Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency

Marija Brgles, Maja Šantak, Beata Halassy, Dubravko Forcic, Jelka TomašicInstitute of Immunology, Research and Development Department, Zagreb, CroatiaBackground: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1–50 and extruded through membranes of 400, 200, and 100 nm. Plasmid DNA encoding green fluorescent protein was used to measure transfection efficiency by flow cytometry. Sizes of liposome/DNA complexes were measured by dynamic light scattering.Results: Liposome size was reduced after extrusion but this was mainly driven by the charge ratio and not by the size of the membrane pores. Reduction of complex size at each charge ratio positively correlated with transfection efficiency. When the size of the complexes was approximately constant, increasing the charge ratio was found to promote transfection efficiency. Cationic lipid N-(1-(2,3-dioleoyloxy)propyl)N,N,N trimethylammonium chloride was used for modulation of positive charge and a cytotoxicity test showed that increasing its amount increases cytotoxicity.Conclusion: It can be concluded that charge ratio dictates the size of the complex whereas overall size reduction and higher charge ratios promote transfection efficiency in vitro.Keywords: transfection efficiency, liposome charge, liposome siz

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

<p>Abstract</p> <p>Background</p> <p>The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous.</p> <p>Results</p> <p>We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo.</p> <p>Conclusion</p> <p>L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.</p

First recorded case of paramyxovirus infection introduced into a healthy snake collection in Croatia

Abstract Background In the present study, we describe the first paramyxovirus infection in a snake collection in Croatia caused by an introduction of new snakes that were not previously tested and didn’t show any signs of disease. Case presentation In less than a month after introduction into a healthy colony, new snakes began to show respiratory symptoms (i.e. mouth opening, wheezing, etc.) and died within a month and a half after antibiotic therapy was applied. The same symptoms and a high mortality rate were then observed in in-contact snakes from other collections belonging to different snake families. Conclusions Two entries of new snakes in different time periods were recorded and recognized as possible sources of infection. We stress the need for veterinary health control and monitoring of snakes prior to transportation as well as implementing obligatory quarantine measures to minimize the risk of infection among newly established snake groups

Mass spectrometry-based investigation of measles and mumps virus proteome

Abstract Background Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs. Methods MUV, MEV and ECVs were purified by ultracentrifugation, hydrophobic interaction chromatography and immunoaffinity chromatography, proteins in the samples were resolved by SDS-PAGE and subjected to identification by MALDI-TOF/TOF-MS. A comparative analysis of HCPs present in all samples was carried out. Results By proteomics approach, it was verified that almost all virus-coded proteins are present in MEV and MUV particles. Protein C in MEV which was until now considered to be non-structural viral protein, was found to be present inside the MeV virions. Results on the presence of HCPs in differently purified virus preparations imply that actin, annexins, cyclophilin A, moesin and integrin β1 are part of the virions. Conclusions All HCPs detected in the viruses are present in ECVs as well, indicating their possible function in vesicle formation, or that most of them are only present in ECVs. Only five HCPs were constantly present in purified virus preparations, regardless of the purification method used, implying they are likely the integral part of the virions. The approach described here is helpful for further investigation of HCPs in other virus preparations

[Carpesium sp.]

原著和名: [記載なし]科名: キク科 = Compositae採集地: 栃木県 日光市 東照宮裏〜雲竜入口 (下野 日光 東照宮裏〜雲竜入口)採集日: 1980/10/10採集者: 萩庭丈壽整理番号: JH027469国立科学博物館整理番号: TNS-VS-977469備考: DB作成協力会による補足あ

Additional file 4: Figure S2. of Common position of indels that cause deviations from canonical genome organization in different measles virus strains

Variability fingerprint constructed using 54 different complete measles genomic sequences. Legend: The variability of each genomic position is quantified by considering the number of different nucleotides that are found at that position (shown in white, light grey, dark grey or black). The rectangle indicates which fingerprint area depicts variability of non-coding region between M and F genes’ open reading frames (1012 nts). (TIF 501 kb

Additional file 1: of Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene

Figure S1. Phylogenetic tree of RSV strains based on HVR2 genomic segment. Tree was generated using maximum-likelihood method, based on the General Time Reversible model and discrete gamma distributed rates across sites. The scale bar indicates the proportion of nucleotide substitutions per site. Numbers are percentages of bootstrap values determined for 1000 iterations, only values above 70% are shown. Strain designations are composed of NCBI GenBank acc. no., name and genotype. (PDF 54 kb

Additional file 2: of Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene

Table S1. Sequences identical at HVR2 or SHseg. (PDF 82 kb