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Acta virologica

Volume 47 / 2003 / number 1

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Immunization with varicella-zoster virus glycoprotein E expressing vectors: comparison of antibody response to DNA vaccine and recombinant vaccinia virus

J. Stašíková, L. Kutinová, M. Šmahel, Š. Němečková*

Department of Experimental Virology, Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2, Czech Republic

Summary. – Immunization with DNA vaccines expressing Varicella-zoster virus (VZV) glycoprotein E (gE) induced formation of specific antibodies in mice. The antibody response correlated with the level of in vitro gE expression if the plasmid was inoculated intradermally (i.d.) with a gene gun but not if intramuscular (i.m.) injection was used. The i.d. vaccination produced a higher antibody level than the i.m. one even though a 100-fold amount of DNA was administered. A plasmid expressing a truncated form of gE was less immunogenic. The magnitude of antibody response induced by immunization with recombinant vaccinia viruses (rVVs) was equivalent to the gene gun vaccination. Administration of DNA by i.m. route or Vaccinia virus (VV) gE by i.d. route resulted in predominance of IgG2a in the response while the gene gun plasmid inoculation usually elicited similar levels of IgG1 and IgG2a. The antibody response elicited by DNA vaccine was boosted by a secondary immunization with rVV. The boosting effect was highest if the virus was administered intraperitoneal (i.p.).

Keywords: Varicella-zoster virus; glycoprotein E; DNA vaccine; Vaccinia virus
Acta virologica 47: 1 – 10, 2003


A.A. KaNSKY1, M. Poljak2*, K. SEME2, B.j. KoCJAN2, N. GALE3, B. LUZAR3, R. GOLOUH4

1Department of Maxillofacial and Oral Surgery, University Medical Center, Ljubljana, Slovenia; 2Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana; Zaloška 4, 1000 Ljubljana, Slovenia; 3Institute of Pathology, Medical Faculty, University of Ljubljana; Ljubljana, Slovenia; 4Institute of Oncology, Ljubljana, Slovenia

Summary. – To elucidate the putative etiologic role of human papillomaviruses (HPV) in oral carcinogenesis, a comparative study was carried out on 62 tissue specimens of oral squamous cell carcinoma (OSCC) and on 62 specimens of histologically normal oral mucosa obtained from the individuals who matched the subjects with OSCC in age, gender, localization of obtained tissue specimens, drinking and smoking habits. Internal control amplification showed that amplifiable DNA was recovered from 59/62 and 61/62 tissue samples of OSCC and normal oral mucosa, respectively. The amplification with two different HPV L1 and one HPV E6 consensus primer sets showed the presence of the HPV DNA genotypes 16, 33, 58 in 5/59 (8.4%) OSCC specimens and HPV genotypes 11, 16, 31, 68 in 4/61 (6.6%) tissue samples of normal oral mucosa tested. In the study in which a comparative examination of the presence of HPV DNA was for the first time performed on the tissue samples of the patients with OSCC and the age- and gender-matched control subjects there was no significant difference in the prevalence of HPV DNA among both study groups. Our results suggest that occasional findings of HPV DNA in OSCC tissue specimens may be the result of an incidental HPV colonization of oral mucosa, rather than of viral infection, and that HPVs play a limited role in the etiopathogenesis of the majority of OSCC.

Key words: human papillomaviruses; oral carcinoma; normal mucosa; Slovenia
Acta virologica 47: 11 – 16, 2003


K. Nikolaou1, L. Varinou1, N. Inoue2, M. Arsenakis1*

1Laboratory of General Microbiology, Section of Genetics, Developmental and Molecular Biology, School of Biology, Aristotle University, Thessaloniki 54006, Greece; 2Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

