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Viral genomic variability and response to interferon therapy in patients with HCV-1B chronic hepatitis
116
Poster Sessions
Abstract 489: Table. Changes on anti-HCV antibody patterns and correlation with HCV RNA in followed subjects First sample
No
Second sample EIA reactive/Riba Indeterm. HCV RNA+ (No)
EIA reactive/Riba pos. HCV RNA+ (No) EIA reactive/Riba positive
38
EIA reactive/Riba Indeterm.
6
EIA negative
24 (22)" 1 (1) 2 (2)
1988
4 (2)b 0 0
EIA negative HCV RNA+ (No) 10 (0) 5 (0) 1986
aStable anti-HCV serologic profiles; hdecreased in intensity of the bands amino acids arginine 339, tfiptophane 368 and lysine 370 could play a critical role in protein function as both cell lysis and permeability changes can be totally blocked by amino acids substitutions in these positions.
'6-9-I DESIGN AND SYNTHESIS OF HEPATITIS C NS3 PROTEINASE INHIBITORS F.X. Wilson, A. Bryson, S. Harris, P.S. Jones, S.T. Kingston, B. Sutton, T.C.I.W. Wilkinson. Roche Discovery Welwyn, UK Hepatitis C Virus (HCV) is the cause of the majority of cases of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV can lead to a range of clinical conditions including an asymptomatic carder state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. Current estimates show ~300 million chronically infected worldwide.
et(; - Pa "P2" Pt ~ PI' "Pz' "etc
COzH
o
-co,.
~
etc. Ps" P2 "HN
~:oz.
L
Electrophlle
I" .,(
'O.'n"
Starting from the observation that the virus possesses a chymotrypsinlike serine proteinase, we have designed potent electrophile-based inhibitors in three structural classes. The design and synthesis of these is detailed, together with the use of rat hepatocyte cultures to correlate in-vitro observations with in-vivo effects on the liver and hence profile compounds for potential hepatotoxicity
-~
INFECTION RATE AND SPONTANEOUS SEROREVERTION OF ANTI-HCV DURING NATURAL COURSE OF HCV INFECTION IN A GENERAL POPULATION OF CENTRAL ITALY
L.A. Kondili 1, p. Chionne 1, A. Costantino 1, U. Villano 1, F. Pannozzo 3, A. Mele 2, S. Gianpaoli 2, M. Rapicetta 1. 1Lab. Virology; 2Lab.
Epidemiology, Istituto Superiore di Sanitc), Rome; 3 USL Latina, Italy Aims: We performed a follow-up study (7 yrs) in a cohort population of 2033 randomly selected subjects, from a Central Italian area, to estimate 1) the prevalence of anti-HCV, 2) the risk of acquiring HCV infection during follow-up 3) the frequency of spontaneous serorevertion during natural course of HCV infection. Methods: EIA 3 (Ortho) RIBA 3; (Chiron, Ortho) and Cobas Amplicor HCV 2.0 (Roche) were applied for anti-HCV and HCV-RNA determinations.
Results: Anti-HCV prevalence was 2.39. HCV infection rate was 1.4/10000 person-years. Conclusions: There is still a permanent risk of HCV spread in general population independently by low HCV endemic level. 26.3% of the subjects with complete R1BA3 reactivity and 83.3% of subjects with indeterminate RIBA3 reactivity gradually lost HCV markers during follow-up. These data does not mean that these patients have totally eradicated HCV with a definitive loss of infectivity.
