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Volume 46 / 2002 / number 2
K. SWITALA-JELEN, K. DABROWSKA, A. GORSKI, L. SLIWA
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Ul. Rudolfa Weigla 12, 53-114 Wrocław, Poland
Summary. – Bacteriophage (phage) T4 belonging to T-even phages is one of the best known phages with a completely deciphered genome sequence. As a model of living systems, T4 phage has many technical advantages. It can be very easily grown in large quantities, manipulated by classical genetics, and engineered by site-directed mutagenesis. Many substances have been first tested for mutagenicity in T-even phages. The results of these tests were very often applicable to higher organisms due to similar mechanisms of mutagenesis. T4 phage is also important in phage therapy, which represents an alternative treatment of bacterial infections since the bacterial resistance to antibiotics has become a serious medical problem. The site-directed mutagenesis is a method that enables to introduce mutations which can influence phage affinity to bacteria and can be a practical technique for enriching phage collections and for widening specificity of phages for new bacterial strains now insensitive to phage therapy.
Key words: bacteriophage
T4; phage therapy; mutation; mutagen; site-directed mutagenesis
Acta virologica 46: 57 – 62, 2002
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J. PTÁČEK, J. ŠKOPEK, P. DĚDIČ, J. MATOUŠEK
Institute for Potato Research, Dobrovského 2366,
580 01 Havlíčkův Brod, Czech Republic;
Department of Molecular Genetics, Institute of Plant Molecular Biology,
Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice,
Czech Republic;
Faculty of Biological Sciences, University of South Bohemia, 370 05 České
Budějovice, Czech Republic
Summary. – Twenty potato virus Y (PVY) isolates were characterized. They represented two strains only, PVYO (three isolates) and PVYN (17 isolates). However, application of serological and molecular genetic methods led to a more complicated characterization. For example, five isolates induced necrotic symptoms on tobacco plants typical of PVYN, despite reacting as PVYO serologically. Moreover, the PVY isolates were not identical according to molecular genetic properties. Typical PVYNTN PCR products were observed for 14 isolates, but five of them (Hr 220-5, Hr 387-7, Nord 242, Syn1Scot, and 41-97) did not produce potato tuber necrotic symptoms in infected cultivars. An immunocapture reverse transcription–polymerase chain reaction (RT-PCR) probing was developed using a set of 24 primer pairs derived from eight regions of the PVY genome. Using this method, five out of seven PVYNTN isolates including the Czech standard PVYNTN from the potato cv. Nicola were found to be identical. However, two PVYNTN isolates and all the other probed PVY samples showed unique patterns, suggesting specific differences at the nucleotide level. This method enabled specific identification of individual isolates variability even within different PVY strains.
Key words: potato
virus Y; Solanum tuberosum L; immunocapture RT-PCR; virus genome
Acta virologica 46: 63 – 68, 2002
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M. VIZVARIOVÁ, S. VAVROVÁ, J. TURŇA
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B2, 842 15 Bratislava, Slovak Republic
Summary. – An in vitro method for cloning and mapping Escherichia coli genes by means of mini-Mu phage and its application to the penicillin G acylase (pac) gene of E. coli PAC2 strain with its flanking regions is described. The gene was marked by insertion of a fragment bearing kanamycin resistance (Kmr). Most of Kmr clones obtained from mini-Mu transductants contained the whole pac gene with its flanking regions. Localization of pac gene to 98.5 min of E. coli PAC2 chromosome was confirmed by an in vivo P1 phage transduction.
Key words: mini-Mu
phage; in vivo cloning; penicillin acylase; E. coli ATCC 11105; E. coli
PAC2
Acta virologica 46: 69 – 74, 2002
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J. ŘÍMAN
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic
Summary. – The deoxyribo-mode expression of primase (Pr) activities of the Pr-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring nucleoprotein (NP) complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (Říman and Šulová, Acta Virol. 41, 181–192 (1997)) is similarly influenced by carbonyldiphosphonate (COMDP) as the ribo-mode expression of Pr activities (Říman, Acta Virol. 45, 109–124 (2001)). In the presence of all four common dNTPs only and dNTPs and rNTPs in the reaction medium, COMDP strongly activates the deoxyribo-mode of Pr activities and again induces a unique phenomenon of primer accumulation. These primers labeled for DNA are up to 90% alkali-resistant and sensitive to DNase I treatment. This suggests that they are constituted mostly of deoxynucleotides (dnts). In contrast to the stimulation of the ribo-mode expression of Pr activities by COMDP, the incorporated radioactivity is in this case more than one order lesser. 1-Mimosine-(alha-amino-beta-[N-(3-hydroxy-4-pyridone)]-propionic acid (MIMO) is again able to substantially eliminate the phenomenon of primer accumulation, suggesting that also in this case the effects of COMDP and MIMO are, at certain reaction conditions, mutually exclusive and that both agents compete for the same active site responsible for mutual coupling of Pr and alpha DNA pol activities.
