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St. Cercet. Virusol. 35 (2) 2005


83 - Influenza virus and primer design 
C.N. Zaharia and Gabriela Anton
Stefan S. Nicolau Institute of Virology, Bucharest, Romania
Abstract. The recent achievements regarding the complete sequencing of the influenza virus type A and especially of the H3N2 subtype genome, in the framework of a project which is under way, initiated by NIAID – USA are pointed out. All the genes of the influenza virus type A are briefly reviewed and the characteristics specific of each of the 8 RNA segments are detailed. The complete sequence of the segment 8 and of the nonstructural protein, with the NS1 and NS2 components which this segment codifies are specified. For the design of primers necessary to the PCR technique, rules, calculation formulae and recommendations for obtain the sequence of the highest performance in the amplification and identification of influenza virus types and subtypes are presented. One section of the work is dedicated to the presentation of commercial or free programs permitting a rapid and efficient primer projection (degenerating primers included) for given or unknown nucleotide sequences. In the annexes are indicated web addresses to several Internet available sources, in order to offer the possibility of accessing the most suitable design programs of primers necessary to the investigations involving the use of the PCR technique. Numerous published primers sequences are presented and accompanied by a series of detailed descriptions.

Keywords: Influenza virus types A, viral genome, nonstructural protein (NS), PCR, primer design, computer programs, primer sequence.


101 - The incidence of genital associated infections in patients with cervical cancer (palillomavirus, cytomegalovirus, Epstein-Barr virus)
Constanta Antipa, Gabriela Anton, Anda Dragodan, Adriana Plesa, M. Stoian
Stefan S. Nicolau Institute of Virology, Bucharest, Romania
Abstract. 53 patients with clinical diagnosis of cervical cancer, ages between 21-70, have been investigated in in the diagnostic laboratory in the Institute of Virology „Stefan S. Nicolau” from Bucharest. Specimens collected from lesions were used to identify the DNA- for: HPV, CMV, and EBV. The patients were divided in three groups according to the age. The results obtained were as follows: in 21-35 age group, HPV was found in 2 from 6 patients (33, 3%), CMV in 3 from 6 patients (50%), and EBV in 3 from 6 patients (50%). In 36-50 age group, HPV was found in 6 from 18 patients (33, 3%), CMV in 11 from 18 patients (61, 1%), and EBV in 10 from 18 patients (55, 5%). In 51-70 age group, HPV was found in 24 from 29 patients (83, 3%), CMV in 19 from 29 patients (65, 5%), and EBV in 17 from 29 patients (58, 6%). Confections founded were HPV/CMV 56%; HPV/EBV 18, 8%; EBV/CMV 16, 9%.

Keywords: papillomavirus, cytomegalovirus, virus Epstein-Barr virus, cervical cancer.

107 - hTERT (Telomerase Catalitic Subunit) expresion in preneoplasic and neoplasic cervical lesions 
Gabriela Anton1, Adriana Plesa1, Coralia Bleotu1, Constanta Antipa1, Elena Popa2, Elena Nistor2
1 Stefan S. Nicolau Institute of Virology, Bucharest, Romania                                                                                   
2 Bucur Hospital, Bucharest, Romania
Abstract. This study was performed to determine a possible correlation between catalytic subunit of human telomerase (hTERT) and (pre)neoplastic cervical lesions. Nucleic acids were isolated from 29 biopsies intraoperatory harvested from patientes presenting (pre)malignant cervical lesions (anatomo-pathologycaly confirmed). DNAs were used in nested- PCR for HPV infection detection and ARNs served as templates for RT-PCR in order to determine hTERT levels.  The semiqantitative hTERT mRNA levels were related to cervical lesions grade and presence of HPV infection with high risk genotypes.  We estimated that hTERT expression is noted from early stages of cervical carcinogenesis but it can not be used as a diagnosis marker.

Keywords: hTERT, HPV, preneoplasic and neoplasic cervical lesions.

