5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>these enzyme activities and exhibited dual inhibition properties in the lowmicromolar range. Interestingly, amino acids mutations in the Non Nucleosideinhibitors binding pocket strongly affected the propenone derivativesRNase H inhibition, suggesting long range RT structural effects, withoutreducing their DNA polymerase inhibition ability. Thus we performedbiochemical and computational analyses in order to clarify their mode <strong>of</strong>action. Notably, these studies indicated that propenone derivatives are characterizedby a novel mechanism <strong>of</strong> action and bind to two different allostericinterdependent pockets. Hence, these results set the basis for the furtherdevelopment <strong>of</strong> RT inhibitors interacting with a novel RT binding site.REF 138Initial study on the mode <strong>of</strong> action <strong>of</strong> castalagin against herpes simplexvirusAngel S. GALABOV 1 , Neli VILHELMOVA 1 , Stephane QUIDEAU 21 The Stephan Angel<strong>of</strong>f Institute <strong>of</strong> Microbiology, Bulgarian Academy<strong>of</strong> Sciences, S<strong>of</strong>ia, BULGARIA; 2 Universite de Bordeaux, Institut desSciences Moleculaires, Pessak Cedex, FRANCEOur previous studies showed that castalagin, a nonahydroxyterphenoylbearing C glycosidic ellagitannin, manifests a significant antiviral activityagainst HSV 1 and HSV 2 strains both sensitive and resistant toACV. Moreover, the combination effect <strong>of</strong> ellagitannins with acyclovirwas shown as a markedly synergistic, especially the effect <strong>of</strong> this combinationtowards the replication <strong>of</strong> HSV 1 sensitive strain [Vilhelmova et al.,Antiviral Res. 89, 2011, 174 181; Antiviral Res. 90/2, 2011, N144, A64A65]. Here we present results <strong>of</strong> the initial step <strong>of</strong> a clearing up the mode<strong>of</strong> antiviral activity <strong>of</strong> castalagin on the replication <strong>of</strong> HSV 1 (Victoria,an ACV sensitive strain) in MDBK cells, including (i) timing <strong>of</strong> additionstudy in the one step growth cycle setup (MOI=1); (ii) the effect on theviral adsorption in MDBK cells, and (iii) the effect on the extracellularHSV virions. The susceptible to castalagin period <strong>of</strong> the virus groth cycleembraces markedly the initial first six hours post virus adsorption, as recordedat the 24th h: an inhibitory effect exceeding 3 logs when the compoundwas added immediately after or at the 1st and 2nd h post virus inoculation,and about 2 logs within the 3 h – 6 h time interval. The compound additionafter the 12th h manifested a negligible activity. Castalagin at the concentration<strong>of</strong> 10 M (MTC) showed a marked effect on virus adsorption: log1.7 on the 30 min, 2.2 logs on the 45 min and 3.2 logs on the 60th min. Awell expressed inhibitory effect <strong>of</strong> 2 logs was registered by 1 M atthe60 min. At a concentration <strong>of</strong> 0.1 M effect the effect was clearly weaker(log 1) and at a concentration <strong>of</strong> 0.01 M a suppression <strong>of</strong> virus adsorptionwas not observed. The compound showed a marked direct inactivatingeffect on extracellular HSV 1, which is markedly temperature dependent.The effect was significantly higher at 37oC: castalagin 10 M decreasedthe infectious virus by log 1.6 at an exposure time <strong>of</strong> 30 min, 1.8 logs at60 min, 2 logs at 90 min, and 2.2 at the 120 min. This activity was stronglydose dependent and exposure time dependent: 30 min at the castalaginconcentrations <strong>of</strong> 10 M and 1 M, 60 min at 0.1 M and 0.01 M.REF 139Association study <strong>of</strong> IL28B: rs12979860 and rs8099917 polymorphismswith response/resistance to standard treatment <strong>of</strong> Moroccanspatients infected with hepatitis C virusNadia KANDOUSSI, Mohamed Rida TAGAJDID, BouchraBELFQUIH, Hicham ELANNAZ, Saâd MRANILaboratory <strong>of</strong> <strong>Virology</strong>, HMIM V, Faculty <strong>of</strong> Medicine and PharmacyMohamed V – Souissi University, Rabat, MOROCCOIntroduction: recently, four genome wide association studies (GWAS)have identified single nucleotide polymorphisms (SNPs) near the IL28Bgene (encoding IFN 3) to be strongly associated with spontaneous andtreatment induced clearance <strong>of</strong> HCV infection. The purpose <strong>of</strong> this studywas to investigate the association between two IL28B polymorphisms(rs8099917 and rs12979860) and response/resistance to standard treatment<strong>of</strong> Moroccan patients infected with hepatitis C virus and allelic frequencies<strong>of</strong> these polymorphisms answered most in a Moroccan population. Materialsand methods: this study was conducted on retrospective data <strong>of</strong> 265adult patients with chronic HCV infection who were diagnosed by antiHCV antibodies (ELISA). Data collection was performed with an Excelbased case report form laboratory, between January 2011 and December2012. All Patients had been treated with bitherapy IFN/RIBAVIRIN. 159patients had a sustained virologic response and 106 patients have persistentHCV viral load. Genotyping for the IL 28B rs12979860 C/T andrs8099917G/T polymorphisms was performed by High Resolution Meltingtechnical “HRM”. Early virological response (EVR) (12 weeks), end<strong>of</strong> treatment response (EOTR) (48 weeks), sustained virological response(SVR) (72 weeks) and relapse were evaluated using conventional andquantitative polymerase chain reaction (PCR) assays. Results and discussion:the frequency <strong>of</strong> the C allele in the population was 39%. Overall, 43%<strong>of</strong> patients experienced SVR. The IL28B CC genotype was significantlyassociated with higher treatment response rates and a lower relapse ratecompared to the other genotypes [84% vs. 58% EVR, 92% vs. 63% EOTR,76% vs. 38% SVR]. Thus, the IL28B genotype appears to be a strong predictor<strong>of</strong> SVR. This study, together with similar research examining otherSNPs, should help to define adequate protocols for the treatment <strong>of</strong> patientsinfected with HCV genotype 1, especially those with a poor prognosis.REF 140Prognostic role <strong>of</strong> Hepcidin level in Cchronic Hepatitis C virus patientson Interferon therapyNashwa KHADR, Hadier OKASHA, Hanan NOUH, Walaa SEMARYFaculty <strong>of</strong> Medicine University <strong>of</strong> Alexandria, Alexandria, EGYPTHCV is one <strong>of</strong> the most important health issues worldwide. Egypt has thehighest prevalence <strong>of</strong> hepatitis C in the world estimated about 15% 20%PEG–IFN plus ribavirin combination therapy became the gold standardin the treatment <strong>of</strong> HCV this treatment enhances sustained virologicalresponse (SVR)There are several host and viral factors aid in predictingresponse to treatment Hepcidine hormone is one <strong>of</strong> these host factors ithas a beneficial role in assessing treatment response during the course<strong>of</strong> therapy Patients with chronic HCV <strong>of</strong>ten have increased liver iron, acondition associated with reduced response to antiviral therapy. The hepatichormone Hepcidine is the major negative regulator <strong>of</strong> iron release inthe circulation. The aim <strong>of</strong> the work is to assess the serum concentration<strong>of</strong> hepcidin in chronic hepatitis C patients and evaluate any possibleassociation with the disease activity (viral load) as well as the efficacy <strong>of</strong>pegylated IFN and Ribavirin therapy. This study was carried on 35 chronicHCV patients attending el kabary hospital to receive peg IFN/Ribavirintherapy and 15 chronic HCV patients not on IFN therapy as a control groupHepcidin hormone levels were measured in sera <strong>of</strong> 35 chronic hepatitis Cpatients before starting therapy (base line) at 12 and 24 weeks <strong>of</strong> therapyusing Hepcidine enzyme immuno assay (USCN life science Inc) Qualitativedetection <strong>of</strong> HCV RNA to asses sustained virologic response (SVR)using reverse transcriptase PCR at week 24 <strong>of</strong> antiviral therapy. Our casesshowed a male predominance (70%) over females (30%) and they showedthey showed a slight elevation <strong>of</strong> AST and Alkaline phosphatase while theother laboratory markers (ALT, Albumin, Bilirubin and Hb) were withinnormal values. The results <strong>of</strong> qualitative PCR for cases after 6 months <strong>of</strong>therapy were in agreement with the RT-PCR results The level <strong>of</strong> hepcidinin all cases was very low before starting therapy and it showed a significantincrease during the course <strong>of</strong> therapy. This rise was detected earlyin responding cases. And it is significantly increased after 6 months <strong>of</strong>therapy as compared to the control group. We found a negative correlationbetween baseline hepcidin level and baseline viral load <strong>of</strong> the cases thatshowed response after 6 months <strong>of</strong> therapy (undetectable viral RNA) wefound no significant relation between degree <strong>of</strong> fibrosis and viral load <strong>of</strong>the studied cases. Conclusion Chronic HCV infection is associated withVi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S157