Key words: alpha DNA
polymerase; butylphenyl deoxyguanosine-5´-triphosphate;
carbonyldiphosphonate; mimosine; Okazaki fragments; primase
Acta virologica 46: 75 – 83, 2002
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F.W. BAI, H.W. ZHANG, J. YAN, Z.C. QU, J. XU, J.G. WEN, M.M. YE, D.L. SHEN
Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, P.R. China
Summary. – Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a non-binding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.
Key words: phage-display
peptide; outer coat protein; Rice black streaked dwarf virus
Acta virologica 46: 85 – 90, 2002
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K. SEME, M. POLJAK, J. BEGOVAC, A. VINCE, J. TOMAŽIČ, L. VIDMAR, T. KNIEWALD
Institute of Microbiology and Immunology, Medical
Faculty, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia;
Dr. Fran Mihaljević University Hospital for Infectious Diseases,
Zagreb, Croatia;
Department of Infectious Diseases, University Medical Center, Ljubljana,
Slovenia
Summary. – The prevalence of hepatitis C virus (HCV) infection in the population of human immunodeficiency virus 1 (HIV-1)-infected individuals from Slovenia and Croatia was determined. One hundred and sixty-six out of a total of 188 Slovenian HIV-1-infected individuals and 120 subjects who were randomly chosen out of a total 342 Croatian HIV-1 antibodies-positive individuals were tested for HCV infection. Detection of HCV antibodies was carried out by a third generation enzyme-linked immunoassay (ELISA) and the positive samples were additionally tested by a third generation immuno-blot assay. Additionally, the presence of HCV RNA was determined in all serum samples by a qualitative polymerase chain reaction (PCR). Twenty-four (14.5%) out of 166 Slovenian and 18 (15.0%) out of 120 Croatian HIV-1-infected individuals were HCV antibodies-positive. Nineteen out of 24 (79.2%) Slovenian and 13 out of 18 (72.2%) Croatian anti-HCV positive individuals were also viremic. HCV RNA was not detected in any of 244 HCV antibodies-negative/HIV-1-infected individual from both countries. A significant difference in the prevalence of HCV infection between blood (77.8% in Slovenia and 66.7% in Croatia) and sexual exposure risk groups (1.6% in Slovenia and 6.6% in Croatia) was found in both countries. In a study carried out on the highest proportion of entire population of HIV-1-infected individuals from a certain country or geographic region, Slovenia and Croatia were identified as countries with the second and third lowest prevalence of HCV infection among HIV-1/HIV-2 infected individuals worldwide.
Key words: HIV-1;
HIV-2; hepatitis C; co-infection; Slovenia; Croatia
Acta virologica 46: 91 – 94, 2002
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K.N. VISWAS, L. MUNIYAPPA, V.V.S. SURYANARAYANA, S.M. BYREGOWDA
Department of Veterinary Microbiology, University
of Agricultural Sciences, Bangalore, India;
Molecular Virology Laboratory, Indian Veterinary Research Institute,
Bangalore, India;
Institute of Animal Health and Veterinary Biologicals, Bangalore, India
Summary. – Two infectious bursal disease virus (IBDV) isolates were obtained from commercial poultry farms with a history of severe outbreaks. A 474-bp product encompassing hypervariable region of IBDV VP2 gene was amplified by reverse transcription–polymerase chain reaction (RT-PCR). The nucleotide sequences of two isolates, VMB1 and VMB2, were determined and compared with those of twenty IBDV strains, including seven very virulent, four classical virulent, four classical attenuated, three antigenic variants and two avirulent serotype 2 strains. The two isolates showed a similarity of 96.5–98.4% with very virulent strains, 84.6–94.6% with classical virulent strains, 90.0–91.4% with classical attenuated strains, 83.0–91.9% with antigenic variants and 65.8–68.7% with avirulent strains. The deduced amino acid sequences of the two isolates showed amino acid substitutions of V256I, N279D, L294I and N299S, specific for very virulent strains. Phylogenetic analysis showed that the two isolates, along with a reported very virulent Indian strain, were closely related to European, Japanese and Chinese very virulent strains indicating their evolutionary origin.
