Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1.

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives.

Automatic detection and morphological delineation of bacteriophages in electron microscopy images.

Automatic detection, recognition and geometric characterization of bacteriophages in electron microscopy images was the main objective of this work. A novel technique, combining phase congruency-based image enhancement, Hough transform-, Radon transform- and open active contours with free boundary conditions-based object detection was developed to detect and recognize the bacteriophages associated with infection and lysis of cyanobacteria Aphanizomenon flos-aquae. A random forest classifier designed to recognize phage capsids provided higher than 99% accuracy, while measurable phage tails were detected and associated with a correct capsid with 81.

Characterization of a lytic cyanophage that infects the bloom-forming cyanobacterium Aphanizomenon flos-aquae.

Vb-AphaS-CL131 is a novel cyanosiphovirus that infects harmful Aphanizomenon flos-aquae. This cyanophage has an isometric head, 97 nm in diameter and a long, flexible non-contractile tail, 361 nm long. With a genome size of ~120 kb, it is the second largest cyanosiphovirus isolated to date.

Functionalization of α-synuclein fibrils.

The propensity of peptides and proteins to form self-assembled structures has very promising applications in the development of novel nanomaterials. Under certain conditions, amyloid protein α-synuclein forms well-ordered structures - fibrils, which have proven to be valuable building blocks for bionanotechnological approaches. Herein we demonstrate the functionalization of fibrils formed by a mutant α-synuclein that contains an additional cysteine residue.

Isolation and characterization of vB_ArS-ArV2 - first Arthrobacter sp. infecting bacteriophage with completely sequenced genome.

This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation.

Klebsiella phage vB_KleM-RaK2 - a giant singleton virus of the family Myoviridae.

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms.

Bacteriophage vB_EcoM_FV3: a new member of "rV5-like viruses".

A proposed new genus of the family Myoviridae, "rV5-like viruses", includes two lytic bacteriophages: Escherichia coli O157: H7-specific bacteriophage rV5 and Salmonella phage PVP-SE1. Here, we present basic properties and genomic characterization of a novel rV5-like phage, vB_EcoM_FV3, which infects E. coli K-12-derived laboratory strains and replicates at high temperature (up to 47 °C).

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained.

Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.

In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively.

Generation of menangle virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds.

Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter.

Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells.

As polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation.

Size and position of truncations in the carboxy-terminal region of major capsid protein VP1 of hamster polyomavirus expressed in yeast determine its assembly capacity.

The hamster polyomavirus major capsid protein VP1 was modified in its carboxy-terminal region by consecutive truncations and single amino acid exchanges. The ability of yeast-expressed VP1 variants to form virus-like particles (VLPs) strongly depended on the size and position of the truncation. VP1 variants lacking 21, 69, and 79 amino acid (aa) residues in their carboxy-terminal region efficiently formed VLPs similar to those formed by the unmodified VP1 (diameter 40-45 nm).

'Candidatus Phytoplasma fragariae', a novel phytoplasma taxon discovered in yellows diseased strawberry, Fragaria x ananassa.

Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawberry (Fragaria x ananassa Duchesne) in Lithuania. Analysis of 16S rRNA gene sequences amplified by PCR indicated that the symptoms were associated with infection by a phytoplasma, designated strawberry yellows (StrawY) phytoplasma. Phylogenetic analysis of 16S rRNA gene sequences indicated that StrawY phytoplasma, 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma asteris', stolbur phytoplasma and 'Candidatus Phytoplasma japonicum' shared a common ancestor, but were mutually distinct.

Generation of Sendai virus nucleocapsid-like particles in yeast.

The gene encoding Sendai virus nucleocapsid protein was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. The high level of recombinant Sendai virus nucleocapsid protein expression (12-14 mg/l of yeast culture) was obtained. The evaluation of recombinant proteins expression in yeast by Western blot analysis revealed specific reactivity with immune sera.

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice.

Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles. Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles. In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced.

Synthesis of the measles virus nucleoprotein in yeast Pichia pastoris and Saccharomyces cerevisiae.

The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology. The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively. Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P.

Development of a measles specific IgM ELISA for use with serum and oral fluid samples using recombinant measles nucleoprotein produced in Saccharomyces cerevisiae.

In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein. Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter. High levels of rMVN (20 mg/litre of yeast culture) were generated.

Synthesis of mumps virus nucleocapsid protein in yeast Pichia pastoris.

The expression of mumps virus nucleocapsid protein in yeast Pichia pastoris was investigated. Viral nucleocapsid proteins usually elicit a strong long-term humoral immune response in patients and experimental animals. Therefore, the detection of antibodies specific to mumps virus nucleoprotein can play an important role in immunoassays for mumps diagnosis.

Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.

Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established.

Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae.

BK and JC viruses are ubiquitous human polyomaviruses that are associated with post-transplant interstitial nephritis (BK virus) and progressive multifocal leucoencephalopathy (JC virus). The use of a yeast system to express the major capsid protein (VP1) of two antigenic variants of BKV (strains SB and AS) and JCV is described. VP1s of AS and JCV expressed in Saccharomyces cerevisiae produced proteins of expected molecular weight as determined by gel electrophoresis whereas that of SB appeared to be lower than anticipated.

First Report of Alder Yellows Phytoplasma in the Eastern Baltic Region.

Alnus glutinosa (alder) is widespread in Europe and is an important component of biological diversity in natural forest ecosystems in the Baltic Region. In 2000, diseased trees of A. glutinosa exhibiting characteristically phytoplasmal disease symptoms of shoot proliferation and leaf yellowing were observed in Aukstaitija National Park, Lithuania.

First Report of Aster Yellows-Related Subgroup I-A Phytoplasma Strains in Carrot, Phlox, Sea-Lavender, Aconitum, and Hyacinth in Lithuania.

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves.

Single and Mixed Phytoplasma Infections in Phyllody- and Dwarf-Diseased Clover Plants in Lithuania.

Naturally diseased plants of clover (Trifolium spp.) exhibiting symptoms of clover phyllody (virescence and phyllody of flowers) or of clover dwarf (abnormally small leaves, shortened internodes, proliferation of shoots, and dwarf growth habit) were observed in fields in Lithuania. Phytoplasma group-specific polymerase chain reactions (PCRs) and restriction fragment length polymorphism (RFLP) analysis of 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas.

Formation of immunogenic virus-like particles by inserting epitopes into surface-exposed regions of hamster polyomavirus major capsid protein.

We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-S1 region of the hepatitis B virus (designated S1; amino acid sequence DPAFR) was introduced into the predicted positions of VP1.