Anti-viral drug resistance

Anti-viral drug resistance

Journal of Clinical Virology 18 (2000) 251 – 269 www.elsevier.com/locate/jcv Anti-7iral drug resistance P-321 An oseltamivir treatment-selected influ...

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Journal of Clinical Virology 18 (2000) 251 – 269 www.elsevier.com/locate/jcv

Anti-7iral drug resistance P-321 An oseltamivir treatment-selected influenza A/N2 virus with a R292K mutation in the neuraminidase gene has reduced infectivity in vivo J. IVES1,*, J. Carr1, N.A. Roberts1, C.Y. Tai2, D.B. Mendel2, L. Kelly3, R. Lambkin3 and J. Oxford3 1 Roche Discovery, 40 Broadwater Road, Welwyn Garden City, Herts, AL7 3AY, UK 2 Gilead Sciences, Foster City, CA, USA 3 www.retroscreen.com *Corresponding author. Tel.: +44-1707-366000; fax: + 44-1707-338297. An influenza A/N2 virus carrying the R292K mutation in the neuraminidase gene has been isolated from a patient on an oseltamivir treatment study. Clinically derived R292K neuraminidase has a two-fold reduction in affinity for (synthetic) substrate, and a greater than 9000-fold reduction in sensitivity to inhibition by Ro 64-0802 (GS4071). The R292K neuraminidase does not impart advantage to the mutant virus in terms of replication in MDCK cells. The infectivity and replicative ability of R292K mutant virus was reduced by at least 100-fold in a mouse model of influenza infection. In the ferret model of influenza infection the infectivity of the mutant virus was reduced by at least 100-fold and its replicative ability was reduced by at least 10 000-fold. Pathogenicity of R292K neuraminidase virus was reduced in parallel with virus titres in ferrets. The compromised infectivity and pathogenicity demonstrated in vivo indicate that virus carrying R292K mutation in the neuraminidase gene is likely to have a reduced risk of transmission in man.

P-322 Monitoring of HIV-1 strains resistant to nucleoside reverse transcriptase inhibitors ´ 1, Milan Reinis1, Jana Vandasova´1 and Marie Stankova2 MARIE BRUCKOVA 1 National Institute of Public Health, Sroba´rova 48, Prague 10, 10042, Czech Republic 2 AIDS Clinical Center, Faculty Hospital Bulovka, Prague, Czech Republic Objecti6e: Monitoring of development of HIV-1 mutants resistant to nucleoside RT inhibitors in HIV-1 infected persons. Methods: In the period from 1998 – 2000, 244 plasma samples from 141 HIV-1 infected individuals were analysed. HIV-1 mutants resistant to treatment with ZDV, ddC, ddl and 3TC were detected by LiPA HIV-1 RT test (Murex/Innogenetics). Variations in codons 41, 69/70, 74/75, 184, 214/215 in the pol gene were identified in this test. 1386-6532/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 1 1 1 - 6

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Results: RT inhibitors resistance was detected in 162 (66,4%) isolates. The majority of strains demonstrated resistance to ZDV (153 samples, 62,7%), 81 strains in combination with resistance to other inhibitors (3TC, ddl, ddC). Resistance to 3TC only was detected in 9 samples (4%). LiPA gave uninterpretable results for 17% of the analysed codons. In general, a good correlation with viral load in studied persons was observed. Conclusion: A significant portion of HIV-1 strains studied was found as resistant to nucleoside RT inhibitors (ZDV, ddC, ddl and 3TC), especially to ZDV. LiPA assay is a fast and convenient method for screening resistance RT inhibitors. However, due to its limitations, it does not fully replace sequence analysis of the RT gene.

P-323 Investigation of mutations in the human cytomegalovirus UL97 phosphotransferase in relation to ganciclovir resistance Mehrdad Mousavi-Jazi1, Roberta Marenzi2, Silva Presi3, Annika Linde1, Paola Cinque2 and MARIA BRYTTING1 1 Department of Virology, Swedish Institute for Infectious Disease Control, and MTC, Karolinska Institute, Solna, Sweden 2 Department of Infectious Disease, San Raffaele Hospital, Milan, Italy 3 Department of Clinical Molecular Biology, San Raffaele Hospital, Milan, Italy Aim: Marker transfer was performed to determine new mutations (H520G, and deletion of 596) in the human cytomegalovirus UL97 phosphotransferase and their roles in inducing ganciclovir (GCV) resistance. Methods: Recombinant viruses were generated by homologous recombination of altered gene fragments into the CMV laboratory strain Towne. The recombinants were selected by growth in medium containing GCV, and the mutations in the recombinants were verified by sequencing. The sensitivities of the recombinants to antiviral drugs were analysed by an antiviral sensitivity assay (solid phase ELISA). Results: The recombinant viruses (H520G and deletion of codon 596) conferred reduced sensitivity to GCV. Alterations involving position 596 have previously been reported to induce GCV resistance. However, the specific deletion of this amino acid has not been fully studied. The alteration H520Q is well known to induce GCV resistance, but the alteration to glycine has not been reported. Conclusions: Two new alterations in UL97 (H520G and deletion at codon 596) were determined to induce GCV resistance.

P-324 Cytomegalovirus strains resistant to ganciclovir ´ 1, G. Moreno1, A. Corte´s1, M.T. Betbese´,1, J. Gavalda´2, A. Roman3 and I. Valle IGNACIO CALICO 1 Microbiology Service, Vall d’Hebron Hospital, P. Vall d’Hebron, 129, Barcelona 08035, Spain 2 Infectious Disease Service, Vall d’Hebron Hospital, Barcelona, Spain 3 Respiratory Service, Vall d’Hebron Hospital, Barcelona, Spain The aim of this study is to investigate the presence of ganciclovir resistant cytomegalovirus (CMV) strains in our medium, observe whether the resistances appear in patients previously treated with gancyclovir and determine its implications in the evolution of CMV infection.

