6th Conference Program (Greek)
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Hellenic Virology
Volume 3, Number 1, 1998
Abstracts
- Emerging plant viruses
P.E. Kyriakopoulou - Evaluation of clinical and laboratory findings in children with infectious mononucleosis
M. Merkouri (1), K. Themeli-Digalaki (2), M. Economou (2), M. Ziva (1), Ch. Koutsia-Karouzou (2), St. Papadakou-Lagogianni (1) - Development of screening methodology for potential antiviral drugs against hepatitis A virus replication in vitro
E. Biziagos - Mechanism of action of selected antiviral substances on hepatitis A virus replication in vitro
E. Biziagos
Laboratory of Plant Pathology, Agricultural University of Athens
Hellenic Virology, 1998, 3(1): 5-25.
The term "emerging virus" was coined in the early 1980's, to express the dramatic situation with the human acquired immunodeficiency syndrom (AIDS) in the last decade. Then, it seemed to fit the epidemiological situation with many human, animal and plant virus diseases. In the present work, definitions of the term "emerging plant virus" are given and three examples are analyzed, cucumber mosaic cucumovirus (CMV), tomato spotted wilt tospovirus (TSWV) and, the most catastrophic of plant viruses, citrus tristeza closterovirus (CTV). These viruses are very widespread geographically, very epidemic and very important economically, both for Greece and worldwide. CTV is a threat for Greece. The first two viruses affect herbaceous crops, vegetables and ornamentals. During the last 15-20 years a severe emergence of these two viruses has occured in wide geographical zones, including Mediterannean countries and Greece. Tomato and other crops are suffering severely, due to their ubiquitous infection and severe symptomatology. Factors such as the very wide host range, genetic plasticity of the virus and easy emergence of new strains, very large number of vector species (CMV) and efficient and massive air vectoring due to massive multiplication and massive flights of aphid (CMV) and thrips (TSWV) vectors, introduction of new virus strains and new efficient vector (TSWV-Frankliniella occidentalis) due to massive importations of ornamental and other plants from affected areas, as well as agricultural intensification and extended monocultures of sensitive species and varieties, are among the factors which boosted and, in many cases, originated the emergence of these viruses. More or less the same factors have caused the catastrophic emergence of CTV in South America in the 1930's, where 30,000,000 trees were wiped-out in the 1940's, and in Spain in the 1950's and 1960's. Massive importations of vegetative citrus propagation material during the first decades of this century and earlier, from South and East Asia, the geographical origin of CTV and its efficient aphid vector Toxoptera citricidus, introduced in these new and agricultutrally expanding areas of America at those times both the virus and its efficient vector in sufficient mass as to build up their volume for the epidemic burstings. The general use of sour orange as rootstock, for avoiding the destructive Phytophthora crown rot, caused the emergence of severe symptomatology, since this rootstock/scion combination, especially with orange or mandarine scions, is extremely sensitive to tristeza (quick or gradual decline). Other less severe forms of tristeza (stem pitting, seedling yellows, lime dieback) prevail in South America and Australia where other rootstocks have been used. High virus genetic plasticity easily creates new strains with differences in symptomatology, vector transmission efficiency etc. Tristeza occurs or has been reported in all Mediterannean countries except Greece. This blessing of Greece, which is due to a high extent to the strict and timely phytosanitary regulations applied since the late 1950's, should be better appreciated, taken advantage-of and diligently protected.
(1) Department of Pediatrics and (2) Department of Microbiology, Peripheral General Hospital "Asklipios", Voula, Athens
Hellenic Virology, 1998, 3(1): 26-31.
Epstein-Barr virus (EBV) is the most frequent cause of mononucleosis (MN) followed by Cytomegalovirus (CMV). The aim of this study was to correlate the clinical and laboratory findings in children suffering from MN with the pathogenetic agent. We present sixty-two children (37 boys, 25 girls) who had been examined at the Pediatric Department of the Asclepeion Voulas Hospital with clinical features of MN. Symptoms like fever, tonsillopharyngitis, cervical lymphadenopathy, hepatomegaly and splenomegaly were evaluated in all children. Laboratory findings such as peripheral white blood cell count, presence of atypical lymphocytes and elevated serum transaminase concentrations were also determined. Specific antibodies to EBV were titrated by immunofluorescence (IFA) while antibodies to CMV were determined by both IFA and enzyme-linked immunosorbent assays (MEIA). Forty (64.5%) of the sixty-two patients were proven to have Epstein-Barr mononucleosis and twenty-two (35.5%) while had cytomegalovirus mononucleosis. Both clinical symptoms and laboratory findings were similar with no significant differences in these two groups. Notable was the fact that 18 (45%) children with EBV had positive heterophil antibodies and that 13 (59%) of the children with CMV mononucleosis were younger than 4 years old. We conclude that the cause of mononucleosis can only be determined by specific serologic methods.
