P 56

VHB INFECTION IN BLOOD DONORS

S. Laitin, L. Negrutiu, I. Marincu
Epidemiology Department from Timisoara “V. Babes” University, Romania

Introduction: In the years before using the markers for etiologic diagnosis of VHB infection, a lot of researches were done in order to find some correlation in clinic, evolutive, biochemical functional and morphological aspects.

Methods: The prevalence of VHB infection in blood donors was determined on a lot of 19257 volunteers at the Regional Blood Transfusion Center between January 1st -December 31st 2001. This has proved the necessity of continuous monitoring of the blood donors. We have analyzed the possibilities to identify the VHB infection using the routine immune markers with the ELISA method.

Results and discussion: From the 19257 blood donors, 11559 (60.02%) came from the urban area and 7698 (39.97%) from the rural area. The prevalence of male sex donors with 15233 (79.10), the female sex donors were 4024 (20.89%) during one year from January 2001 to December 2001, the activity peak was in December (1842 donors) compared to January, when was the lowest activity (1524 donors). From 19527 blood donors, 148 were AgHBs positive, representing 0.76% from this lot with high risk in VHB infection.

Conclusion: Permanent monitoring of the blood donors for the immune markers of VHB and VHC allows safe measures of transfusion. This will decrease the incidence of the post-transfusion hepatitis and the incidence of VHB infection. The need of some human blood products, which will avoid the transmission of the post-transfusion viral infection.

P 57

TRANSDERMAL DELIVERY EFFICACY OF SOME ORIGINAL PHENOL-CONTAINED COMPOUNDS WHEN EXPERIMENTAL INFLUENZA INFECTION WAS MODELED

V. Larionov, I. Kravchenko, V. Lozitsky, R. Lozytska, A. Alexandrova, N. Ovcharenko, T. Gridina
Odessa National University, Odessa, Ukraine
Ukrainian I.I. Mechnikov Research Anti-Plague Institute, Odessa, Ukraine
A.V. Bogatsky Physics-Chemical Institute of NAS of Ukraine, Odessa

Transdermal delivery of drugs is a novel method of pharmacotherapy. The main advantages of this method are prolongation of the drug action, absence of the drug concentration hopping, reducing of the adverse reactions. This method of administration has to be viewed for prevention and therapy of influenza infection. Transdermal rimantadine delivery has shown high anti-influenza effectiveness during experimental infection (I. Kravchenko et al., 2003). The aim of this research was studying of the transdermal delivery efficacy of original bis-phenol substitutied compounds with different structure of “bridges” when experiment influenza was modeled. Substances were administrated in the hydrogel matrix (formed from 1.2-propylene glycol and polyvinyl alcohol) in the doses 1 or 2 mg/mouse applied on the shaved back of mice 1 day before infection. Animals of experimental and control groups were infected intranasally with highly pathogenic for mice influenza virus strain A/PR/8/34 (H1N1). Challenge was carried out using 4 animals for each virus dilution within the range of 10-1 to 10-6. Deaths of animals were recorded for 14 days.
The results of our investigations had shown high anti-influenza efficacy of three from four studied compounds when transdermal delivery was used. Fourth compound had high toxicity in conditions of experiments. Difference of LD50 between control and received the most active compound groups averaged 1.6 log10 when its dose was 1 mg/mouse and was similar to rimantadine efficacy in the same experimental conditions. So the most of synthesized by us for the first time compounds have established anti-influenza efficacy when transdermal delivery was used.

P 58

INSERTION IN THE CODON 36 HIV-1 PROTEASE CODING REGION: CLINICAL AND ANTIRETROVIRAL TREATMENT IMPLICATIONS

A.L. Carvalho, V. Duque, C. Faro, A.F. Fernandes, I. Ramos, J. Oliveira, S. da Cunha, A. Meliço-Silvestre
Virology Laboratory of the Infectious Diseases Department/ Coimbra University Hospital
Molecular Biology and Biotechnology Department of Center for Neuroscience and Cell Biology/ Coimbra University, Portugal

Background: HIV-1 subtype B protease structures are known and were used as models to create protease inhibitor drugs (PIs). Mutations that confer resistance to these antiretroviral drugs are well characterized and some of them are associated with viral fitness impairment.