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Patients and methods: One hundred twenty-three strains of CMV corresponding to the following 94 patients were studied: 17 breast feeding children of healthy parents who were negative controls (sensitive strains), 43 organ transplant recipients, 29 AIDS patients and 2 other immunodeficiences and 3 children with intrauterine infection. 17 patients were studied due to the insidious course despite treatment. The remaining were random. The technique used was that of growth inhibition of the strains seeded on different gradients of ganciclovir. The strains presenting an inhibitory doses 50% greater than 10 mM were considered as resistant. Results: Eighty-two strains presented an ID 10% lower than 5 mM, 24 from 5 to 10 mM, and in the 17 remaining strains corresponding to 12 patients, the ID 50% was greater than 10 mM. All patients had undergone previous treatment with gancyclovir except 2: one treated with acyclovir 15 days before and the other was an infant of an HIV mother who had received the drug. The evolution of these 12 patients was of death in 8. All were immunosupressed. One had a chronic evolution, and 3 with better immunity became cured. Conclusions: The previous use of ganciclovir, and in one case of acyclovir, appears to be implicated in the appearance of resistance. The evolution of the immunodepressed patients with infection by resistant strains was mortal except where their immunity was improved. P-325 Prevalence of baseline mutations in Irish HIV-infected antiretroviral-naive patients SUZIE COUGHLAN, A. Conroy, S. Dooley and W.W. Hall Virus Reference Laboratory, University College Dublin, Belfield, Dublin 4, Ireland Objecti6e: To determine the prevalence of resistance associated mutations in the reverse transcriptase and protease genes of Irish HIV-infected antiretroviral-naive patients. Methods: Genotypic data was collected from newly diagnosed patients with no previous exposure to antiretroviral therapy. Samples from an equal number of males and females from various exposure categories were chosen for the study. All had viral loads greater than 1000 copies/ml. HIV RNA was extracted from frozen plasma samples and a nucleotide fingerprint of the protease and reverse transcriptase genes was generated using TruGene HIV-1 sequencing assay from Visible Genetics Inc. Results: Sequence data was obtained from 50 patients. Although resistance related mutations were present in 90% of specimens tested at baseline, levels of primary mutations were much less notable. However, reverse transcriptase related mutations were present at a significant level of 10% of the test population. These mutations conferred resistance to some of the first antiretroviral drugs available, Zidovudine and Lamivudine. No primary protease mutations were observed despite levels of secondary mutations reaching as much as 60% at particular amino acid sites (L63P). Conclusion: Of the 50 newly diagnosed HIV-infected patients in this retrospective analysis approximately 10% had drug resistant virus. Therefore results of this study support the routine use of genotypic analysis (at least for the reverse transcriptase gene) in the Irish newly diagnosed population. P-326 Phenotypic and genotypic assay of influenza virus neuraminidase indicates a low incidence of viral drug resistance during treatment with oseltamivir EMMA COVINGTON3, D.B. Mendel1, P. Escarpe1, C.Y. Tai1, K. Soderbarg2 and N.A. Roberts3 1 Gilead Sciences, Foster City, CA, USA 2 PGL, Uppsala Science Park, Uppsala, Sweden

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Roche Discovery, Roche Products Ltd., Broadwater Road, Welwyn Garden City, AL7 3AY, UK

GS4071, the active metabolite of oseltamivir, is a potent and specific inhibitor of influenza A and B virus neuraminidases. The incidence of neuraminidase resistant to GS4071 in virus from oseltamivir treatment studies has been assessed using a sensitive and reproducible neuraminidase phenotypic assay and was found to be low. Only 4/418 patients, all of whom responded normally to treatment, were found to carry GS4071-resistant virus. All resistant neuraminidases were in A/H3N2 virus and the mutations were identified as R292K (3 cases) and E119V (1 case). Neuraminidase genotyping of pre- and post-expansion viruses (405 sequences from 115 patients) indicated that the phenotypic assays had detected most mutant neuraminidases. Haemagglutinin genotyping (n = 80) revealed no evidence of treatment-related mutations. Thus, the incidence of viral resistance during oseltamivir treatment is low and there is no evidence of clinical or virologic deterioration. Additionally, the mutant viruses are at least 100-fold less infectious than wild-type.

P-327 Line immuno probe assay and DNA sequencing for the detection of resistance to protease inhibitors FEDERICO GARCIA GARCIA, M. Alvarez, M. Martı´nez, J. Herna´ndez, F. Garcı´a Jr., M.C. Bernal, M.C. Maroto and G. Pie´drola Microbiology Department and Infectious Diseases Unit, University School of Medicine ‘H. San Cecilio’, Av. de Madrid 11, Granada E-18102, Spain Introduction: Mutation detection may help on the therapeutic management of HIV patients. We have evaluated the performance of INNO-LIPATM HIV PROTEASE (Innogenetics) to detect the most important mutations of the protease gene and compared it with DNA sequencing. Methods: 18 patients were included in the study, 2 were paediatric (one naive). The rest 16 patients showed the following characteristics: mean age: 36,3!10,5; mean CD4 count: 324!354; mean viral load (log10): 4,69!4,98; 68% IVDUs and 68% naive for treatment with PIs. All patients were studied by the Innogenetics test, consisting of an RT-PCR and specific nested PCR for the codons 30–84 and 90, followed by reverse hybridisation on LIPA strips. Additionally, 12 patients were studied with a semi-automated DNA sequencing method (Visible Genetics). Results: using the LIPA assay, mutations conferring genotypic resistance were found for 3/18 patients (V/182A, 3 patients; L90M, 1 patient). All three patients were not naive for PIs (HAART). DNA sequencing on two of these 3 patients detected V/182A (two patients) and L90M (one patient), as well as other mutations without clinical relevance; the sample from the third patient could not be amplified. Conclusion: INNO-LIPATM HIV KEY PROTEASE assay is easy to perform and suits the requirements of the clinical laboratory. A good correlation with DNA sequencing has been observed, although further comparative studies need to be performed.

P-328 Genetic heterogeneity of TT virus isolates in patients under interferon treatment M. Martı´nez, FEDERICO GARCIA GARCIA, M. Alvarez, P. Morata, J. Herna´ndez, F. Garcı´a Jr., M.C. Bernal, M.C. Maroto and G. Pie´drola Microbiology Department and Infectious Diseases Unit, University School of Medicine ‘H. San Cecilio’, Av. de Madrid 11, Granada E-18102, Spain

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The aim of our study was to evaluate genetic variability of TT virus, a novel DNA virus related with hepatitis, on isolates from patients under interferon treatment. Patients: 7 patients positive for TTV DNA were studied. Patients were coinfected with hepatitis C virus (HCV) and were treated with r-a Interferon (IFN) for their chronic type c hepatitis with 3 MU three days weekly for one year. Samplings were at the onset of therapy (basal), 4 and 8 months later, at the end of treatment (12 months) and a year after (24 months). TTV DNA was amplified from the nucleic acids extracted from 100 ul of serum with semi-nested primers from ORF1 of TTV genome. A 270-bp fragment was visualised and analysed using Single Stranded Conformation Polymorphism (SSCP) followed by PAGE and silver staining. Results: Two patients showed the same viral quasispecies at the basal and 24 month sample. Four patients showed a different SSCP pattern at basal and revaluation samples (three patients cleared viral quasispecies at revaluation and one showed an additional quasispecies); for one patient TTV DNA was cleared from the serum at revaluation. Conclusion: Overall, TTV isolates show genetic differences at revaluation when compared to basal samples; this differences in the quasispecies pattern may be due to point mutations selected by IFN or to re-infection by new viruses

P-329 Characterisation of the herpesvirus saimiri ORF 73 gene product KERSTEN HALL1, Mathew S. Giles1, Thomas F. Schulz2 and Adrian Whitehouse1 1 Molecular Medicine Unit, University of Leeds, St. James’s University Hospital, Leeds LS9 7TF, UK 2 Department of Medical Microbiology, University of Liverpool, Liverpool, UK The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localises with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, we have recently shown that ORF73 is expressed in a stably transduced human carcinoma cell line where the HVS genome persists as a non-integrated circular episome. Here we describe characterisation of the HVS ORF73 protein and mapping of its functional domains. Our results suggest that the ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the aminoterminus contains two functional nuclear localisation signals and the carboxy-terminus is required for the distinctive speckled nuclear distribution pattern observed with both the HVS and KSHV ORF73 proteins. Furthermore, the yeast 2-hybrid system was employed to identify interacting cellular proteins using ORF 73 as bait. Results identified an interaction with RING3, a homolog of the Drosphila female sterile homeotic gene, which has previously been shown to interact with KSHV ORF 73 (Platt et al., J. Virol. 73, 9789–9795). GST pulldown experiments have been performed to map the domains responsible for this interaction.