Summary. – Human herpesvirus 6 (HHV-6) isolates can be classified into two variants, A and B. Comparison of genomic sequences of these variants has highlighted sequence variability in the region spanning U86 to U100. This region includes the immediate early A (IE-A) locus that was defined as positional homologue of the major IE locus of Human cytomegalovirus (HCMV) with little recognizable sequence homologies. A 3.5 kb transcript, one of the four spliced transcripts identified in the IE-A locus, is derived from the U90/89 ORF encoding the IE1 protein. We expressed six Escherichia coli fragments spanning the HHV-6A U90/89 ORF as IE1 fusion proteins. The bacterially expressed fusion protein was used to raise monospecific polyclonal antiserum for detection and identification of the IE1 protein product(s). Using this antiserum we detected 165, 190, and >190 K proteins in HHV-6A- and HHV-6B-infected cells and the 165 K protein in cells transfected with an IE1 cDNA construct. The IE1 proteins exhibited perinuclear and cytoplasmic localization in infected cells. There was a correlation between the expression level of IE1 and the degree of permissiveness for virus growth in various cell lines. In transient expression experiments a 140 bp fragment from the upstream IE-A region was shown to possess promoter activity. The C-terminal region of IE1 delineated by amino acids (aa) 588 to 636 showed a DNA binding activity in Southwestern blot analysis.

Key words: Human herpesvirus 6; immediate early gene; U90/89; U90; IE-A region
Acta virologica 47: 17 – 26, 2003

Identification of a novel Human SAND family protein in HUMAN FibroblastS induced by HERPES SIMPLEX VIRUS 1 binding

S. Dong, C. Dong, L. Liu, Y. Che, M. Sun, F. Hu, J. Li, Q. Li*

Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, 379 Jiaoling Rd., Kunming 650118, P.R. China

Summary. – Studies on interaction between Herpes simplex virus 1 (HSV-1) and human fibroblasts KMB-17 have demonstrated that HSV-1 binding to the cell surface could induce a specific gene response. In this study, the HSV-1 stimulation-related gene 1 (HSRG1), a new so far unknown gene function of cellular response induced by a specific stimulation with HSV-1, was cloned from the cDNA library established from mRNA of early gene response of KMB-17 cells. The gene product consisted of 547 amino acids and had a significant homology in six eukaryotic species. On the basis of its structure it was identified as a member of the SAND protein family. The HSRG1 protein was fused with glutathione S-transferase (GST) and expressed in Escherichia coli DHPa strain under the control of T7 promoter. An antibody to HSRG1 raised in mice was used to detect expression of the HSRG1 protein in KMB-17 cells stimulated by HSV-1 by an immunoprecipitation assay. It was found that the HSRG1 protein was induced in these cells by HSV-1 at high level.

Key words: Herpes simplex virus 1; SAND protein family; signal transduction
Acta virologica 47: 27 – 32, 2003

Demonstration of bovine respiratory syncytial virus RNA in peripheral blood leukocytes of naturally infected cattle

V. ValentovÁ*, I. PŠikal, K. KovaŘČÍk

Veterinary Research Institute, Hudcova 70, Brno 621 32, Czech Republic

Summary. – RNA of Bovine respiratory syncytial virus (BRSV) was found in peripheral leukocytes and nasal mucosa of infected cows by nested reverse transcription–polymerase chain reaction (nRT-PCR). We suppose that this finding obtained in the convalescent phase of infection indicates possible persistence of the virus in cells of the immune system.

Key words: Bovine respiratory syncytial virus; cattle; leukocytes; nasal mucosa; persistent infection; serum antibodies; RT-PCR
Acta virologica 47: 33 – 36, 2003

nucleotide sequenceS of coat protein coding regionS of six Potato mop-top virus isolates


Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce 1a, 160 00 Prague 6, the Czech Republic

Summary. – Coat protein (CP) coding regions of six Potato mop-top virus (PMTV) isolates from the Czech Republic and Denmark (54-10, 54-11, 54-15, 54-19, Korneta and Pacov) were sequenced. Comparison of the obtained nucleotide sequences as well as alignment of the deduced amino acid sequences were performed. The obtained results showed that the isolates from different parts of Europe seem to have highly conserved coding regions which is unexpected for a viral RNA genome known for its high mutation rate. Thus considerable differences in virulence and significant variation in biological properties of these isolates should not be attributed to CP but to some other part of the genome.