[-~
ASSESSMENT OF PREDICTIVE FACTORS FOR TREATMENT OF CHRONIC HEPATITIS C IN HIV-INFECTED CARRIERS
M. Perez-Olmeda, M. Romero, F. Munoz, J. Garcia-Samaniego, J. Gonzalez-Lahoz, V. Soriano. Infectious Diseases, Hospital Carlos 111,
Spain Five different parameters have been recognized as independent predictors of treatment response of chronic hepatitis C. Low fibrosis in liver histology, younger age at HCV infection, female gender, HCV genotypes 2/3, and serum HCV load < 800,000 IU/ml have all been associated with a better response to interferon plus ribavirin. We have assessed the profile of a large group of HCV-HIV coinfected patients referred to our institution for treatment of chronic hepatitis C. From total of 175 individuals, HCV genotypes were distributed as follows: HCV-1 (n -- 87, 49.7%), HCV-2 (n = 2, 1.1%), HCV-3 (n = 57, 32.5%), HCV-4 (n = 7, 4%), and HCV-6 (n = 1, 0.5%). Infection with multiple HCV genotypes were recognized in 21 (12%). Mean serum HCV load was 1,159,938 IU/ml and 133 (76%) individuals harboured an HCV load > 800,000 IU/ml. Mean age of subjects with HCV-HIV coinfection was 36.7 years-old (76% males). More than 80% of them were former IDUs and had acquired HCV infection through needle sharing. Conclusion: chronic hepatitis C among HIV+ carders would show a better prognostic profile for treatment response than among HIV-negative population, when considering the relative higher rate of HCV genotype 2/3, and the younger age of patients. However, male predominance and high HCV-RNA might counteract this benefit. In the mean time, up to one third of subjects (those carrying HCV genotypes 2/3) might be treated with shorter periods of interferon plus ribavirin (6 months) which might favour a better tolerance in subjects on antiretroviral drugs.
-~
VIRAL GENOMIC VARIABILITYAND RESPONSE TO INTERFERON THERAPY IN PATIENTS WITH HCV-1B CHRONIC HEPATITIS
G. Squadrito, G. Raffa, T. Restuccia, T. Pollicino, G. Raimondo.
Dipartimento di Medicina Interna, Universitd di Messina, Italy Reports suggested that the NS5A gene of the HCV may be involved in the poor responsiveness to IFN treatment of genotype lb-infected patients because of possible interference with the cellular interferon-induced PKR. In particular, the NS5A aa sequence 2209-2274 comprising the ISDR and the V3 region of this protein were supposed to be impor-
Category 5: Viral hepatitis: basic aspects tant. Additional reports suggested that the aa sequence 659--671 of the HCV-E2 protein might also be involved in the interferon resistence via the PKR elF2~. The high variability of each of these regions was supposed to correlate with favourable response to IFN treatment. We analysed the NS5A gene and the E2659-671 region in isolates from pretreatment serum samples of 17 HCV-lb infected patients. Ten of them were non-responders to IFN therapy (NR) while 7 showed a sustained biochemical and virological response persisting from 4 to 6 years after stopping therapy (SR). The E2659-671 region and the carboxi-terminal half of NS5A gene (including both PKR binding and V3 regions) were amplified and directly sequenced in all the cases. In addition, the entire NS5A gene was amplified, cloned and sequenced in 6 cases (3 NR and 3 SR). The E2659--671 region was quite completely conserved in all the cases. The sequencing analysis of the NS5A carboxi-terminal region showed difference neither in the NS5A PKR binding nor in the V3 regions between NR and SR. Cloning analysis of full length NS5A showed no difference also in the amino-terminal part of the gene and absence of quasispecies diversity between the two studied groups. Conclusions: Pretreatment analysis of the NS5A and E2 genomic variability has not value in predicting response to IFN therapy in patients with HCV-lb chronic infection.
-I LYMPHOTROPIC HCV QUASISPECIES ARE MORE FREQUENT DURING MILD LIVER DISEASE AND AFTER LIVER TRANSPLANTATION C. Feray, A.M. Roque, R. Kara, M. Gigou, J.J. Jiang, C. Guettier, E. Dussaix, D. Samuel. CHB-Virology and Pathology, INSERM9941-Hopital Paul Brousse, CHB, France HCV quasispecies are not randomly distributed in serum, liver and lymphocytes. Apparently specific quasispecies are detected in peripheral blood lymphocytes (PBL) raising the possibility that some quasipecies have an extra-hepatic tropism and possibly affect virus/host interactions. The relations between these lymphotropic quasispecies and the natural course of HCV is unknown. Patients: 100 patients chronically infected by HCV with a known duration of infection. 45 were liver transplanted and have recurrent infection. None had been treated by interferon. PBL and serum were obtained when liver biopsy was performed. 