P-330 An oseltamivir treatment-selected influenza A/Wuhan/359/95 virus with an E119V mutation in the neuraminidase gene has reduced infectivity in vivo JANE IVES1, J. Carr1, N.A. Roberts1, C.Y. Tai 2, D.B. Mendel2, L. Kelly3, R. Lambkin3 and J. Oxford3

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1

Roche Discovery, 40 Broadwater Road, Welwyn Garden City, Herts, AL7 3AY, UK Gilead Sciences, Foster City, CA, USA 3 www.retroscreen.com 2

Clinically derived influenza A/Wuhan/359/95 (H3N2) E119V neuraminidase had a two-fold increase in affinity for synthetic substrate, and a 20-fold reduction in sensitivity to inhibition by Ro 64-0802 (GS4071). E119V mutant virus had no advantage over wild-type virus in terms of growth in vitro. In vivo, the E119V mutation compromised virus infectivity in both mice and ferrets, by at least 10–100-fold, and at least 100 – 1000-fold, respectively. Pathogenicity in ferrets was reduced in parallel with the reduction in infectivity exhibited by mutant virus. The long lag phase preceding E119V mutant viral replication observed in ferrets on day 6 (at lower challenge doses) was not observed in the only patient in an oseltamivir treatment study carrying this mutant virus. The E119V mutation was not stable over 6 days repeated passage in vivo, as indicated by identification of revertant wild-type virus from E119V virus-infected ferrets at day 6. These data indicate that virus carrying E119V neuraminidase is likely to have a reduced risk of transmission in man. P-331 Laboratory monitoring of CMV infection in allogeneic peripheral blood stem cell transplantation ZIPORA KRA-OZ, Judith Satinger and Valentina Vaksman Virology Laboratory, Rambam Medical Center, Bat-Galim, Haifa 31096, Israel Background: Pre-emptive antiviral therapy is an important strategy for the prevention of CMV disease in BMT. This approach is highly dependent on early detection of CMV infection by sensitive laboratory methods. Objecti6e: To assess the efficacy of routine weekly monitoring of allogeneic peripheral blood stem cell recipients (all-PBSCT) by qualitative nPCR and pp65 antigenemia to detect early CMV replication. Design: The patients were examined once a week during the first 3-4 months and in lower frequency later on. Leukocytes from blood samples were tested by qualitative nested PCR, by antigenemia assay and by shell vial culture (SVC). Urine samples were tested by PCR and by SVC. Other samples like BAL, stoo and biopsies were examined according to clinical symptoms. Results: 46 allo-PBSCT were monitored between 1996–1999. The surveillance period ranged from 21 daysto 22 months. 31 patients experienced episodes of CMV infection, at least once, and 7 of them had episodes of high antigenemia (\20 positives/6 × 105 cells). The mean time for the first CMV infection was 40 days (range 14–100 days). The results of the antigenemia assay and the nPCR, but not viral shedding in urine, were in good correlation to clinical symptoms. None of the patients had died from CMV disease albeit episodes of symptomatic CMV infection. Conclusions: Routine monitoring of allo-PBSCT by antigenemia and qualitative nPCR is effective in preventing CMV disease by pre-emptive therapy. It is still needed to recognize patients likely to develop CMV disease, quantitative PCR may provide such a tool. P-332 An in vivo and in vitro evaluation of antiviral and cytostatic activity of N-benzoylphenylisoserinates of sesquiterpenes-analogs of taxol EWA KRAWCZYK1, Maciej Przybylski1, Miroslaw Kobus1, Piotr Kopczacki2, Miroslaw Luczak1 and Wlodzimierz Daniewski2

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Department of Medical Microbiology, Medical University in Warsaw, 5 Chalubinski Street, Warsaw 02-004, Poland 2 Institute of Organic Chemistry, Polish Academy of Sciences, Poland The study comprised 19 newly synthesised sesquiterpenoid analogs of taxol i.e. N-enzoylphenylisoserinates of sesquiterpenoid alcohols. The synthesis of the compounds was performed at the Institute of Organic Chemistry, Polish Academy of Sciences. Sesquiterpenes were selected on the basis of their biological activity, detected in our previous experiments. NMRI mice were infected intramuscularly with a Moloney murine sarcoma virus (MoMSV). Tested compounds were administered to the mice intravenously on the day of virus inoculation and thereafter for three consecutive days. The doses of the compounds-administered to the animals-were selected on the basis of results of toxicity tests, performed on two-week-old mice. In MoMSV-infected mice dynamics of tumour progression and regression was assessed, as well as a mean time interval of tumour disappearance. Cytostatic activity of the compounds was measured using cultures of A-549, PA-1, SKOV 3 and U-373 cell lines of neoplasmic origin. The dynamics of cell culture growth for particular concentrations of the tested compounds was evaluated. A formazan method was used and the results of colour reactions were read in a photometer. Among the compounds tested: 13-O-[(2%R,3%S)-N-benzoyl-3-phenylisoserinate]-5-oxo-2a,9a-H-marasm7(8)-en-13-ol, 8-O-[(2%R,3%S)-N-benzoyl-3-phenylisoserinate]-5-oxo-2a,9a-H-lactaran-6(7)-en-3a, 8b-diol and 8-O-[(2%R,3%S)-N-benzoyl-3-phenylisoserinate]-5-oxo-2a,9a-H-isolactaran-3b,8b-diol significantly inhibited the development of tumours and shortened the time of their total regression in the course of MoMSV infection. These substances also showed a potent inhibitory activity on the proliferation of differentiated cells, depending on the concentration of the compounds and type of cell line used in the culture.

P-333 A cell-based assay with virus-inducible secreted protein for rapid diagnosis and antiviral susceptibility testing of herpes simplex virus SZU-HAO KUNG1, Chun Wang1 and Wu-Tse Liu1,2 1 Faculty of Medical Technology/Institute of Biotechnology in Medicine, National Yang-Ming University, Li-nong St. Sec. , c 155, Taipei 112, Taiwan, ROC 2 Division of Clinical Virology, Veterans General Hospital-Taipei, Shih-Pai, 112, Taipei, Taiwan, ROC A genetically modified cell line (VERO-ICP10-SEAP) was established for detection of herpes simplex virus (HSV) by a sensitive, rapid and quantitative method. The cell line was constructed by stable transfection of Vero cell with a plasmid encoding the secreted alkaline phosphatase (SEAP) driven by the promoter of the HSV-2 ICP10 gene. Following infection with either HSV-1 or HSV-2, the level of the SEAP activity in the supernatant was measured with a highly sensitive chemoluminescence-based assay. The system permitted detection of the viral titer as low as a single plaque-forming unit (PFU), with a linear range up to the equivalent of 2.5×10(4) PFU inoculum 24 h post-infection. Moreover we analyzed a total of 212 clinical specimens with VERO-ICP10-SEAP line, and detected twenty-one HSV-positive specimens in 24 h post-infection; ten of the HSV-positive samples resulted in cytopathic effect (CPE) three or more days post-infection. PCR analysis with HSV-specific primer confirmed the results with the stable line. The stable line was also used to develop a system for antiviral susceptibility testing. The system mimics the conventional plaque reduction assay (PRA) with the levels of SEAP activity reflects the numbers of

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plaque. Evaluation with two acyclovir-sensitive and two acyclovir-resistant HSV isolates demonstrated that the 50% inhibitory concentrations (IC50) as determined by this method correlated with those from the PRA. Taken together, this novel SEAP reporter system is a sensitive means for rapid diagnosis and drug susceptibility testing for HSV, with potential to the development of an automated assay.