Methods: Automated direct sequencing/phylogenetic analysis of HIV-1 protease and RT coding regions have been done in all samples collected from a cohort of antiretroviral inexperienced patients (n=104) enrolled at our infectious diseases clinic during the year 2000. All sequences are registered at Genbank sequence database. Three dimensional structure of these HIV-1 protease enzymes are being studied by virtual modelling with interaction with protease inhibitors, and by vector protein recombination methods for catalytic efficiency either with or without interaction with protease inhibitors.

Results: Two sequences (< 2 %) from two patients infected in Portugal were found to have an insertion at codon 36. Response to antiretroviral treatment including PIs was good (HIV-1 RNA < 50 copies/ml and sustained rise in CD4+ cell count). No signs of clinical progression have been observed since the year 2000. Three dimensional model shown different interaction between protease enzyme and PIs. Enzyme catalytic efficiency is being studied.

Conclusion: Codon 36 insertion seems to be a very rare event detected in HIV-1 drug naïve patients. In the short term no unfavourable impact could be assigned to this insertion in terms of clinical or viral response. A different interaction between the enzyme and PIs was documented. However, the functional impact is not known and is being studied.

P 59

NEW AUTOMATED MONITOR FOR DIRECT VIRUS-CELL INTERACTION MONITORING

O.P. Fedchuk, A.O. Fedchuk, A.S. Fedchuk, P.O. Fedchuk
I.I. Mechnikov Odessa National University, Odessa, Ukraine
Ukrainian I.I. Mechnikov Anti-Plague Research Institute, Odessa, Ukraine
Odessa State Academy of Refrigeration, Odessa, Ukraine

Influenza virus A2/Hong Kong/1/68 was chosen to be the model object of the present study. The preparations with various haemagglutinating infectious activities (HIA) were under investigation. Chicken embryos were infected by this virus. After 48 hours the allantoic liquid was collected and new viral generation HIA was checked up through HA reaction. The virus-containing material was titrated on chorioallantoic membranes’ fragments and the infectious activity TID50 was evaluated. We have measured the fractal dimension (FD) of the virus containing preparations using the fractal diffraction patterns monitor (FDPM). The DP images’ frames were registered by digital VZM 1000 Color LabVideo system and processed with the use of original software FractImagePro elaborated in our group. It was demonstrated experimentally for the first time that FD is the reliable, objective and precise numerical parameter that describes the presence as well as HIA of a given viral material. The addition of anti-influenza immunoglobulin to the virus containing preparations has led to the significant changes of their FD. These changes, most probably, are due to the reaction of antigen-antibody type that takes place. The proposed FDPM method allows to detect the virus-cell interaction occurrence without any coloring and at the minimal virus-containing concentrations some minutes after the reaction has started. The application of FDPM method could be successfully performed even in the case of enveloped viral particles detection. We have shown experimentally for the first time that the FDPM method and device could be used for express diagnostics in drug design and clinical practice.

P 60

NEW NUMERICAL IN-LINE DESCRIPTION OF VIRUS-CELL INTERACTION AT EVERY STAGE

A.O. Fedchuk, O.P. Fedchuk, A.S. Fedchuk, P.O. Fedchuk
I.I. Mechnikov Odessa National University, Odessa, Ukraine
Ukrainian I.I. Mechnikov Anti-Plague Research Institute, Odessa, Ukraine
Odessa State Academy of Refrigeration, Odessa, Ukraine

We have used for the first time the computer-assisted analysis of diffraction patterns (CAADP) obtained during laser irradiation of cell culture being attacked by Herpes simplex virus (HSV) for virus-cell interaction monitoring in real time. Samples of Hep-2 cells cultivated for 24 and 48 hours were used as a substrate for further infection with HSV of US-1 strain. E-aminocaproic acid (E-ACA) was used to modify the early stage of virus-cell interaction by inhibition the stage of proteolysis. The E-ACA treated and non-treated substrates both were investigated. The main experimental result was that all DP have shown a definitely fractal structure which could be numerically described by fractal dimension value D being strongly specific for every type of specimen as well as the prehistory of its preparation. We have registered also that almost all the information about the rate of virus-cell interaction and structural changes of virus-cell dynamic system was contained in the central self-shadowed part of the fractal cluster. The changes of D values for the space occupied by particles and inter-particle space were discussed. The changes of the smallest element of self-organized tenuous fractal cluster were registered while the concentration of E-ACA added was in the vicinity of the toxic dosage. The proposed method that uses fractal approach has demonstrated in experiment the highest possible sensitivity and could be used widely for the purposes of antiviral drug design and also as the feedback signal source at the stage of viral infectious diseases treatment for drug type and dosage correction.