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>reduced level <strong>of</strong> serum hepcidin hormone. The reduced serum hepcidinin chronic HCV patients is fully reversible after IFN/RBV therapy Initialrise in serum hepcidin concentration could be used as one <strong>of</strong> indicators<strong>of</strong> patient response to interferon therapy. We could not find a significantrelation between baseline viral load and degree <strong>of</strong> liver fibrosis or liverenzymes. No significant relation was found between hepcidin levels andneither degree <strong>of</strong> liver fibrosis nor liver enzymes.REF 141Determination <strong>of</strong> antiviral resistance mutations in chronic hepatitis Bsubjects used antiviral therapy by pyrosequencing methodSevin KIRDAR 1 , Sibel ÖZSU 2 , Alpay ARI 3 , Emine Deniz BAYRAM 21 Adnan Menderes University, Medicine Faculty, Department <strong>of</strong> MedicalMicrobiology, Aydin, TURKEY; 2 Medical Microbiology Laboratory, IzmirBozyaka Training and Research Hospital, Izmir, TURKEY; 3 Department <strong>of</strong>Infectious Disease, Izmir Bozyaka Training and Research Hospital, Izmir,TURKEYIntroduction and aim: Chronic hepatitis B virus (HBV) infection is aserious public health problem in the worldwide. Antiviral therapy usingnucleos(t)ide analogues (NAs) is an effective treatment. However theyneed long term treatment. The existence <strong>of</strong> HBV variants with primaryantiviral resistance may be important for treatment choice. In this studywe aimed to show the HBV rt mutants in chronic hepatitis B patients whohave received nucleos(t)ide analogues therapy.Method: Fifty serum samples obtained from 50 chronic hepatitis Bpatients who used antiviral therapy from January 2010 to March 2013.The mean age <strong>of</strong> the patients (15 female, 35 male) was 45.75 (range:2873) years. Serum samples <strong>of</strong> the patients were analysed by pyrosequencingmethod and HBV DNA was quantitated with real time PCR.Results: HBV rt mutations were detected in 13 samples (26% <strong>of</strong> the cases)and the remaining 37 samples (74% <strong>of</strong> the cases) showed wild type (WT)isolates. The distribution <strong>of</strong> HBV RT mutations was detected as 204 V/I in8 cases(61.5%), L180 M in three cases (23.7), A181 V in one case (7.6%).Both <strong>of</strong> 204 V/I and 1 L180 M were found in one case (7.6%). The one <strong>of</strong>the cases detected L180 M mutation converted from wild type.Conclusion: Pyrosequencing is a rapid, specific, and sensitive tool thatmay be useful in detecting and quantifying subpopulations <strong>of</strong> resistantviruses. In this study, it was concluded that this method may be a convenienttool for monitoring HBV infected patients receiving nucleos(t)ideanalogues therapy.Key words: antiviral therapy, mutation, Hepatitis B virusREF 142Intracellular co factors for HCMV replication – novel targets forantiviral therapyIra NAPIERKOWSKI, Elke BOGNERInstitute <strong>of</strong> <strong>Virology</strong>, Charité Medical School, Berlin, GERMANYHuman cytomegalovirus (HCMV) is a Herpesvirus that establishes lifelong latency in the host after primary infection. While HCMV infection isusually asymptomatic in immunocompetent people, primary infection andreactivation in individuals with a compromised or undeveloped immunesystem can cause life threatening disease. Although antiviral compoundsfor treatment <strong>of</strong> HCMV infection and disease are available resistance andtoxicity are major problems. Therefore, the development <strong>of</strong> new antiviralcompounds with novel drug targets is imperative. One potential new antiviraltarget is the HCMV terminase complex consisting <strong>of</strong> a large subunit,pUL56, and a small subunit, pUL89. The terminase is required for cleavingand translocating concatemeric HCMV DNA and packaging unit lengthgenomes into procapsids – an essential process for viral replication andtherefore a promising target for new antivirals. This study aims to identifycellular proteins involved in viral replication and packaging using theYeast Two Hybrid System, followed by confirmation <strong>of</strong> found interactionsby other methods. Possible inhibitors based on the identified hostproteins will then be designed, synthesised, and characterised regardingtheir antiviral activity. So far, Yeast Two Hybrid library screens were performedfor the terminase subunits, the portal protein pUL104 and pUL77,which is also involved in DNA packaging. Potential cellular interactionpartners could be found for pUL77 and have been investigated further byretransformation into yeast and co immunoprecipitation.REF 143Transmission <strong>of</strong> drug resistant HIV 1 strains among injecting drugusers from the Greater Lisbon, PortugalJoão PIEDADE 1,2 , Sandra VIDEIRA E. CASTRO 1 , Carina SOUSA 1 ,Elizabeth PÁDUA 3 , Ricardo PARREIRA 1,2 , Aida ESTEVES 1,21 Grupo de Virologia, Unidade de Microbiologia Médica, Instituto deHigiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, POR-TUGAL; 2 Unidade de Parasitologia e Microbiologia Médicas (UPMM),Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,Lisbon, PORTUGAL; 3 Lab. Nac. Referência IST VIH/SIDA e Hepatites Be C, Dep. de Doenças Infecciosas, Instituto Nacional de Saúde Dr. RicardoJorge, Lisbon, PORTUGALHIV 1 drug resistance information is useful in the clinical setting, as itcan guide the choice <strong>of</strong> antiretrovirals, also providing data on the circulation<strong>of</strong> resistant strains. Viral RNA, purified from blood samples fromHIV 1 infected injecting drug users, was amplified by RT nested PCR.DNA sequencing <strong>of</strong> protease (PR), reverse transcriptase (RT), integrase(IN) and C2V3C3 gp120 amplicons originated fragments <strong>of</strong> 297, 598, 813and 516 bp, respectively, for resistance analysis. The Stanford GenotypicResistance Interpretation Algorithm was used for PR, RT and IN. Aftertranslation, C2V3C3 sequences were compared to a subtype B consensusto search for the presence <strong>of</strong> genetic polymorphisms associated tomaraviroc (MVC) resistance. A total <strong>of</strong> 123 PR, RT or IN sequences wasobtained. Overall, 15 resistance associated mutations were found, with adistribution <strong>of</strong> 1 3 mutations/sequence (4 minor in PR; 3 high level, 1 lowlevel and 2 polymorphisms in RT; 5 in IN, 1 major and 4 minor). Half<strong>of</strong> the subjects infected with these strains (n=11) were drug naïve, showingtransmission <strong>of</strong> resistant viral strains. Based upon published data,we observed 12 different single mutations on V3 loop, as well as 2 mutationalpatterns, in 35 sequences, at variable numbers. The presence <strong>of</strong> thepatterns 11S+26 V and 20F+25D+26 V, in 3 sequences, is relevant, sincethey have been associated with MVC resistance in vivo. Our results willbe useful for therapy implementation and monitoring <strong>of</strong> infection in thispopulation, having also an impact on the clinical management <strong>of</strong> HIV 1drug experienced individuals.REF 144In silico approach <strong>of</strong> the influence <strong>of</strong> HBV envelope glycoproteinson HBs antigen clearance under anti HBV treatmentEvelyne SCHVOERER 1,2 , Aurelie VELAY 1,2 , FrançoisGOEHRINGER 1,3 , Hélène JEULIN 1,2 , Mouni BENSENANE 4 , ThierryMAY 3 , Fabien ZOULIM 5 , Jean Pol FRIPPIAT 1 , Jean PierreBRONOWICKI 41 Université de Lorraine, EA 7300 « Stress, IMmunité, PAthogènes », Vandoeuvreles Nancy, FRANCE; 2 Centre Hospitalier Universitaire de Nancy,Laboratoire de Vi<strong>rologie</strong>, Vandoeuvre les Nancy, FRANCE; 3 Centre HospitalierUniversitaire de Nancy, Service des Maladies Infectieuses etTropicales, Vandoeuvre les Nancy, FRANCE; 4 Centre Hospitalier Universitairede Nancy, Service d’Hépato Gastroenté<strong>rologie</strong>, Vandoeuvreles Nancy, FRANCE; 5 Université de Lyon, Unité Inserm UI1052, Lyon,FRANCES158 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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