5'NC region, core and HVR were amplified and analyzed through SSCP. Viremia was quantified with Taqman real-time PCR. Compartmental Distribution (CD) was defined by difference of SSCP patterns between serum or PBL. Results: CD was observed in 5' non coding region (33%), core (31%) and HVR (74%). CD and number of quasispecies were more pronounced in 5' non-coding region (p < 0.01) but not in core or HVR in transplanted patients. Minimal lesions were correlated to CD in HVR and activity of hepatitis to the CD in core (p = 0.02 and p = 0.01). CD in 5'NC were inversely correlated to the duration of infection. The CD defined in core, HVR and 5'NC were not correlated to HCV genotypes or the level of viremia. Levels of viremia was correlated to the numbers of serum quasispecies defined in HVR. Conclusion: Lymphotropic HCV quasispecies are frequent during chronic hepatitis, inversely correlated to the severity and the duration of infection. These findings suggest that compartmental distribution of quasispecies in PBL is important during the course of hepatitis C both in transplanted and non-transplanted patients
- ~
NON-STRUCTURAL 5A (NS5A) PROTEIN AND HCV GENOTYPE 3a SENSITIVITY TO INTERFERON (IFN)-ALPHA THERAPY
L. Castera, M. Leroux, A. Soulier, R. Brillet, I. Lonjon, D. Dhumeaux, J.M. Pawlotsky. Virology, Hopital Henri Mondor, France Hepatitis C virus (HCV) NS5A protein mediates HCV resistance to IFN
117
alpha in vitro. However, HCV resistance to IFN in patients in vivo is multifactorial, and the role of viral proteins, if NS5A plays a role in IFN sensitivity or resistance of HCV genotype 3a. 10 patients infected with HCV genotype 3a treated with 3 to 6 MU of IFN alpha-2a tiw for 6 to 12 months were studied. 4 of them achieved a sustained HCV RNA clearance (SVRs), whereas 6 did not respond or relapsed (non-SVRs). A 567-bp fragment spanning the transcriptionnal activation domain of NS5A was amplified by PCR before treatment in all patients, and at the time of relapse in 2 non-SVRs. A 200-bp fragment containing HVR1 was studied in the same samples. Overall, a total of 500 clones (i.e. 20 to 30 clones per sample) were sequenced and extensive genetic and phylogenetic analyses were performed. Results: NS5A and HVR1 were distributed as quasispecies at any time point. Phylogenetic analyses showed no distinct clustering of the NS5A and HVR1 quasispecies variants isolated from the SVRs and the non-SVRs, respectively. The SVRs had significantly lower nucleotide sequence complexity (as assessed by the entropy, p < 0.04) than the non-SVRs in NS5A but not HVR1. In 2 non-SVR patients, a significant genetic evolution of both NS5A and HVR1 quasispecies was observed at the time of relapse. In contrast with HVR1, the rate of accumulation of synonymous mutations was significantly higher than the rate of accumulation of non synonymous mutations in NS5A. In smnmary, no specific NS5A protein sequence appears to be associated with HCV genotype 3a sensitivity or resistance to IFN alpha. These data do not support a role for HCV genotype 3a NS5A protein in sensitivity or resistance to IFN.
- ~
REAL TIME RT-PCR QUANTIFICATION: A NEW METHOD TO STUDY HEPATITIS C VIRUS ADSORPTION ON VERO CELLS
R. Germi 1, J.M. Crance 2, D. Garin 2, j. Guimet 1' C. Rothlisberger 2, J.P. Zarski 3, E. Drouet 1.1 Laboratoire de virologie, Univ J. Fourier (Faculte de Medecine-Pharmacie); 2 CRSSA Emile Parde; 3 CHU Albert MichaUon, Grenoble, France HCV binding on cells is critical for pathogenesis. We previously developed Vero and mosquito AP61 cell systems, which are able to bind and replicate HCV. To assess HCV binding on Vero cell-receptors, we developed a real time RT-PCR fluorimetric assay. (i) Virus adsorption assay: cell inoculation (2 h at 20°C) with HCV-positive plasma (genotype lb; 2 x 10 e 6 copies/mL) was followed by 6 washes. (ii) Cells and the last wash were submitted to quantification assay: real time one step RT-PCR (Light Cycler®) using forward primer (2CH = AAC TAC TGT CTI" CAC GCA GAA), reverse primer (ITS = GCG ACC CAA CAC TAC TCG GCT), TaqMan ® probe (CRSSAc = C6-Fam-AAC CCG CTC AAT GCC TGGA-Tamra). Standard curve derived from known virus concentrations have a linear relationship over 3.5 logs from 50 to 10 e 5.2 viral copies. After inoculation of 10 e 4 HCV RNA copies on 10 e 6 cells, we detected 500 copies on ceils. The last wash remained negative. Reducing inoculum amount, bound virus number decreased. Anti-HCV E2 antibodies (J. Dubuisson, Lille, France) led to 50% reduction of HCV binding (p < 0.05, quintuplicate assay). This work reports quantitative analysis of HCV cell adsorption. This technique could be used to screen ligands involved in the first step of HCV infection and to identify molecules blocking virus binding on cells.