111 - Alteration of control mechanisms in HPV infection
Coralia Bleotu
Stefan S. Nicolau Institute of Virology, Bucharest, Romania
Abstract. The significance of viruses in carcinogenesis remains the most interesting problems for oncology and biology. The human papillomavirus (HPV) supply impressive information on the mechanisms of tumor growth induction. The oncogenic potential of HPV is based on their capacity to alter the cell cycle control, establishing the accumulation and transmission of genetic alteration. These abilities are not sufficient to produce carcinoma, frequently occurring spontaneous clearance of HPV infection. On the other hand, a lot of diference between HPV tipes probably are implicated in the long delay between the onset of persistent infection and emergence of the malignancy.

Keywords: Human papillomaviruses, oncogenesis, E6, E7, E5.


127 - Procalcitonin and ceruloplasmin - new serum markers for the differential diagnosis of bacterial and viral meningitis
Florica Toparceanu1, Rodica Rusu2, Loretta Ichim1, Camelia Augustina Mihai2, P.I. Calistru2
1Stefan S. Nicolau Institute of Virology, Bucharest, Romania                                                                          
2Victor Babes Infectious Diseases Clinical Hospital, Bucharest, Romania
Abstract. In order to establish a more precisely and precocious diagnosis which might discriminate between bacterial meningitis (BACT_M) and viral meningitis (VIR_M), the utilization of biological markers additional to the CSF analysis, such as the acute-phase proteins, was proposed. In this work, the accuracy of differential diagnosis values between BACT_M and VIR_M of the newly proposed markers, procalcitonin (PCT) and ceruloplasmin (CP), in comparison with the value of the classical marker, the C-reactive protein (CRP) was evaluated. Our results obtained on a series of 25 children, of whom 12 diagnosed with BACT_M and 13 with VIR_M, show that PCT and CRP are earlier markers than CP. PCT which is detectable in BACT-M and undetectable in VIR-M seem to be a more conclusive marker for the differential diagnosis of meningitides than CRP, which may have moderate concentrations both in some BACT-M and in some VIR-M. The increased value of CP may be very useful in the diagnosis both of late-hospitalized BACT-M, when the PCT and CRP concentrations are significantly diminished under the partial treatment with antibiotics, and of complicated   VIR-M (multiple etiologies). On the other hand, PCT, CRP and CP may be the same degree markers of the severe prognosis.

Keywords: acute meningitis, differential diagnosis, viral/bacterial infection, procalcitonin, ceruloplasmin, C-reactive protein.


141 - Different aspects of cell death receptors-induced apoptosis 
D. Hotnog, Mihaela Daniela Simioana, Viviana Roman
Center of Immunology, Institute of Virology Stefan S. Nicolau, Bucharest, Romania
Abstract. Apoptosis represent one of the most studyed process in this moment, and all signaling pathway in witch apoptosis is implicated may serve in the future to find new solution for therapy for many diseases and also to improve substantial the life of afected patients. The study of cell death receptors-induced apoptosis (TNF family) is very important for finding of different therapeutic target (like TRAIL receptor) and also for finding new strategy for treatment of patients with different forms of cancer. The study of the molecules implicated in death-receptors pathway (like caspases, viral inhibitors of caspases, kB nuclear factor) could lead to new therapeutical targets for some diseases caused by apoptosis process dysfunctions.

Keywords: apoptosis, TNF, caspases, NF-kB, FLIP.


149 - Correlations between the structure and functions of some complement regulatory proteins 
Mihaela Daniela Simioana, Mariana Claudia Constantin, D. Hotnog, Lorelai Irina Brasoveanu
Center of Immunology, Institute of Virology Stefan S. Nicolau, Bucharest, Romania
Abstract. Complement system contributes to the elimination of invading microorganisms, immune complexes and transformed cells from tissues and biologic fluids. Complement activation is an irreversible phenomenon initiatedby three distinct pathways (classical, alternative and lectin) having as a result the lysis of target cell. For an efficient immune surveillance the complement system is continuously activated at a low level by the alternative pathway, which might become dangerous for host tissues. Therefore, the strict regulation of complement activation is essential in order to prevent nonspecific damages. This function is due to complement regulatory proteins (CRP) that protect host cells against autologous complement attack, by controlling different steps of activation. Concerted action of C regulators induces the resistance of own cells to complement mediated lysis. In addition, several viruses (CMV, HTLV-1, HIV-1) take off some CRP (DAF, MCP or CD59) from the host cell membrane, and use them in order to protect themselves and avoid C-mediated lysis, whilst other (HPV) synthetize proteins which mimic human CRP.