P 61

HEPATITIS C THERAPY IN A COHORT OF HIV COINFECTED

J.E. Serra
Hospital Universidade Coimbra, Portugal

Both HIV and HCV infections share the same risk of transmission. Coinfection with both viruses is common, averages 35%. After introduction of HAART for HIV therapy in last years the morbidity and mortality due to chronic HCV infection had acquired most relevance. Today all HIV/HCV-coinfected patients should be evaluated for liver disease and for HCV treatment. A retrospective study was done in a cohort of twenty-two chronic hepatitis C HIV coinfected treated patients.
The aim of the study was to analyze some factors considered of most prognostic relevance in this HVC and consider the same aspects in the HIV coinfected patients.
HIV eligible patients could be on HAART and must have CD4 > 200 /mm³ and HIV RNA < 50 000 copies/ml, without current AIDS related disease.
The HCV factors analyzed were: sex and age of patients, HCV genotype, viral load, duration of current HCV infection and liver function and histology. All patients were on HCV bi-therapy, for a period of 24 to 48 weeks. The therapy consisted of pegylated interferon alfa 2b 1.5 µg sc/Kg/week or interferon alfa 2b 3MU sc thrice a week and Ribavirine per os 400 mg bid, each 9 patients group and on pegylated interferon alfa 2a 180 µg sc/Kg/week with Ribavirine per os 400 mg bid in 4 cases. A response was obtained in 68.1% of cases, that was sustained (SR) in 45% (10 patients) and recurrent (R) in 22.7% (5 patients). Nonresponse (NR) occurred in the remained 31% (7 patients).
In SR group of 10 patients, 90% belonged to CDC HIV early phases, 60% being male, the mean age was 40.4±4 years old. Only one patient was genotype 1 and had high viral load; the evolution of HCV infection was 16.3±7.8 years, 20% of them with cirrhosis in liver histology. The therapy consisted of pegylated interferon A in 70% of cases during 48 weeks in 60%.
Others have reported that a SVR can also be achieved in persons with HIV/HCV coinfection, as illustrated in this case study.

P 62

DETECTION OF ANTIRETROVIRAL RESISTANCE MUTATIONS BY REVERSE HYBRIDISATION AND SEQUENCING: A COMPARATIVE STUDY ON HIV-1 STRAINS ISOLATED FROM PATIENTS WITH VIROLOGICAL AND/OR IMMUNOLOGICAL FAILURE

M. Dinu, S. Manaila, M. Tinischi, D. Banica, D. Otelea
“Prof. Dr. Matei Bals”-Institute for Infectious Diseases, Bucharest, Romania

The genotypic resistance of 20 HIV-1 strains to antiretroviral treatment has been assessed by two different techniques. The HIV-1 virus strains originated from patients with virological and/or immunological failure and a history of treatment with several antiretroviral drugs. The extracted RNA used for viral load determination was used to amplify by RT-PCR specific genomic segments which were subsequently analysed for the presence of resistance mutations either by sequencing or by reverse hybridisation. The sequencing and data interpretation were performed with the ViroSeq™ (Applied Biosystems) genotyping kit and the hybridisation with the Versant™ HIV-1 Protease and RT Resistance assays (LiPA) (Bayer).
In addition to raw data comparison, resistance interpretation algorithms were performed to generate two sets of resistance reports in order to assess the impact of the groups of results on the clinical decision.
In our hands both techniques have displayed elements of strength and pitfalls. The sequencing has generated data on mutations in positions which could not be analysed by the reverse hybridisation because of either the lack of specific probes or the lack of reactivity of the probes to our DNA targets. On the other hand the reverse hybridisation was apparently more apt to detect some mixtures of resistance and wild-type mutations. The genotyping data were correlated with the clinical data and the predicted phenotypic resistance of the strains.