-1 CHARACTERISATION OF INTERFERON ALFA-2A AND PEGYLATED INTERFERON ALFA-2A IN COMBINATION WITH RIBAVlRIN, MYCOPHENOLIC ACID OR VX-497 AS INHIBITORS OF HCV REPLICON REPLICATION T.C.I. Wilkinson, C. Laxton, E. Hobbs, R. Bartenschlager, M. Graves, R. Devos, I. Najera. Roche Discovery Welwyn, UK Background: The current treatment of HCV infection relies on alpha interferon alone or in combination with ribavirin. Until recently, the lack
Poster Sessions
Abstract 489: Table. Changes on anti-HCV antibody patterns and correlation with HCV RNA in followed subjects First sample
No
Second sample EIA reactive/Riba Indeterm. HCV RNA+ (No)
EIA reactive/Riba pos. HCV RNA+ (No) EIA reactive/Riba positive
38
EIA reactive/Riba Indeterm.
6
EIA negative
24 (22)" 1 (1) 2 (2)
1988
4 (2)b 0 0
EIA negative HCV RNA+ (No) 10 (0) 5 (0) 1986
aStable anti-HCV serologic profiles; hdecreased in intensity of the bands amino acids arginine 339, tfiptophane 368 and lysine 370 could play a critical role in protein function as both cell lysis and permeability changes can be totally blocked by amino acids substitutions in these positions.
'6-9-I DESIGN AND SYNTHESIS OF HEPATITIS C NS3 PROTEINASE INHIBITORS F.X. Wilson, A. Bryson, S. Harris, P.S. Jones, S.T. Kingston, B. Sutton, T.C.I.W. Wilkinson. Roche Discovery Welwyn, UK Hepatitis C Virus (HCV) is the cause of the majority of cases of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV can lead to a range of clinical conditions including an asymptomatic carder state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. Current estimates show ~300 million chronically infected worldwide.
et(; - Pa "P2" Pt ~ PI' "Pz' "etc
COzH
o
-co,.
~
etc. Ps" P2 "HN
~:oz.
L
Electrophlle
I" .,(
'O.'n"
Starting from the observation that the virus possesses a chymotrypsinlike serine proteinase, we have designed potent electrophile-based inhibitors in three structural classes. The design and synthesis of these is detailed, together with the use of rat hepatocyte cultures to correlate in-vitro observations with in-vivo effects on the liver and hence profile compounds for potential hepatotoxicity
-~
INFECTION RATE AND SPONTANEOUS SEROREVERTION OF ANTI-HCV DURING NATURAL COURSE OF HCV INFECTION IN A GENERAL POPULATION OF CENTRAL ITALY
L.A. Kondili 1, p. Chionne 1, A. Costantino 1, U. Villano 1, F. Pannozzo 3, A. Mele 2, S. Gianpaoli 2, M. Rapicetta 1. 1Lab. Virology; 2Lab.
Epidemiology, Istituto Superiore di Sanitc), Rome; 3 USL Latina, Italy Aims: We performed a follow-up study (7 yrs) in a cohort population of 2033 randomly selected subjects, from a Central Italian area, to estimate 1) the prevalence of anti-HCV, 2) the risk of acquiring HCV infection during follow-up 3) the frequency of spontaneous serorevertion during natural course of HCV infection. Methods: EIA 3 (Ortho) RIBA 3; (Chiron, Ortho) and Cobas Amplicor HCV 2.0 (Roche) were applied for anti-HCV and HCV-RNA determinations.
Results: Anti-HCV prevalence was 2.39. HCV infection rate was 1.4/10000 person-years. Conclusions: There is still a permanent risk of HCV spread in general population independently by low HCV endemic level. 26.3% of the subjects with complete R1BA3 reactivity and 83.3% of subjects with indeterminate RIBA3 reactivity gradually lost HCV markers during follow-up. These data does not mean that these patients have totally eradicated HCV with a definitive loss of infectivity.