Keywords: complement, regulatory protein, DAF, MCP, CD59.


159 - The selection of biological active compounds and drug development.
Florica Nicolescu1, T.O. Nicolescu2, Carmen Purdel1
1University of Medicine and Pharmacy „Carol Davila” Bucharest, Faculty of Pharmacy DepartmentToxicology                                                                                                                                
2University of Medicine and Pharmacy „Carol Davila” Bucharest, Faculty of Pharmacy, Department of  Organic Chemistry
Abstract. Along time were discovered numerous cures which properties were made evident expecially through direct observation, often undesigned, and more later were selected and clasified based on structure - biological activity relation. These days, the research on drug field imply quality’s assurance, understanding by this the ability to bring a therapeutic inovation through discovering new entity - biological active molecules, with new mechanism of action, or by changing a known class of compounds. Also, an important stage on drug’s research is represented by bioechivalence studies related to pharmaceutical form in which active substance is insert. The development of drug, after establishing a new structural entity through, involves two stages: preclinical development (preformulation, formulation and optimise) and clinical development.

Keywords: Relation structure - activity, pharmacological screening, bioechivalence, drug’s development.

169 - Susceptibility to acyclovir of herpes simplex virus isolates obtained between January – March 2001 
IrinaAlexiu, Mihaela Chivu, Carmen Diaconu, Coralia Bleotu, Anda Dragodan, C. Cernescu
Stefan S. Nicolau Institute of Virology, Bucharest, Romania
Abstract. Acyclovir (ACV) has been used for more than 15 years in the management of herpes simplex virus infections. The present survey was undertaken to assess the level of HSV susceptibility of the clinical isolates from general population. Four isolates obtained between January – March 2001 and a HSV strain from the Institute of Virology reference collection were tested for sensitivity to ACV. The mean susceptibility of HSV was 0.28±0.20, confirmed by the 50% inhibitory concentration (IC50). All the clinical isolates showed sensitivity to ACV in a dose-dependent manner. In line with previous studies assessing a low prevalence of resistance amoung isolates in general population we have found no resistant strain. Infectivity and susceptibility to ACV of clinical isolates are similar to the reference strain.

Keywords: herpes simplex virus, acyclovir, susceptibility.


173 - Location of structural symmetry molecules in plane lipid bilayers.
D. Popescu1, C.N. Zaharia2, Ecaterina Maries1
1Bucharest University, Faculty of Biology, Bucharest, Romania
2Stefan S. Nicolau Institute of Virology, Bucharest, Romania
Abstract. The lipid matrix is one of the main constituents of biomembranes. The passage mechanism of molecules across this matrix is very significant for any cell-membrane related process, for example in the study of drug absorption. The theoretical and molecular simulation researches on the lipid matrix of its traversing mechanism permit the demonstration of important aspects, some of which are impossible to be obtained experimentally, but are confirmed indirectly by evaluations of the biological activity at the cell level. The study presented is performed on plane lipid bilayers in which hypericin molecules are inserted. In this environment, the inserted molecules, which have an amphiphilic character, are oriented towards the polar zones of the bilayer. The data obtained show that the molecules are in relation with the lipid matrix, most probably in two positions, which are called asymmetrical and symmetrical, owing to the structural symmetry of hypericin. In the asymmetrical position, the symmetry axis of the molecules is parallel to the median plane of the lipid bilayer, whereas the symmetry axis of the molecule symmetrically positioned is perpendicular on the bilayer. These results were obtained by dynamic molecular simulation.

Keywords: hypericin, structural symmetry, lipid bilayer, dynamic molecular simulation.


179 - A new cellular automata model with applications in epidemiology.
C.N. Zaharia1 and Z. Haschi2
1Stefan S. Nicolau Institute of Virology, Bucharest, Romania
2 Politehnica University of Bucharest, Faculty of electronics and telecomunications, Bucharest, Romania
Abstract. For viral epidemic modeling a model based on cellular automata and multi-agent systems is proposed. The basic paradigms of the model are described and the characteristics of the cellular automaton state, movement and agent perception are detailed. Geographic restrictions are also taken into account in the model. The model implementation is achieved according to an original ArtVirus programe, written in C++ language.

Keywords: cellular automaton, multi-agent system, epidemiology, model, geographic restrictions.