P-334 Antiviral properties of carbonyl conjugated pentaene, roseophungin SVETLANA LEVANDOVSKAYA, S.S. Khudyakova, V.P. Tolmacheva, O.S. Protsyshina, E.T. Nikitina, A.M. Daurenbekova, A.P. Bogoyavlenskiy and V.E. Berezin Institute of Microbiology and Virology, Bogenbay batyr Str., 103, Almaty 480100, Kazakhstan Two different modes of entry have been described for enveloped animal viruses: direct fusion of the viral lipid envelope with the plasma membrane and internalisation of virus particles in endosomes, followed by fusion of the viral and the endosomal membranes. Therefore using compounds destroying membranes for inhibiting of viruses are very interesting. This paper reports on the examination of roseophungin antiviral activity, a polyenic macrolide antibiotic. It was shown that the virus inhibiting activity of roseophungin against ortho- and paramyxoviruses was sufficiently high. In a range of roseophungin concentrations between 3 and 12 micrograms/embryo virus production was drastically reduced. The activity of roseophungin against influenza A virus did not differ from that of remantadine, the most active inhibitor of influenza virus reproduction. A protective effect of roseophungin was shown on the chickens with experimental influenza. Our findings suggest that roseophungin may have potential to develop as antiviral drugs in the future.

P-335 Screening for the susceptibility to Zanamivir of clinical influenza isolates BRUNO LINA, Miche`le Aymard, Olivier Ferraris, Lionel Gerentes, Jeanine Jolly and Nicole Kessler Centre National de Reference pour la Grippe (France-Sud), Laboratoire de Virologie, Domaine Rockefeller, 69373 Lyon cedex 08, France When antineuraminidase agents became available for the treatment and prophylaxis of influenza infections, reproducible and sensitive assays were established for the determination of the susceptibility of influenza viruses to these agents. The neuraminidase inhibition testing (NI) using fetuin as substrate is an easy, cheap and reproducible assay. Using NI assays on the different types, subtypes and variants of influenza susceptible prototype viruses, the concentration of Zanamivir reducing by 50% the Neuraminidase activity (IC50 NI) varied from 0.3 to 3 ng/ml. When influenza viruses are isolated in MDCK cells, isolates may have poor or no neuraminidase activity. As a result, these isolates can not be checked for antineuraminidase susceptibility using a NI assay. As an alternative method, a microneutralization assay (Nt) was developed and used to determine the concentration of Zanamivir reducing by 50% the multiplication of viruses isolated from MDCK cells (IC50 Nt). In this Nt assay, the growth of the influenza isolates was determined by measuring by ELISA the amount of NP antigen released after a 48 h-incubation period. With this Nt assay, the IC50 Nt values ranged from 10 ng/ml to 10 mg/ml of Zanamivir, depending to the inoculum size, strain and subtype.

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Some of the recent (1999 and 2000) and previous (1993 to 1995) viruses collected from patients presenting with acute respiratory infections, and isolated in MDCK cells were processed by both assay, showing discrepancies between values obtained with NI and Nt tests. Moreover, some isolates collected from patients that did not receive antiviral therapy were not neutralised with up to 100 mg/ml Zanamivir when using the Nt assay. To assess the reliability of this Nt assay, we have to further analyse the strains with reduced susceptibility by determining the sequences of both Neuraminidase and Haemagglutinin genes of the putative resistant isolates, and check for mutations that might be related to resistance.

P-336 Effect of marasm-triol on experimental infections with herpes simplex virus type 1 (HSV-1) MIROSLAW L * UCZAK1, Miroslaw Kobus1, Katarzyna Wajs1 and Wlodzimierz Daniewski2 1 Department of Medical Microbiology, Medical University in Warsaw, 5 Chalubinski Street, Warsaw 02-004, Poland 2 Institute of Organic Chemistry, Polish Academy of Sciences, Poland A Vero cell line was used for in vitro studies. In the experiments the cell cultures were established in plastic plates (Nunc) in the amount of 2,5× 103 cells/well. The cell cultures were infected with HSV-1 (McIntyre strain) and its titre was expressed in TCID50/ml at particular stages of the experiments. The tested compound was added in different concentrations to the cell culture during or after viral infection. The activity of viral DNA polymerase was assessed in the infected cell cultures, exposed to the particular compound, using a DNA polymerase assay (Behringer). For in vivo experiments NMRI mice were infected intranasally and the tested compound was administered intraperitoneally every day. In in vitro studies, marasm-triol (5,10a, 13-trihydroxy-2a,9a-H-marasm-7(8)-en) statistically significantly inhibited proliferation of the virus, when administered after viral inoculation. It did not show, however, any significant effect if administered only during viral adsorption. The compound showed a significant inhibition of the activity of viral DNA polymerase. In in vivo experiments, marasm-triol showed a significant effect on the course of HSV-1 infection in mice, decreasing their mortality. The strongest inhibitory effect—about 60% — was recorded when administration of the substance was started on the day of infection.

P-337 An in vivo and in vitro evaluation of antiviral and cytostatic activity of N-benzoylphenylisoserinates of sesquiterpenes-analogs of taxol MIROSLAW L * UCZAK1, M. Przybylski1, M. Kobus1, P. Kopczacki2, E. Krawczyk1 and W. Daniewski2 1 Department of Medical Microbiology, Medical University in Warsaw, 5 Chalubinski Street, Warsaw 02-004, Poland 2 Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland The study comprised 19 newly synthesised sesquiterpenoid analogs of taxol i.c. Nbenzoylphenylisoserinates of sesquiterpenoid alcohols. The synthesis of the compounds was performed at the Institute of Organic Chemistry, Polish Academy of Sciences. Sesquiterpenes were selected on the basis of their biological activity, detected in our previous experiments. NMRI mice were infected intramuscularly with a Moloney murine sarcoma virus (MoMSV). Tested compounds were administered to the mice intra-

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venously on the day of virus inoculation and thereafter for three consecutive days. The doses of the compounds—administered to the animals — were selected on the basis of results of toxicity tests, performed on two-week-old mice. In MoMSV-infected mice dynamics of tumour progression and regression was assessed, as well as a mean time interval of tumour disappearance. Cytostatic activity of the compounds was measured using cultures of A-549, PA-1, SKOV 3 and U-373 cell lines of neoplasmic origin. The dynamics of cell culture growth for particular concentrations of the tested compounds was evaluated. A formazan method was used and the results of colour reactions were read in a photometer. Among the compounds tested: 13-0-[(2%R,3%S)-N-benzoyl-3-phenylisoserinate]-5-oxo-2a,9a-Hmarasm-7(8)en-13-ol,8-0-[(2%R,3%S)-N-benzoyl-3-phenylisoserinate]-5-oxo-2a,9a-H-lactaran-6(7)-en-3a,8p-diol and 8-0[(2%R,3%S)-N-benzoyl-3-phenylisoserinatel-5-oxo-2a,9a-Hisolactaran-3p,8p-diol significantly inhibited the development of tumours and shortened the time of their total regression in the course of MoMSV infection. These substances also showed a potent inhibitory activity on the proliferation of differentiated cells, depending on the concentration of the compounds and type of cell line used in the culture.