[-~
ASSESSMENT OF PREDICTIVE FACTORS FOR TREATMENT OF CHRONIC HEPATITIS C IN HIV-INFECTED CARRIERS
M. Perez-Olmeda, M. Romero, F. Munoz, J. Garcia-Samaniego, J. Gonzalez-Lahoz, V. Soriano. Infectious Diseases, Hospital Carlos 111,
Spain Five different parameters have been recognized as independent predictors of treatment response of chronic hepatitis C. Low fibrosis in liver histology, younger age at HCV infection, female gender, HCV genotypes 2/3, and serum HCV load < 800,000 IU/ml have all been associated with a better response to interferon plus ribavirin. We have assessed the profile of a large group of HCV-HIV coinfected patients referred to our institution for treatment of chronic hepatitis C. From total of 175 individuals, HCV genotypes were distributed as follows: HCV-1 (n -- 87, 49.7%), HCV-2 (n = 2, 1.1%), HCV-3 (n = 57, 32.5%), HCV-4 (n = 7, 4%), and HCV-6 (n = 1, 0.5%). Infection with multiple HCV genotypes were recognized in 21 (12%). Mean serum HCV load was 1,159,938 IU/ml and 133 (76%) individuals harboured an HCV load > 800,000 IU/ml. Mean age of subjects with HCV-HIV coinfection was 36.7 years-old (76% males). More than 80% of them were former IDUs and had acquired HCV infection through needle sharing. Conclusion: chronic hepatitis C among HIV+ carders would show a better prognostic profile for treatment response than among HIV-negative population, when considering the relative higher rate of HCV genotype 2/3, and the younger age of patients. However, male predominance and high HCV-RNA might counteract this benefit. In the mean time, up to one third of subjects (those carrying HCV genotypes 2/3) might be treated with shorter periods of interferon plus ribavirin (6 months) which might favour a better tolerance in subjects on antiretroviral drugs.
-~
VIRAL GENOMIC VARIABILITYAND RESPONSE TO INTERFERON THERAPY IN PATIENTS WITH HCV-1B CHRONIC HEPATITIS
G. Squadrito, G. Raffa, T. Restuccia, T. Pollicino, G. Raimondo.
Dipartimento di Medicina Interna, Universitd di Messina, Italy Reports suggested that the NS5A gene of the HCV may be involved in the poor responsiveness to IFN treatment of genotype lb-infected patients because of possible interference with the cellular interferon-induced PKR. In particular, the NS5A aa sequence 2209-2274 comprising the ISDR and the V3 region of this protein were supposed to be impor-
Category 5: Viral hepatitis: basic aspects tant. Additional reports suggested that the aa sequence 659--671 of the HCV-E2 protein might also be involved in the interferon resistence via the PKR elF2~. The high variability of each of these regions was supposed to correlate with favourable response to IFN treatment. We analysed the NS5A gene and the E2659-671 region in isolates from pretreatment serum samples of 17 HCV-lb infected patients. Ten of them were non-responders to IFN therapy (NR) while 7 showed a sustained biochemical and virological response persisting from 4 to 6 years after stopping therapy (SR). The E2659-671 region and the carboxi-terminal half of NS5A gene (including both PKR binding and V3 regions) were amplified and directly sequenced in all the cases. In addition, the entire NS5A gene was amplified, cloned and sequenced in 6 cases (3 NR and 3 SR). The E2659--671 region was quite completely conserved in all the cases. The sequencing analysis of the NS5A carboxi-terminal region showed difference neither in the NS5A PKR binding nor in the V3 regions between NR and SR. Cloning analysis of full length NS5A showed no difference also in the amino-terminal part of the gene and absence of quasispecies diversity between the two studied groups. Conclusions: Pretreatment analysis of the NS5A and E2 genomic variability has not value in predicting response to IFN therapy in patients with HCV-lb chronic infection.
