess the adaptation and attenuation cytopathology, virus titration, sequencing of both F and N genes and live animal experimentation were done. Different cellular alterations produced by PPR virus in infected Vero cells including syncytia formation, development of both intranuclear and intra cytoplasmic inclusion bodies and finally cell degradation are the indications of adaptation. The virus titre was found 2.5, 3.31, 3.55, 4.44, 4.71 and 6.5 Log10 TCID50/ml at 10th, 20th, 30th, 40th, 50th and 60th passages level respectively. In F gene sequence analysis it has been observed that few nucleotide (nt) and mino acid (aa) has been substituted as the effects of serial passaging of PPR virus in Vero cells. TCF at 60th passage level was found effective to produced protective antibody (Ab) titre in live animal experimentation. It is concluded that serially passaged and Vero cells adapted PPR virus TCF could be used as a vaccine candidate for further use to develop a potent & effective vaccine against PPR diseases.

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Pathotypic and genotypic characterization of two Bangladeshi isolates of Newcastle disease virus of chicken and pigeon origin.

Two Bangladeshi isolates of Newcastle disease virus (NDV), one from a chicken and one from a pigeon, were characterized in this study. Pathogenicity of the isolates was evaluated on the basis of intracerebral pathogenicity index (ICPI). Both the isolates were found to be of velogenic pathotype having ICPI of 1.83 and 1.51 for the chicken and pigeon isolate, respectively. Genotype of the isolates was determined by phylogenetic analysis based on partial F gene sequences. A 766-bp genome fragment spanning partial M and F gene was amplified by RT-PCR and sequenced. The first 354 bp of the coding region of F gene and corresponding deduced amino acid sequences (residues 1-118) of these two NDV isolates were aligned with that of other NDV strains retrieved from GenBank. A phylogenetic tree constructed from the alignment showed that the chicken isolate (BD-C162) belonged to the newly described genotype XIII and the pigeon isolate (BD-P01) to genotype VI. Both the chicken and pigeon isolates possessed a virulent-like fusion protein cleavage site (112) RRQKRF(117) .

Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012.

In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. There have so far been seven human cases of H5N1 HPAI infection in Bangladesh with one death. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. Partial or complete nucleotide sequences of all eight gene segments of two chicken isolates, five gene segments of a duck isolate and the haemagglutinin gene segment of 18 isolates from Bangladesh were established in the present study and subjected to molecular analysis. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates available in GenBank were included in the analysis. The analysis revealed that the first introduction of clade 2.2 virus in Bangladesh in 2007 was followed by the introduction of clade 2.3.2.1 and 2.3.4 viruses in 2011. However, only clade 2.3.2.1 viruses could be isolated in 2012, indicating progressive replacement of clade 2.2 and 2.3.4 viruses. There has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where H5N1 virus acquired the PB1 gene from a H9N2 virus. Point mutations have accumulated in Bangladeshi isolates over the last 5 years with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh.

Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus.

A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD-3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication.

Synergistic effect of PKC activation and actin filament disruption on carbonate apatite-facilitated lymphocyte transfection.

Leukemia and lymphoma cells are potential targets for genetic manipulation in cancer therapy. On the other hand, genetically modified autologous lymphocytes expressing a chimeric antigen against a receptor overexpressed in tumor cells or tumor vasculature are promising cell-based therapeutics for cancer.However, the lack of a smart device for efficient transgene delivery to the lymphocytes poses the major obstacle to the successful clinical applications of these attractive approaches. Recently, we developed a carbonate apatite-based nanocarrier system for effective intracellular delivery and release of DNA molecules, achieving very high level of transgene expression in both primary and cancer cells. Although its efficacy in human T leukemia cells is relatively poor, immobilization of fibronectin and/or chimeric E-cadherin-Fc on particle surface could enhance transgene delivery in presence of an actin filament disrupter. Here, we report for the first time that simultaneous stimulation of human T leukemia cells by a protein kinase C (PKC) activator, a Ca(2+) ionophore and an actin filament disrupter dramatically accelerated carbonate apatite-mediated transgene delivery in the cells, resulting in over 100-fold more efficacy than commcercially available lipofectamine.

New introduction of clade 2.3.2.1 avian influenza virus (H5N1) into Bangladesh.

Since the first outbreak of highly pathogenic H5N1 avian inafluenza (HPAI) in Bangladesh in February 2007, a total of 519 disease events have been reported till 22 October 2011. Partial HA gene sequences of 11 selected H5N1 HPAI isolates of 2007 to 2011 were determined and subjected to phylogenetic analysis. The study revealed a recent introduction of clade 2.3.2 and 2.3.4 viruses into Bangladesh in 2011 in addition to clade 2.2 viruses that had been in circulation since 2007. Clade 2.3.2 virus isolates from Bangladesh are phylogenetically related to the newly designated clade 2.3.2.1 viruses, reported recently from Asia and Eastern Europe.

Differentiation of infectious bursal disease virus (IBDV) genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis.