P-338 Codon changes and phenotypic resistance of HIV-1 isolates to Abacavir HORST-GUENTER MAXEINER, N. Wiese, H. Maxeiner, F. Hein, H. Petersen, U. Schmitz and Th. Fenner City Laboratories Dres. Fenner,c/o Maxeiner, Bergstraße 14, D-20095 Hamburg, Germany Objecti6e: Codon changes contributing to phenotypic resistance to Abacavir. Methods: Sequencing codon 40 to 228 of rt gene and recombinant virus assay for phenotyping in 26 patients. Results: A week correlation between the total number of codon changes and the degree of resistance to Abacavir was observed. (R2 =0,21) 6 out of 14 sensitive (B 4 fold decrease of sensitivity) strains harboured a M41 L mutation whereas 11 out of 12 resistant ( \ 4 fold decrease of sensitivity) strains displayed the codon 41 change. Codon 67 was changed to 67D in 3 out of 14 sensitive stains and in 10 out of 12 resistant strains. A K166R change was observed in 33% of resistant strains. The mutation M184V, which is linked to 3TC resistance, was present in 29% of the sensitive strains and in 67% of the resistant strains. Strains harbouring a M184 V mutation loss 5,4 fold sensitivity (s: 5, range 1–18). Without M184V the loss was 4,6 fold (s: 4, range 1 – 11). Three out of 13 highly resistant 3TC strains were highly resistant (more than 10 fold) against Abacavir, 3 out of 5 highly resistant Abacavir strains were resistant against 3TC. Conclusions: M184V is not a major resistance mutation. Codon 41 and 67 changes were associated with resistance. Codon 166R mutation is a minor resistance mutation.

P-339 Changes in codon 184 and 118 and phenotypic resistance of HIV-1 isolates to 3TC and AZT HORST-GUENTER MAXEINER, N. Wiese, F. Hein, H. Petersen, U. Schmitz and Th. Fenner

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City Laboratories Dres. Fenner,c/o Maxeiner, Bergstraße 14, D-20095 Hamburg, Germany Objecti6e: Reversal of AZT resistance by M 184 mutation and 3TC resistance. Methods: RTgene sequencing (codon 40 – 228); phenotypic resistance by recombinant virus assay of 29 patients. Results: Increase in resistance to 3TC was 16,5 fold (s: 17,8; range 0,8 to 54 fold) in the presence of M184V (n= 16). Without M184V (n = 13) increase was 8,2 fold (s: 6,9; 1 to 22 fold). In 3 out of 6 patients displaying a \10 fold decrease in sensitivity and lacking a M184V mutation a V1181 change was observed, whereas none of 8 sensitive (B 10-fold) harboured a V1181 mutation. Strains (n = 12) expressing M184V and at least one AZT associated mutation the increase in resistance to AZT was 38 fold (s: 34; 1 – 109); without codon 184 change (n= 9) the increase was 30 fold (s: 38; 1 – 117). Conclusions: (1) M184V mutation doubles the degree of resistance to 3TC. (2) No differences in the average loss of sensitivity to AZT was found in strains with/without the M184V mutation. However, comparing paired strains with identical AZT-resistance mutations, we observed in 2 out of 5 AZT resistant strains an up to 20 fold increase in sensitivity in the presence of a M184V mutation. This indicates that some patients may benefit from the 3TC linked M184V mutation for AZT therapy. A V1181 mutation contribute to 3TC resistance as a minor resistance mutation

P-340 Scrapie spreads from the gastrointestinal tract to the CNS along autonomic and sensory relays of the peripheral nervous system PATRICIA McBRIDE1, Maura Donaldson1 and Michael Beekes2 1 Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF, UK 2 Robert-Koch Institute, Berlin, Germany

Scrapie, a naturally-occurring disease of sheep, is the most studied member of a group of transmissible, neurodegenerative diseases known collectively as transmissible spongiform encephalopathies (TSE) or prion diseases. The group includes BSE of cattle and ‘variant’ CJD of man. Although the ultimate target of TSE infection is the CNS, the peripheral nervous system (PNS) appears to play a fundamental role in pathogenesis. In several peripherally-infected rodent models of scrapie, spread of infection to the brain and spinal cord shows a pattern consistent with transport along nerves supplying the viscera. We used immunocytochemistry to identify the location and temporal sequence of pathological accumulations of a host protein PrP, in the CNS and PNS of hamsters orally challenged with 263K scrapie. Specific PNS components of parasympathetic/sensory (vagal) or sympathetic/sensory (splanchnic) nervous systems were examined along with brain and spinal cord with which they form relay circuits. Enteric ganglia were included as potential ‘first stop’ sites of infection. Deposition was quantified to compare relative amounts of protein in different tissues. Results showed that pathological PrP accumulated in sequence, in target sites that accurately reflected known autonomic and sensory relays. Deposition was seen in enteric, sympathetic and parasympathetic ganglia prior to sensory ganglia. These findings suggest that, in this scrapie model, after uptake from the gastrointestinal tract, the infectious agent primarily uses synaptically-linked autonomic ganglia and efferent fibres of the splanchnic and vagus nerves to reach CNS target sites.

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P-341 Characterization of drug resistance associated mutations in the human cytomegalovirus DNA polymerase MEHRDAD MOUSAVI-JAZI, A. Linde, B. Wahren and M. Brytting Swedish Institute for Infectious Disease Control, and MTC, Karolinska Institute, SE-171 82 Solna, Sweden Aim: To determine the role of mutations in human cytomegalovirus (HCMV) DNA polymerase (UL54) inducing drug resistance. Methods: By in Vitro mutagenesis recombinant viruses were generated. A segment of the UL54 gene from the clinical isolate was transferred into laboratory strain Towne by homologus recombination. Recombinant viruses were selected by selective growth in antiviral pressure. The presence of mutations in the recombinants were verified by sequencing. The sensitivities of the recombinants to antiviral drugs were analysed by a solid phase ELISA. Results: The recombinant viruses containing V7811 and D588N in UL54 showed 5- and 8-fold reduced sensitivity to foscarnet, and 2-and 8-fold reduced sensitivity to ganciclovir, respectively. We are now investigating whether the alterations E756K, and V787L in HCMV DNA polymerase are correlated to foscarnet resistance. Further, the growth phenotype of the recombinant containing the alteration V7811 in the DNA polymerase gene was studied. This mutation did not change the growth phenotype of Towne. Conclusions: Identification of viral genome nucleotide variation of importance for drug resistance and rate of replication will facilitate the analysis of antiviral drug treatment.