Virology Laboratory, Diagnostic and Therapeutic Medical Center, Rethymnon, Crete
Hellenic Virology, 1998, 3(1): 32-43.
In this work we developed a methodology for screening substances with potential antiviral activity against hepatitis A virus (HAV) replication in the PLC/PRF/5 cell line. The methodology was the following: A) Examination of the toxicity of each drug at various concentrations for a 15-day period by daily cell examination under a phase-contrast microscope to discard concentrations damaging the cell layer and determination of the minimum toxic concentration of each drug that induced a microscopically visible alteration of normal cell morphology. On day 15 (or on day 3 for deoxythymidine incorporation experiments), cytotoxicity of the retained concentrations for each compound was further evaluated by (a) the determination of cell viability (viable cell counts after staining with trypan blue) and cell proliferation ( re-culture of cells following the action of drugs and estimation of cell growth), (b) the determination of total cell protein (measurement of protein quantity by Lowry's procedure), and (c) the determination of the inhibition of cellular DNA, RNA and protein biosynthesis (measurement of the methyl-[3H]deoxythymidine, [3H]uridine and [3H]leucine incorporation respectively). With these measurements, we determined the maximum nontoxic concentration, the 50% cytotoxic dose and the 50% inhibitory dose for each drug. B) Examination of the antiviral activity of each drug on the viral antigen expression for determining the 50% effective dose and thus the selectivity indices, and determination of the true antiviral activity of each drug at various concentrations (until the maximum nontoxic dose determined above) on the HAV infectivity. Using this procedure, more than fifty compounds were screened for inhibotory effect on HAV multiplication in PLC/PRF/5 cells. Eleven of them (amantadine, atropine, glycyrrhizin, interferon a-2a, interferon a-2b, iota-carrageenan, kappa-carrageenan, lambda-carrageenan, protamine, pyrazofurin and ribavirin) caused concentration-dependent reductions on the HAV antigenicity and infectivity with selectivity indices >5, and emerged from the present study as promising candidates for chemotherapy of acute hepatitis A.
Virology Laboratory, Diagnostic and Therapeutic Medical Center, Rethymnon, Crete
Hellenic Virology, 1998, 3(1): 44-59.
Atropine (ART) , glycyrrhizin (GL) and protamine (PRO) were tested for their effects on hepatitis A virus (HAV) replication in cell culture, in order to determine their mechanism of action. PLC/PRF/5 hepatoma cell line was infected by cell culture-adapted HAV strain CF53 at several multiplicities of infection (MOIs) and experiments were carried-out for 4 and 15 days according to MOIs used, in order to achieve one-step and multiple-step growth conditions. The yields of infectious HAV after 4 and 15 days were markedly reduced by each drug at its maximum non-toxic concentration, especially at the lowest MOI. The observed reductions in the HAV infectivitywere inoculum size-dependents. The activity of each drug was irreversible. ATR, GL and PRO were active when added as late as 2, 4, and 6h respectively after inoculation with HAV. Their action was not virucidal because we have no measurable effects on the extracellular virus. ATR had a prophylactic effect and inhibited HAV adsorption as measured by the kinetic of adsorption of [3H] uridine-labeled virions to PLC/PRF/5 cells. GL had no measurable effect on the adsorption of radiolabelled virions but inhibited HAV penetration on the plasma membrane as measured by the amount of infective virus no longer neutralizable by specific antibody over time. PRO did not induce any anti-HAV state by treating cells with this drug prior to inoculation and did not act on the adsorption and/or penetration of HAV on the plasma membrane. It acts at an early step of the HAV replicative cycle, probably on a transcriptional event, such as the beginning of accumulation of total viral RNA or the transcription of positive-strand RNA into the negative strands that are used as templates for positive-strand RNA neosynthesis. The combination of ATR and PRO resulted in an enhanced anti HAV effect. We concluded that these drugs affect separate steps in the HAV replicative cycle in PLC/PRF/5 cells.