Molecular characterization of IBDV usually relies on the analysis of segment A of the bi-segmented, double-stranded RNA genome. Although segments B of classical and very virulent IBDVs differ significantly, re-assortment of genome segments does occur, and molecular epidemiological studies demand the analysis of both segments. An RT-PCR and restriction enzyme analysis for molecular discrimination between genome segment B of classical and very virulent IBDVs is described. Tested on eight IBDV strains/isolates, the protocol successfully identified very virulent and classical IBDVs as well as a segment reassortant. This approach is a valuable tool for molecular epidemiological studies on IBDV.

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA.

Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Nuclear targeting of viral and non-viral DNA.

The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.

Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles.

Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments.

Self-assembly of DNA and cell-adhesive proteins onto pH-sensitive inorganic crystals for precise and efficient transgene delivery.

Intracellular delivery of a functional gene or a gene-silencing DNA or RNA sequence is expected to be a powerful tool for treating critical human diseases very precisely and effectively. One of the major hurdles to the successful delivery of a nucleic acid with nanoparticles is the transport across the plasma membrane. The existence of various and numerous cell surface receptors with potential capability of being internalized by cells upon ligand binding unveils the ways of overcoming the barrier by targeting the nanoparticles to specific receptor. This review will reveal the current progress on utilizing the cell adhesion molecules as targeting receptors for transgene delivery, with a special focus on the design of bio-functionalized inorganic nanocrystals using both naturally occurring and genetically engineered cell adhesive proteins for high efficiency transfection of embryonic stem cells. Self-assembly of both DNA and cell-adhesive proteins, such as fibronectin and E-cadherin-Fc into the growing nanocrystals of carbonate apatite leads to their high affinity interactions with fibronectin-specific integrins and E-cadherin in embryonic stem cell surface and accelerates transgene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced transgene delivery with a value notably higher than that of commercially available lipofection system. Activation of protein kinase C (PKC) dramatically enhances transgene expression probably by up-regulating both integrin and E-cadherin. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

High performance DNA nano-carriers of carbonate apatite: multiple factors in regulation of particle synthesis and transfection efficiency.

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.

Effective delivery with enhanced translational activity synergistically accelerates mRNA-based transfection.

mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Current technologies for mRNA delivery are based on cationic lipids with DOTAP being the most efficient one. We previously reported on the synthesis of an inorganic-organic hybrid carrier by embedding inorganic nano-particles of carbonate apatite onto liposomal carrier DOTAP and demonstrated its high transfection potency of luciferase mRNA both in mitotic and non-mitotic cells. Here we show that in addition to the carrier design for effective endocytosis and release of mRNA to the cytoplasm, enhancement of mRNA translation efficiency is a prerequisite for maximum protein expression. We used the modified cap analog (ARCA) during in vitro transcription of luciferase DNA for proper cap orientation and demonstrated that transfection with ARCA-mRNA resulted in higher protein expression than the mRNA with usual cap structure for both DOTAP and DOTAP-apatite complex. Secondly, exogenous poly(A) was co-delivered with mRNA by the DOTAP-apatite, resulting in very significant expression compared to mRNA delivery only. Finally, when combined both of the effects of smart carrier and the modifications at mRNA translational level, a notable enhancement (100 times) was achieved as compared to the existing DOTAP-based liposome technology. Our findings, therefore, unveiled a novel approach that an effective delivery system can be developed by the improvement of the gene expression level in combination with the enhancement of the carrier potency.

pH-sensitive nano-crystals of carbonate apatite for smart and cell-specific transgene delivery.

The treatment of a human disease at a genetic level by either providing a cell with a functional gene or a nucleic acid sequence to precisely silence a harmful gene, is a powerful approach that could revolutionise clinical medicine. Despite the existence of both genetically engineered viral vectors and synthetically designed lipid- or polymer-based nanocarriers, an ideal delivery system in terms of safety and efficacy is still lacking. This editorial reports on the development of biocompatible, inorganic nanoparticles of carbonate apatite, which has the unique features essentially required for smart delivery, as well as for the expression of a genetic material in a mammalian cell.

Surface functionalization of inorganic nano-crystals with fibronectin and E-cadherin chimera synergistically accelerates trans-gene delivery into embryonic stem cells.

Stem cells holding great promises in regenerative medicine have the potential to be differentiated to a specific cell type through genetic manipulation. However, conventional ways of gene transfer to such progenitor cells suffer from a number of disadvantages particularly involving safety and efficacy issues. Here, we report on the development of a bio-functionalized inorganic nano-carrier of DNA by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high affinity interactions with embryonic stem cell surface and accelerated trans-gene delivery for subsequent expression. While only apatite nano-particles were very inefficient in transfecting embryonic stem cells, fibronectin-anchored particles and to a more significant extent, fibronectin and E-cadherin-Fc-associated particles dramatically enhanced trans-gene delivery with a value notably higher than that of commercially available lipofection system. The involvement of both cell surface integrin and E-cadherin in mediating intracellular localization of the hybrid carrier was verified by blocking integrin binding site with excess free fibronectin and up-regulating both integrin and E-cadherin through PKC activation. Thus, the new establishment of a bio-functional hybrid gene-carrier would promote and facilitate development of stem cell-based therapy in regenerative medicine.

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.