P-342 Contrasting patterns of response to lamivudine monotherapy R.A. de Man1, L.M.M. Wolters1, A.B. van Nunen1, A.D.M.E. Osterhaus2 and HUBERT G.M. NIESTERS2 1 Department of Hepatogastroenterology, Erasmus University Hospital, Rotterdam, The Netherlands 2 Department of Virology, Erasmus University Hospital Rotterdam, Dr Molewaterplein 40, Rotterdam 3015 GD, The Netherlands We describe a cohort of patients treated with lamivudine, a reverse transcriptase inhibitor with a strong virus suppressive effect on the hepatitis B virus. Eighty-nine patients were included in the intention to treat analysis. Evaluation with the Kaplan-Meier method was based on response to therapy of HBV DNA levels as well as normalisation of transaminases. Subgroup evaluation based on the per protocol data was performed on two groups within this cohort based on the HBV DNA level at the end of therapy. During a 52 weeks treatment period, the chance of being HBV DNA negative at one point in time as measured by the Digene Hybrid Capture 11 assay (HCS) or the quantitative PCR assay (Q PCR), or the chance of normalisation of transaminases at one point in time is 78%, 57% and 66% respectively. Nineteen out of 73 patients who had continuing active viral replication after at least 24 weeks of lamivudine therapy were evaluated for the emergence of mutations which are resistant to lamivudine. In 3 out of 19 patients a mutation in the highly conservative YMDD region of the polymerase gene was detected. Baseline viral load in this group was significantly higher compared to the other 54 patients who were treated for 24 weeks or longer. Thirty-one out of 73 patients (46%) became negative by Q PCR. HBV DNA level at start of treatment was significantly lower compared to the 42 patients who stayed HBV DNA positive. Eleven consecutive patients within this group who became negative by qualitative PCR (limit of detection 400 geq/ml) were evaluated to obtain characteristics for lamivudine withdrawal. Ten out of eleven patients

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became HBeAg negative with anti-HBe in 6. HBcAg in the liver biopsy was negative in ten out of eleven patients, nine out of ten obtained biopsies were positive for HBV DNA, indicating low-level viral replication. In two patients in whom lamivudine was withdrawn, rebound of virus occurred. In conclusion, the response to lamivudine using sensitive HBV DNA assays is more variable as previously described. Criteria to stop lamivudine therapy need further development.

P-343 Algal antiviral activity (review) PAVEL PROSSELKOV and Shoshana Arad Institute for Applied Biosciences, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel Since the pioneer work of Pratt and Fong in 1940 [1] first demonstrated the antibacterial activity of an aquatic microalgae a huge amount of publications appeared proving that algal antiviral activity does really exist. First observations did not generate much interest because the antiviral action of the extracts tested was considered to be only non-specific. Current investigations show that algal compounds have indeed highly specific in vitro or in vivo activity against a wide range of viruses including herpes viruses, paramyxoviruses, retroviruses, rhabdoviruses, togaviruses, and many others. Among the active antiviral compounds isolated from algae may be mentioned the following groups of them: alkaloids, peptides (cyanovirin-N), indolocarbazoles, peyssonols, polyphenols, steroids, sulfated polysaccharides (calcium spirulan, carrageenans, fucoidan, galactan sulfate, mannan sulfate, etc.), ulfoglycolipids, th iazolo-iso indol in ones (welwistatin). Some of them exhibit a unique inhibitory activity against different strains, which suggests differences in the target molecules with which these compounds interact. As a promising source of antiviral agents which may act on different stages in virus replication cycle the isolated compounds act at concentrations as low as 0.1 to 0.01 mg/ml without cytotoxicity at concentrations up to 2.5-10 mg/ml, in evarage. There are no any direct correlations between the phylogenetic position of a specific group of algae and their exhibited antiviral activity. The same activity may be presented by different groups of algae with distinct mechanisms of antiviral action. Algal antiviral compounds are not applied immediately in the treatment of viruses-causes disorders. Most of the pharmacopeia of the active compounds in algae with antiviral activity is probably still left unknown and its discovery is only a challenge for the future. [1] Pratt, R., and Fong, J. (1940) Studies on Chlorella 6ulgaris. Further evidence that chlorella cells form a growth inhibiting substance. Am. J. Bot. 27: 431–436.

P-344 Detection of mutations in the HCMV UL97 gene associated with ganciclovir resistance J. PSOHLAVEC1, M. Forstl1,2, L. Plı´sˇkova´1,2 and J. Hora´ek1,2 1 Institute of Clinical Microbiology, Faculty of Medicine of Charles University, University Hospital, Sokolska’ul, 500 05 Hradec Kralove, Czech Republic Institute of Clinical Biochemistry, Department of Detection of Extrahuman Genome, Faculty of Medicine of Charles University, University Hospital, Hradec Kralove, Czech Republic.

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Long-term treatment of HCMV-infected immunocompromised patients, including kidney transplant recipients, with ganciclovir may result in failure of therapy. Resistance to ganciclovir is related to mutations in the UL97 region of the viral genome and/or mutations in the viral DNA polymerase (UL54). UL97 gene encoding a phosphontransferase which is essential for monophosohorylation of ganciclovir. We analyzed UL97 gene of clinical HCMV strains isolated from renal transplant recipients. These clinical isolated obtained from 30 blood samples of thirty renal transplant recipients were amplified by polymerase chain reaction and analyzed by restriction enzyme analysis. The specific point mutations conferring with ganciclovir resistance, including mutations in codons 460, 520, 591, 592, 594, 595, 599, 603 and 607 were investigated. We did not find any sensitive isolates carried any of these mutations. RFLP detection of point mutations in HCMV UL97 gene conferring with ganciclovir resistance may be a great contribution for rapid detection of ganciclovir resistance.

P-345 Prediction of the emergence of drug-resistant HBV strains during lamivudine therapy by monitoring the viral load ELISABETH PUCHHAMMER1, C.W. Mandl1, J. Kletzmayr2, H. Holzmann1, A. Hofmann1, S.W. Aberle1, F.X. Heinz1 and H. Hofmann1 1 Institute of Virology, University of Vienna, Kinderspitalgasse 15, Vienna A-1095, Austria 2 Division of Nephrology and Dialysis, Department of Medicine, University of Vienna, Vienna, Austria The development of drug-resistant hepatitis-B virus (HBV) strains in the course of lamivudine treatment has been repeatedly described. To investigate whether the selection of such resistant HBV strains can be predicted in an early phase of therapy the HBV-DNA load in serum has been monitored in 11 renal transplant patients under lamivudine treatment at 3-month intervals using a newly developed quantitative HBV PCR assay. Lamivudine resistance was determined by sequence analysis. Five of the patients developed resistance to lamivudine in the 12–15 month follow-up period. In all of them a viral load of 1000 HBV-DNA copies or more was still detectable after 3 months of therapy. This was statistically significantly different from those patients in whom no lamivudine resistant HBV strains did emerge within the observation period and who all had no HBV DNA detectable after 3 month of treatment (P= 0.0022). Thus our data indicate that viral load testing using a sensitive PCR assay allows the early prediction of the emergence of lamivudine resistant HBV-strains.