P 63

AMIXIN BINDING LEVELS IN CHRONIC VIRAL HEPATITIS C PATIENTS

E.V. Nikitin, L.N. Velichko, E.I. Dragomeretskaya, A.V. Bogdanova, K.L. Usichenko, A.A. Kalashnikov, A.S. Buiko
Medical University and V.P. Filatov Institute of Eye Diseases and Tissue Therapy, Odessa, Ukraine

Application of recombinant interferon for treatment chronic viral hepatitis C patients can be inhibited because of various side effects. Part of HCV patients have low cell sensitivity to interferon. Successful alternatives for recombinant interferon are interferon inductors. There are a lot of questions in treatment with imunnoinductors. Difference in efficiency of initial and repeated treatment courses is possible.

Aims: To study amixin (tyloron) binding levels with lymphocyte receptors in initial and repeated treatment courses in chronic viral hepatitis C patients. We have studied amixin (tyloron) binding level with lymphocyte receptors in 100 patients with chronic HCV. It was determined by rosette test after incubation with amixin. Different levels of binding were found in chronic HCV patients. Low level (2-8%) of binding was found in 10 patients, middle level (10-14%) in 40 patients and in 50 patients there was found high binding level (> 18%).
Patients with middle and high levels were treated with amixin for 2 years.Treatment included 10 courses (amixin 125 mg two times weekly for 5 weeks) with a month break between courses.
Amixin has not shown addicting property. Lymphocyte sensitivity to amixin was stable during the treatment. Normalization of Nk-cells cytotoxic activity and biochemical markers was shown.

Conclusion: Considering different sensitivity for amixin will improve its therapeutic efficiency. Amixin can be used in long-term therapy since it has no addiction and sensitivity to it is stable.

P 64

EFFICIENCY OF COMBINED APPLICATION OF ANTIVIRAL DRUGS AND SPECIFIC IMMUNOGLOBULIN FOR TREATMENT OF A CENTRAL NERVOUS SYSTEM LESIONS CAUSED BY EPSTEIN-BARR VIRUS

N. Nesterova, N. Dyachenko, A. Rudenko, K. Kurischuk, L. Ryadskaya,
L. Muravskaya, S. Ribalko, T. Berestova, E. Andreeva, S. Zagorodnaya, G. Baranova, V. Vinokurova
Inst. of Microbiology & Virology Nat. Acad. Sci. of Ukraine,
State Kyiv Enterprise for Production of Bacterial Medicines “Biopharma”,
Inst. of Epidemiology & Infectious Diseases Acad. Med. Sci. of Ukraine, Kyiv, Ukraine

For treatment of CNS lesions, caused by EBV (encephalomyelitis, arachnoencephalitis, meningoencephalitis), we have used gancyclovir or zovirax in combination with a new drug – the specific immunoglobulin against EBV, which is produced by “Biopharma”. Gancyclovir was injected intravenously at a rate of 10 mg/kg of a body weight; zovirax – at a rate of 20-30 mg/kg during 10-14 days. The immunoglobulin was injected intramuscularly for 5 times by 4.5 ml. The EBV-etiology of diseases has been confirmed by PCR results and also by detection of IgM and IgG to EBNA, EA and VCA antigens EBV with the help of ELISA.
It was established that as a result of a combined therapy it was observed the reduction of duration of some neurologic signs in patients in comparison with control group obtaining the only chemical antiviral drugs. In patients of the first group it was observed improvement of indexes of laboratory etiological diagnostics by the end of treatment (disappearence of viral DNA in liquor or blood, tendency to decrease of IgM levels and increase the IgG levels, in particularly, to VCA antigen EBV).
The interferonogenese features for both patients groups have been investigated.
The obtained data testify the high effectiveness of the specific immunoglobulin at CNS lesion treatment, caused by EBV.

P 65

THE EFFICACY OF ANTIVIRAL THERAPY IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

O.N. Egorova, R.M. Balabanova
The Institute of Rheumatology of RAMS, Moscow, Russia