Key words: infectious
bursal disease virus; virus isolates; RT-PCR; nucleotide sequence; amino
acid sequence; phylogenetic analysis
Acta virologica 46: 95 – 101, 2002
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M.J. DE SIERRA, A.M. SÁNCHEZ, L. QUIRICCI, A. DIAMONT, G. RODRIGUEZ, H. CHIPARELLI, A.M. FERRARI, J. RUSSI, J. ARBIZA
Sección Virología, Facultad de Ciencias,
Universidad de la República, Igua 4225, Montevideo, Uruguay;
Departamento de Bacteriología y Virología, Facultad de Medicina,
Universidad de la República; Montevideo, Uruguay;
Centro de Asistencia del Sindicato Médico del Uruguay (CASMU),
Montevideo, Uruguay
Summary. – Electropherotypes of human rotavirus isolates from infants with acute diarrhea belonging to two populations with different clinical features were determined. Thirteen electropherotypes were identified in total 69 isolates; 46 (66.6%) isolates had long RNA migration patterns and 23 (33.3%) isolates had short migration pattern. One of the long-pattern electropherotypes (47.82% of the total electropherotypes) was predominant. It was detected in both populations almost throughout the whole period of the study, while other electropherotypes were found only occasionally. The co-circulation of long and short electropherotypes was not frequent.
Key words: rotavirus;
RNA electropherotypes; infantile diarrhea
Acta virologica 46: 103 – 106, 2002
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T.L. LIN, C.C. LOA, C.C. WU
Department of Veterinary Pathobiology, Purdue University, 1175 ADDL, West Lafayette, Indiana 47907-1175, USA
Summary. – A segment of genomic RNA extending from the 3´-end of the membrane (M) protein gene to the 5'-end of the nucleocapsid (N) protein gene of Turkey coronavirus (TCV) was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The primers were derived from the corresponding sequences of Infectious bronchitis virus (IBV). The PCR products were cloned and sequenced and their nucleic acid structure and similarity to the published sequences of IBV were analyzed. Gene 5 containing two overlapping open reading frames (ORFs), 5a and 5b, was localized between M and N genes of TCV. The overall nucleotide sequences of the amplified regions from TCV isolates shared 88.4% to 91.8% similarity to the corresponding region of IBV strains. The consensus transcription-associated sequence of IBV, CTTAACAA, was highly conserved in the TCV genome with regard to nucleotide sequence and location in terms of the initiation codons of the genes 5 and N. The similarities between the predicted amino acid sequences of ORFs 5a and 5b of TCV isolates and the homologous genes of IBV strains were 85.4% to 94.0%. The results indicate the existence of gene 5 in the genome of TCV and a close relatedness of the TCV gene 5 to the IBV gene 5 in location and nucleotide sequence.
Key words: avian
infectious bronchitis virus; turkey coronavirus; genomic relatedness,
gene 5
Acta virologica 46: 107 – 116, 2002
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M. SLÁVIKOVÁ, P. KOCÁKOVÁ, M. SLOVÁK, I. VANČOVÁ, V. HAJNICKÁ, J. GAŠPERÍK, N. FUCHSBERGER, P.A. NUTTALL
Institute of Virology, Slovak Academy of Sciences,
Dúbravská cesta 9, 842 45 Bratislava, Slovak Republic;
Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovak
Republic;
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava,
Slovak Republic;
CEH Institute of Virology and Environmental Microbiology, Oxford OX1 3SR
United Kingdom
Summary. – A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.
Key words: vesicular
stomatitis virus; nucleocapsid protein; IL-8; tick salivary gland;
Dermacentor reticulatus
Acta virologica 46: 117 – 120, 2002
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P. DOMINGUES, P. PALKOVIČ, R. TOMAN
Department of Chemistry, University of Aveiro,
Aveiro, Portugal;
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9,
842 45 Bratislava, Slovak Republic
Summary. – Phospholipids extracted from the Coxiella burnetii strain Nine Mile virulent phase I and low-virulent phase II cells were directly analyzed by fast atom bombardment mass spectrometry (FAB-MS). Constant neutral loss (CNL) scanning mass spectra (MS) were acquired to identify various phospholipids within phospholipid classes. Phospholipids from the phase I C. burnetii cells were much more complex than those from the phase II cells. Moreover, in the latter, the absence of phospholipids of the phosphatidylinositol class could be noticed. The results indicate that CNL scanning of phospholipid samples provides a rapid and simple method for identification of the phase state of the bacterium.
Key words: Coxiella
burnetii; phospholipids; FAB-MS; phase state
Acta virologica 46: 121 – 124, 2002
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K. PETRZIK, D. KUBELKOVÁ, I. MRÁZ
Department of Plant Virology, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic
Key words: strawberry
vein banding virus; recombinant coat protein; Pichia pastoris;
Escherichia coli; DAS-ELISA
Acta virologica 46: 125 – 126, 2002
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