P-346 Immunisation of women at risk of rubella — opportunities missed PHILIP RICE, K.R. Devi and M. Viagappan Department of Medical Microbiology and Virology, St. George’s Hospital, Blackshaw Road, London, SW17 0QT, UK Purpose of the study: In developed countries rubella vaccination programmes have resulted in the almost complete disappearance of congenital rubella syndrome (CRS). To prevent its re-emergence, every occasion to opportunistically administer rubella vaccine in susceptible women of child-bearing age, particularly from developing countries, must be used. The most conspicuous occasions arise when women contact obstetric or gynaecological (O+ G) services. We therefore assessed the effectiveness of the UK

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Department of Health’s recommendation for opportunistic rubella immunisation in women who received antenatal care at St. George’s Hospital, London in 1998. Methods: All rubella susceptible women who had booking bloods in 1998 were identified and details were collected on country of birth, rubella vaccination history and contact with UK O+G services. Results: Of 3500 such women, 111 (3.2%) were susceptible to rubella; 79 (71.1%) were from developing countries. The highest susceptibility rate was among Sri Lankans (23/142[16.2%]), compared with 16/195 (8.2%) from the Indian sub-continent, 22/276 (7.9%) from Africa and 20/2220 (0.9%) from the UK. Of note, Sri Lankan women had the lowest rubella vaccination rate, 2/23 (8.6%). A total of 60 opportunities for rubella immunisation had been missed in 35/79 (44.3%) women from developing countries who had received O+ G care in the UK before 1998. The figures for the developed world were no better: 41 opportunities among 18/32 (56.3%) women being missed. Conclusion: These data re-emphasise the need to continue educating women’s healthcare providers of the need to ensure that every chance to immunise women at risk of rubella must be used.

P-347 Impact of dual HIV-1/HIV-2 infections on disease management and routine diagnostics MARTIN SCHUTTEN1, M.E. van der Ende1 and H.G.M. Niesters2 1 Department of Virology, University Hospital Rotterdam, Dr Molewaterplein 40, Rotterdam 3015 GD, The Netherlands 2 A.D.M.E. Osterhaus University Hospital Rotterdam, Department of Virology, Rotterdam, The Netherlands In Europe, approximately 500 asymptomatic and symptomatic HIV-2 seropositive individuals have been reported in France, Portugal, Spain, United Kingdom, The Netherlands and Germany. Most individuals have direct or indirect links with West Africa. A plasma HIV-2 RNA load assay is however only available in a limited number of virological diagnostic laboratories. Routine diagnostics of HIV suspected patients include HIV antibody EIA, HIV-1/2 western blot and subsequently a quantitative plasma HIV-1 RNA assay. In dually HIV-1 and HIV-2 seropositive individuals with a measurable plasma HIV-1 RNA load, who are offered therapy, the possibility of dual infection is in general disregarded. Using a newly developed quantitative plasma HIV-2 RNA assay we have recently identified three patient with a plasma HIV-1 and HIV-2 RNA load, in our cohort of twelve HIV-2 seropositive patients receiving triple therapy. In these patients the plasma HIV-1 RNA load decreased after start of therapy and remained undetectable since. In all three patients however, an initial decrease of the plasma HIV-2 RNA load was followed within several months by a rebound to levels higher or equal to those observed before start of therapy. These data indicate that in dually HIV-1 and HIV-2 seropositive individuals who are offered therapy, the possibility of dual infection should be taken in account. Especially since non nucleoside reverse transcriptase inhibitors and several protease inhibitors have been shown to be ineffective against HIV-2.

P-348 A randomised, placebo-controlled, single-blind pilot study to evaluate the efficacy of tea tree oil gel (6%) in the treatment of herpes labialis DAVID SMITH2, C.F. Carson1, L. Ashton1, L. Dry1 and T.V. Riley2

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1

University of Western Australia, Nedlands, Australia Division of Microbiology, Western Australian Centre for Pathology and Medical Research, Locked Bag 2009, Nedlands 6009, Australia 2

Recurrent herpes labialis (HL), or cold sores, affects up to 20% of the general population, but treatment options are limited. There is a need for cheap, effective alternatives. Anecdotal and in vitro evidence suggests that tea tree oil (TTO), the essential oil of the Australian native plant Melaleuca alternifolia, may be useful. Twenty volunteers with HL lesions were randomised to receive TTO gel (6%) or placebo gel applied up to five times daily. They were seen daily (except Sundays) for clinical assessment and swabbing for tissue culture and DNA detection by PCR. This continued until re-epithelialisation had occurred and two consecutive PCR results were negative. One of the TTO group who was subsequently negative for herpes simplex virus and did not develop a cold sore was withdrawn due to a possible adverse reaction. The treatment (n= 9) and placebo (n= 10) groups did not differ significantly in terms of mean age, gender or days to presentation. Outcomes for the TTO and placebo groups measured were: days to crust 3.79 0.7 versus 4.69 3.3, days to re-epithelialisation 9.993.6 versus 12.0 92.6, days PCR positive 6.59 2.0 versus 7.99 4.9. All of these outcomes favoured the TTO group, but did not reach statistical significance using the student’s t-test due to the small sample size. Analysis of viral titres is underway to determine the effect on viable virus. A larger study is warranted to verify these results.

P-349 Feasibility of TaqMan real time quantitative PCR for antiviral susceptibility testing of HSV strains DAVID SMITH, Merjo Bovenhorst, Charles A.B. Boucher, Anton M. van Loon and Rob Schuurman Division of Microbiology, Western Australian Centre for Pathology and Medical Research, Locked Bag 2009, Nedlands 6009, Australia The widespread and prolonged use of acyclovir has promoted the emergence of acyclovir-resistant HSV strains. Despite this fact the clinical labs do not routinely perform antiviral susceptibility testing. This is mainly due to the fact that the currently used golden standard method, plaque reduction assay (PRA), is labor intensive, time-consuming and gives subjective endpoint; the results therefore are of little use for the clinicians. We describe the application of a fluorogenic probe-based PCR assay (TaqMan; Perkin Elmer Corp./ Applied Biosystems) for the evaluation of the sensitivity of HSV to antiviral drugs. Virus was cultured on Vero cells in the presence of the increasing concentrations of acyclovir. 48 hours after infection, the DNA in the culture supernatants was quantified directly by real time PCR. The virus sensitivity was expressed as an IC50 value, which was the concentration of antiviral drug reducing the number of DNA copies by 50%. We investigated the kinetics of the assay, the influence of the multiplicity of infection on the antiviral effect of acyclovir, the reproducibility and precision of the assay. Well-characterised HSV strains as well as clinical isolates were tested. Parallel PRA was performed for comparison. The data from the pilot study indicate, that the established detection system based on TaqMan real time quantitative PCR combined with the short-time culture could be used as a readout system for antiviral susceptibility testing of HSV clinical isolates.