Infectious agent involved in pathogenesis of SLE is actively searched to the last years. 32 SLE patients (according to the ACR criteria) (mean age 35.3±10.1 years; mean disease duration 7.79±5.32 years) were examined. The sera of patients were investigated for IgG and IgM antibody to CMV, EBV, HSV 1 and 2, Chlamydia pneumoniae. Anti-CMV IgG was detected in 87.8%, anti-VCA(EBV) - 78.8%), anti-EA(EBV) - 72.7%; anti-CMV IgM - 37.5%; anti-HSV-2 (IgM and IgG) - 36.4% and anti-Chlamydia pneumoniae IgG - 30.9%. Associated production of IgM anti-CMV and anti-EA EBV was detected in 27.2% patients. Correlation with disease duration was not observed. IgM antibody and CMV-Ag production were dominated in young patients (44.4% and 50% respectively), in older 30 years patients – anti-CMV (73.3%) and anti-VCA EBV IgG (60%) (p < 0.01). The negative correlation was observed between IFN-gamma, IFN-alpha production and anti-CMV and anti-VCA VEB IgG (r = -0.82; p < 0.01). This data has allowed expecting the chronic viral infection as result secondary immunodeficient. These patients used glucocorticosteroids (in dose 10 - 22.5 mg/day). They were assigned to receive during 3 months aciclovir (1 g/day for 7 days by the repeated course in 14 days – 11 pts), IFN-alfa-2b (10 - pts ), cyclophosphamide 200 mg/week or azathioprin 100 mg/day (11 - pts). Antiviral therapy has reduced the number of patients with erythema (36.36%), enanthema (9.09%), arthralgia (48.48%) and signs of CNS involvement (12.12%). Cases of lupus nephritis were not observed. The positive influences of antiviral therapy (particularly in IFN-alfa-2b received pts) were correlated with increasing of the antiviral antibody production, detecting of family Herpes viridae antigens, and increasing of IFN production. The use of antiviral therapy can be considered as therapy of SLE component.

P 66

INFLUENCE OF 6-AZACYTIDINE AND ACiCLOVIR ON SYNTHESIS OF EPSTEIN-BARR VIRUS DNA AND APOPTOSIS OF B-LYMPHOCYTES

N. Dyachenko, N. Nesterova, I. Alexeeva, S. Zagorodnaya, G. Baranova, O. Povnitsa, L. Palchikovskaya, S. Sidorenko, L. Shlapatskaya
Inst. of Microbiology & Virology Nat. Acad. Sci. of Ukraine;
Inst. of Molec. Biology and Genetics NAS of Ukraine; Inst. of Exp. Pathol., Oncol. & Radiobiol. NAS of Ukraine, Kyiv, Ukraine

Modified nucleosides have proved to be the useful substances for antiviral drugs design. The objective of the present investigation was the comparative studying of the activity of 6-azacytidine and aciclovir against Epstein-Barr virus (EBV) – lymphotropic and oncogenic virus from Herpes viridae family and expression of CD 95-mediated apoptosis in B-lymphoblastoid cells.
6-Azacytidine (2-b-D-ribofuranosyl-5-amino-1,2,4-triazin-3(2H)-on; 6-AC) is an original structural cytidine analogue. Aciclovir [9-(2-hydroxyethoxymethyl)guanine] is the acyclic analogue of deoxyguanine. It is known as effective inhibitor of herpes viruses, but there is no precise proof of its activity against EBV. As a model of EBV-infection in vitro we used the line of Raji cells, which was infected by EBV. An inhibition of reproduction of EBV in cell culture by 6-AC and aciclovir was determined by reduction of a number of genome-equivalents of EBV DNA on a cell, which were revealed by the quantitative PCR with the use of primes and reagents “AMPLY-Senc-100R” (Russia). CD 95-mediated apoptosis of Raji cells was studied with the help of Mab IPO-4 to CD 95 and staining of cells on Hoechst.
The concentrations of 6-AC which inhibited the quantity of alive cells on 50% (ID50) were equal to 125 mg/ml. The minimal inhibiting concentration (MIC) of 6-AC was equal to 0.5 µg/ml, because the amount of genome-equivalents of DNA EBV on a cell was reduced with 6.0 up to 3.1. Hence, the index of selectivity (IS) was equal to 250 for 6-AC. Aciclovir has shown the considerably smaller activity against EBV; in concentration of 125-500 µg/ml it reduced the quantity of DNA EBV up to 6.5-4.8 genome-equivalents on a cell (7.12 without the inhibitor). It was investigated the influence of 6-AC on CD95-mediated apoptosis in Raji cells infected by EBV as well as uninfected. It was shown that 6-AC in concentration 32 125 µg/ml strengthened the expression of CD 95-mediated apoptosis. The presented investigation was partially supported by INTAS Grant Program (INTAS Grant N 011-2382).