Key words: Potato mop-top virus; virus isolates; coat protein; gene; protein; amino acid sequence; nucleotide sequence
Acta virologica 47: 37– 40, 2003


D. Bystřická1,2, O. Lenz2, I. Mráz1, P. Dědič4, M. Šíp1,3,*

1Department of Plant Virology, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, 2Faculty of Biological Sciences, University of South Bohemia, České Budějovice, the Czech Republic; 3Faculty of Health and Social Studies, University of South Bohemia, Jírovcova 24, 370 05 České Budějovice, the Czech Republic; 4Potato Research Institute, Havlíčkův Brod, the Czech Republic

Summary. – DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.

Key words: DNA microarray assay; potato viruses; diagnostics
Acta virologica 47: 41– 44, 2003

Experimental transmission of Chikungunya virus by Anopheles stephensi mosquitoes

P. Yadav, M.D. Gokhale, P.V. Barde, D.K. Singh, A.C. Mishra, D.T. Mourya*

Microbial Containment Complex, National Institute of Virology, Indian Council of Medical Research, Sus Road, Pashan, Pune 411 021, India

Summary. – The Aedes aegypti mosquito has been considered the principal vector of Chikungunya (CHIK) virus. As CHIK epidemics usually occur in urban regions and Anopheles stephensi is another highly endophilic and anthropophilic mosquito, there is a very high probability of this mosquito to feed on CHIK virus-infected patients, to pick up and transmit the virus. Therefore the present study was conducted to test the CHIK virus transmission capability for the A. stephensi mosquito. The obtained results showed that this mosquito species is capable of transmitting CHIK virus. It is surmised that during any epidemic of febrile illness CHIK virus isolation attempts should also be made from this mosquito species.

Key words: Aedes aegypti; Anopheles stephensi; Chikungunya virus; mosquitoes; RT-PCR; suckling mice; susceptibility; transmission
Acta virologica 47: 45– 47, 2003


M. Glasa1,*, G. Labonne2, J.-B. Quiot2

1Department of Plant Viruses, Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovak Republic; 2UMR Biologie et Génétique des Interactions Plante-Parasite pour la Protection Intégrée, INRA-ENSAM, Montpellier, France

Summary. – One of the key factors of progress of an epidemic is the duration of virus availability for a vector in plants, which could be influenced by temperature. Using five epidemiologically different isolates of Plum pox virus (PPV) we studied the effect of temperature on the virus infectivity, intensity of disease symptoms and virus accumulation in Nicotiana benthamiana plants as determined by a double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). No differences in infectivity and intensity of disease symptoms between the five isolates were observed at 17°C. However, they differed in their capacity to infect and multiply in the plant at higher temperatures. The temperature of 32°C was inhibitory to the multiplication of all the five PPV isolates studied. Fewer plants were infected and a significantly decreased amount of virus antigen was detected at 30°C. The natural PPV recombinant BOR-3 isolate showed a greater temperature tolerance compared to other PPV isolates tested. We conclude that adaptation to higher temperatures may favour the epidemiological impact of PPV.

Key words: sharka; potyvirus; effect of temperature; ELISA; epidemiology
Acta virologica 47: 49– 52, 2003

Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride iN experimental murine influenza A virus infection

J. Serkedjieva1*, I. Roeva1, M. Angelova1, P. Dolashka2, W.g. Voelter3

1Institute of Microbiology, Bulgarian Academy of Sciences, 26 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria; 2Institute of Organic Chemistry, Center of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria; 3Abteilung fur Physikalische Biochemie des Physiologisch-Chemischen Instituts der Universität, Tübingen, Germany

Summary. – The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.

Key words: fungus Humicula lutea; superoxide dismutase, rimantadine hydrochloride, combined protective effect, murine influenza virus infection
Acta virologica 47: 53–56, 2003


IN SITU Reverse Transcription–Polymerase Chain Reaction: a Novel Technique for Detection of Rabies Virus RNA in Murine Neuroblastoma Cells


1Department of Animal Biotechnology and 2 Department of Bacterial Vaccines, Tamil Nadu Veterinary and Animal Sciences University, Madras Veterinary College, Chennai 600 007, India

Acta virologica 47: 57– 59, 2003


Structure-Function Relationships of Human Pathogenic Viruses

A. Holzenburg and E. Bogner (Eds): Structure-Function Relationships of Human Pathogenic Viruses. Kluwer Academic/Plenum Publishers, New York, 2002, 528 pp.

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