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P-350 Prevalence of antiretroviral-drug resistance amongst treatment naı¨ve HIV-1 infected patients in Merseyside and South London ANDREW STREET1, P.B. Carey2, B. Peters3 and C.Y.W. Tong4 1 Department of Virology, Royal Liverpool University Hospital, Prescott Street, Liverpool, L7 8XP, UK 2 Department of Genitourinary Medicine, Royal Liverpool University Hospital, Liverpool, UK 3 Department of Genitourinary Medicine, St. Thomas’ Hospital, London, UK 4 Department of Infection, St. Thomas’ Hospital, London, UK Introduction: The introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 infection has been associated with a sharp decline in mortality and morbidity amongst this patient group. However, the development of viral resistance to these drugs is an important cause of treatment failure and it has been hypothesised that the widespread use of HAART will result in the increased transmission of drug resistant virus. Objecti6e: To measure antiretroviral-drug resistance in treatment naive HIV-1 infected patients by a genotypic method. Methods: RT-nested PCR amplification of the reverse transcriptase and protease genes of HIV-1, was followed by DNA sequencing. The genotypic resistance profiles of thirty-seven patients, twenty-two from Merseyside and fifteen from South London, were studied. Results: Nineteen out of twenty-two patients from Merseyside had wild-type reverse transcriptase sequences. Two patients had non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations (V1081, V179D) and one patient had a secondary multiple nucleoside resistance mutation (A62V). No primary protease inhibitor resistance mutations were observed, however secondary resistance mutations were detected in fourteen (63%) patients (1 to 3 mutations per patients). No primary resistance mutations were detected in either the reverse transcriptase or protease genes of the fifteen South London patients. Secondary mutations in protease gene were detected in eleven (73%) patients (1 to 6 mutations per patient). Conclusions: Pre-treatment anti-retroviral drug resistance existed in a minority of patients in this cohort. Although uncommon at present, continuous monitoring of pre-treatment drug resistance is necessary with the more widespread use of antiretroviral combination therapy.

P-351 Protease activity of a novel human retrovirus CECILE VOISSET1, P.J.W. Venables2, R. Weiss1 and D. J. Griffiths1 1 Wohl Virion Centre, University College London, Windeyer Institute, UCL, 46 Cleveland Street, London, W1 6DB, United Kingdom 2 Kennedy Institute of Rheumatology, 1 Aspenlea Road, London W6 8LH, UK A new human retrovirus has previously been identified in tissues from patients with autoimmune inflammatory diseases using a degenerate primer PCR strategy (Griffiths et al., 1997). We have provisionally designated this new exogenous virus Human Retrovirus-5 (HRV-5) as it is the fifth retrovirus described in humans. HRV-5 displays sequence similarities with B and D-type retroviruses and presents several features typical of infectious retroviruses, such as open reading frames in gag, protease and pol regions, and sequence variation in isolates from the same individual and from different individuals. Studies examining the association between HRV-5 infection and rheumatic disease using a nested PCR assay for viral DNA revealed that HRV-5 was present in approximately 50% of DNA from synovial tissue

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biopsies from patients with inflammatory joint disease (Griffiths et al., 1999; Brand et al., 1999). HRV-5 DNA was also found in 12% of peripheral blood samples from patients with rheumatoid arthritis and 15% of patients with systemic lupus erythematosus. In contrast, the virus was not detectable in normal synovia and only rarely found in affected tissues of other inflammatory disease patients. We have studied the HRV-5 protease activity expressed in E. coli. HRV-5 protease displays an autocatalytic activity, and an activity in trans on homologous Gag proteins. Moreover, HRV-5 protease activity is inhibited by pepstatine A, confirming it belongs to the aspartic protease family. Further studies will examine the effects of other inhibitors of retroviral proteases on HRV-5 enzyme, as well as the activity of other enzymes of this novel human retrovirus.

P-352 Genotypic and phenotypic analysis of HIV-1 protease from Czech patients treated with protease inhibitors: towards a multipotent protease inhibitor JAN WEBER1, J. Sponarova1, L. Machala2, M. Reinis3, J. Litera1, M. Hradilek1, M. Soucek1, M. Bruckova3, M. Stankova2 and J. Konvalinka1 1 Institute of Organic Chemistry and Biochemistry, Academy of Science of Czech Republic, Flemingovo n. 2, Prague 166 10, Czech Republic 2 AIDS Center, Bulovka Hospital, Prague, Czech Republic 3 National Reference Laboratory on AIDS, National Institute of Public Health, Prague, Czech Republic Protease (PR) inhibitors became very important class of antiretroviral agents for the treatment of HIV infection. One of the most serious obstacles to the successful clinical use of protease inhibitors (PI) is the emergence of drug-resistant mutants. In present study we monitored 20 patients receiving highly active antiretroviral therapy, including various combinations of reverse transcriptase inhibitors together with saquinavir, indinavir, ritonavir and nelfinavir, in Czech Republic. HIV-1 protease genes have been isolated and amplified from peripheral blood mononuclear cells and/or patient plasma followed by dideoxynucleotide terminator cycle sequencing. HIV genotypes corresponding with promotion of HIV-1 PR drug resistance have been found in 11 patients. We have cloned, expressed and characterized HIV-1 PR variants exhibiting high number of genotype changes associated with PI resistance. We used them for determination of the inhibition constants for commercially available PIs in vitro. We have found that these mutated PRs bind the inhibitors as much as 70 times more weakly in comparison with the wild type PR. We have also observed cross-resistance among the mutants towards other inhibitors. We have tested these mutants with a novel pseudopeptide inhibitor of HIV PR developed in our institute. QF34, the most efficacious inhibitor with the scissile bond replaced by nonhydrolysable diastereomeric hydroxyethylene isostere, is a subnanomolar inhibitor of all PR variants tested up to date. The structural basis for this wide substrate specifity of the highly potent inhibitor will be explained using X-ray structure of the QF34 complexed with the HIV-PR. The implications for the design of multipotent inhibitors of HIV, active against various resistant strains, will be discussed.

P-353 Phenotypic HIV-1 drug resistance analysis using a self-inactivating virus vector system BENEDIKT WEISSBRICH1, G. Jarmy1, M. Heinkelein1, B. Weissbrich1, C. Jassoy1 and A. Rethwilm2

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Institute of Virology and Immunobiology, University of Wuerzburg, Versbacher Straße, Wuerzburg 97078, Germany 2 Institute of Virology, Carus Technical University, 01069 Dresden, Germany Background: Drug resistance is a major problem in antiretroviral therapy of HIV-1 infection. Within the last years phenotypic assays have been developed to detect and measure emerging resistance of HIV against antiviral drugs. Most of these assays are time consuming and require propagation of infectious virus. Design: A self-inactivating replication-defective vector system based on HIV-1 was constructed. It contains a reporter gene for rapid quantification of HIV vector-transduced target cells. Sequences encoding the protease (PR) and reverse transcriptase (RT) genes derived either from HIV proviral clones, laboratory or patient virus isolates were inserted into the vector plasmid. Plasmids were transfected into 293T cells to produce a pool of recombinant vectors. Antiviral agents were added upon transfection and transduction of target cells with the vectors. Results: Resistance to nucleoside and non-nucleoside RT inhibitors as well as PR inhibitors could readily be quantified by this test system. Results were obtained within two weeks and were highly reproducible. Conclusion: The assay system represents a rapid and convenient phenotypic method for resistance testing against currently used anti-HIV drugs. Since propagation of infectious virus is not required, this phenotypic resistance assay can be performed at biosafety level 2.

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