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 81.35% accuracy. Automatically derived morphometric measurements of phage capsids and tails exhibited lower variability than the ones obtained manually. The technique allows performing precise and accurate quantitative (e.g. abundance estimation) and qualitative (e.g. diversity and capsid size) measurements for studying the interactions between host population and different phages that infect the same host.

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). In contrast, VP1 derivatives with carboxy-terminal truncations of 35 to 56 aa residues failed to form VLPs. VP1 mutants with a single A336G aa exchange or internal deletions of aa 335 to aa 346 and aa 335 to aa 363 resulted in the formation of VLPs of a smaller size (diameter 20 nm). These data indicate that certain parts of the carboxy-terminal region of VP1 are not essential for pentamer-pentamer interactions in the capsid, at least in the yeast expression system used.

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. METHODS: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. RESULTS: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. CONCLUSION: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.

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. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) Jomantiene et al. Phytopathology 90:S39, 2000. (3) Staniulis et al. Plant Dis. 84:1061, 2000.

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. In other parts of Europe, alder is affected by a phytoplasmal disease known as alder yellows, which is characterized by symptoms that include yellowing and reduced leaf size, die-back of branches, and decline of trees (2,3). Proliferation of shoots has not been previously reported with this disease. An association between alder yellows and infection by a phytoplasma has been reported in A. glutinosa in Germany and Italy, and a phytoplasma has been found in A. glutinosa in France and Hungary (2,4). We examined symptomatic alder from Lithuania using nested polymerase chain reaction (PCR) (1), primed by P1/P7 and followed by R16F2n/R16R2 (F2n/R2), for amplification of phytoplasmal ribosomal (r) DNA. The results indicated the presence of a phytoplasma, designated ALY-L, in the diseased alder. We classified the ALY-L phytoplasma through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA. A 1.2-kbp fragment (F2n-R2 segment) of rDNA, amplified in PCR primed by F2n/R2, was analyzed using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, Sau3AI, BfaI, and ThaI. On the basis of collective RFLP patterns, phytoplasma ALY-L was classified as a member of group 16SrV (group V, elm yellows group), subgroup C. The amplified 16S rDNA was cloned in Escherichia coli and sequenced, and the sequence was deposited in the GenBank data library (Accession No. AY028789). Nucleotide sequence alignment revealed that 16S rDNA from phytoplasma ALY-L shared 100% sequence similarity with 16S rDNA (GenBank Accession No. Y16387) from a phytoplasma associated with alder yellows (ALY) disease in Italy. The results support the conclusion that a strain of ALY phytoplasma is present in Lithuania. Phytoplasmas belonging to groups 16SrI (aster yellows phytoplasma group) and III (X-disease phytoplasma group) have been found in herbaceous plant species in Lithuania. This report records the first finding of a group V phytoplasma, and the first finding of a phytoplasma in a tree species in the eastern Baltic Region. These findings contribute knowledge about the diversity of phytoplasmas in the Baltic Region and the distribution of ALY phytoplasma in Europe. Apparently, A. glutinosa may be infected by the phytoplasma but not develop obvious disease symptoms, as has been reported elsewhere (3). Thus, it is possible that ALY-L phytoplasma is widespread, but as yet undetected, throughout the geographic range of alder in the Baltic Region. This possibility is supported by the finding of the monophagous leafhopper vector (Oncopsis alni) of ALY phytoplasma throughout Europe (cited in Maixner and Reinert [3]). Further research is needed to assess the impact of phytoplasmal infections such as those by ALY-related phytoplasma strains on trends in biological diversity in the natural forest ecosystems of the Baltic Region and elsewhere in Europe. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) W. Lederer and E. Seemüller. Eur. J. For. Pathol. 21:90, 1991. (3) M. Maixner and W. Reinert. Eur. J. Plant Pathol. 105:87, 1999. (4) R. Mäurer et al. Phytopathology 83:971, 1993.

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. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57Bl6 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3. Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development.

First Report of Phytoplasmas in Soybean, Alfalfa, and Lupinus sp. in Lithuania.

Plants of cultivated soybean (Glycine max) and alfalfa (Medicago sativa) in Dotnuva and of wild Lupinus sp. in Ledakalnis, Lithuania, exhibited symptoms that suggested phytoplasmal infections. Soybean plants were of normal growth habit but exhibited veinal necrosis. Alfalfa and Lupinus plants exhibited stunting, abnormally small leaves, and witches'-broom symptoms. Diseases in the plants were termed soybean veinal necrosis (SVN), alfalfa stunt (AlfS), and Lupinus stunt (LupS), respectively. The presence of phytoplasmas in diseased plants was assessed using polymerase chain reaction (PCR) for amplification of phytoplasma-specific 16S rDNA. A phytoplasma-characteristic 1.2-kbp DNA fragment was amplified from all diseased plants but not from known healthy plants in nested PCRs in which the first DNA amplification was primed by primer pair P1/P7 and reamplification of DNA was primed by primer pair F2n/R2 (2,4). Products from the nested PCR primed by F2n/R2 were subjected to restriction fragment length polymorphism (RFLP) analysis, and the RFLP patterns obtained were compared with patterns previously published (1-4). On the basis of AluI, HaeIII, HhaI, HpaI, KpnI, MseI, and RsaI RFLP patterns, the SVN and LupS phytoplasmas were classified in group 16SrIII (peach X-disease phytoplasma group), subgroup B (III-B, type strain clover yellow edge phytoplasma), and the AlfS phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (I-B, type strain aster yellows phytoplasma). Nucleotide sequences were determined for 16S rDNA fragments amplified from SVN and AlfS phytoplasmas in nested PCRs primed by F2n/R2. The sequences were deposited in GenBank under Accession nos. AF177383 for SVN and AF177384 for AlfS. Sequence similarity between the 16S rDNAs of SVN and Canadian clover yellow edge (strain CYE-C, GenBank Accession no. AF175304) phytoplasmas was 99.8%; sequence similarity between 16S rDNAs of AlfS and aster yellows (strain SAY, GenBank Accession no. M86340) phytoplasmas was 99.6%. The SVN phytoplasma 16S rDNA shared 100% sequence similarity with a 16S rDNA from the Lithuanian clover yellow edge (CYE-L, GenBank Accession no. AF173558) phytoplasma. The nucleotide sequence data supported the conclusion that the SVN and AlfS phytoplasmas were closely related to strains classified in subgroups III-B and I-B, respectively. Our findings extend the known geographic ranges of phytoplasma subgroups I-B and III-B to northern Europe, including Lithuania, and expand the known plant host ranges of these pathogens. References: (1) R. E. Davis et al. Int. J. Syst. Bacteriol. 47:262, 1997. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) R. Jomantiene et al. HortScience 33:1069, 1998. (4) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Yeast cells allow high-level expression and formation of polyomavirus-like particles.

Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania.

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5' and 3' parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with "universal" Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.