-I LYMPHOTROPIC HCV QUASISPECIES ARE MORE FREQUENT DURING MILD LIVER DISEASE AND AFTER LIVER TRANSPLANTATION C. Feray, A.M. Roque, R. Kara, M. Gigou, J.J. Jiang, C. Guettier, E. Dussaix, D. Samuel. CHB-Virology and Pathology, INSERM9941-Hopital Paul Brousse, CHB, France HCV quasispecies are not randomly distributed in serum, liver and lymphocytes. Apparently specific quasispecies are detected in peripheral blood lymphocytes (PBL) raising the possibility that some quasipecies have an extra-hepatic tropism and possibly affect virus/host interactions. The relations between these lymphotropic quasispecies and the natural course of HCV is unknown. Patients: 100 patients chronically infected by HCV with a known duration of infection. 45 were liver transplanted and have recurrent infection. None had been treated by interferon. PBL and serum were obtained when liver biopsy was performed. 5'NC region, core and HVR were amplified and analyzed through SSCP. Viremia was quantified with Taqman real-time PCR. Compartmental Distribution (CD) was defined by difference of SSCP patterns between serum or PBL. Results: CD was observed in 5' non coding region (33%), core (31%) and HVR (74%). CD and number of quasispecies were more pronounced in 5' non-coding region (p < 0.01) but not in core or HVR in transplanted patients. Minimal lesions were correlated to CD in HVR and activity of hepatitis to the CD in core (p = 0.02 and p = 0.01). CD in 5'NC were inversely correlated to the duration of infection. The CD defined in core, HVR and 5'NC were not correlated to HCV genotypes or the level of viremia. Levels of viremia was correlated to the numbers of serum quasispecies defined in HVR. Conclusion: Lymphotropic HCV quasispecies are frequent during chronic hepatitis, inversely correlated to the severity and the duration of infection. These findings suggest that compartmental distribution of quasispecies in PBL is important during the course of hepatitis C both in transplanted and non-transplanted patients
- ~
NON-STRUCTURAL 5A (NS5A) PROTEIN AND HCV GENOTYPE 3a SENSITIVITY TO INTERFERON (IFN)-ALPHA THERAPY
L. Castera, M. Leroux, A. Soulier, R. Brillet, I. Lonjon, D. Dhumeaux, J.M. Pawlotsky. Virology, Hopital Henri Mondor, France Hepatitis C virus (HCV) NS5A protein mediates HCV resistance to IFN
117
alpha in vitro. However, HCV resistance to IFN in patients in vivo is multifactorial, and the role of viral proteins, if NS5A plays a role in IFN sensitivity or resistance of HCV genotype 3a. 10 patients infected with HCV genotype 3a treated with 3 to 6 MU of IFN alpha-2a tiw for 6 to 12 months were studied. 4 of them achieved a sustained HCV RNA clearance (SVRs), whereas 6 did not respond or relapsed (non-SVRs). A 567-bp fragment spanning the transcriptionnal activation domain of NS5A was amplified by PCR before treatment in all patients, and at the time of relapse in 2 non-SVRs. A 200-bp fragment containing HVR1 was studied in the same samples. Overall, a total of 500 clones (i.e. 20 to 30 clones per sample) were sequenced and extensive genetic and phylogenetic analyses were performed. Results: NS5A and HVR1 were distributed as quasispecies at any time point. Phylogenetic analyses showed no distinct clustering of the NS5A and HVR1 quasispecies variants isolated from the SVRs and the non-SVRs, respectively. The SVRs had significantly lower nucleotide sequence complexity (as assessed by the entropy, p < 0.04) than the non-SVRs in NS5A but not HVR1. In 2 non-SVR patients, a significant genetic evolution of both NS5A and HVR1 quasispecies was observed at the time of relapse. In contrast with HVR1, the rate of accumulation of synonymous mutations was significantly higher than the rate of accumulation of non synonymous mutations in NS5A. In smnmary, no specific NS5A protein sequence appears to be associated with HCV genotype 3a sensitivity or resistance to IFN alpha. These data do not support a role for HCV genotype 3a NS5A protein in sensitivity or resistance to IFN.
- ~
REAL TIME RT-PCR QUANTIFICATION: A NEW METHOD TO STUDY HEPATITIS C VIRUS ADSORPTION ON VERO CELLS
R. Germi 1, J.M. Crance 2, D. Garin 2, j. Guimet 1' C. Rothlisberger 2, J.P. Zarski 3, E. Drouet 1.1 Laboratoire de virologie, Univ J. Fourier (Faculte de Medecine-Pharmacie); 2 CRSSA Emile Parde; 3 CHU Albert MichaUon, Grenoble, France HCV binding on cells is critical for pathogenesis. We previously developed Vero and mosquito AP61 cell systems, which are able to bind and replicate HCV. To assess HCV binding on Vero cell-receptors, we developed a real time RT-PCR fluorimetric assay. (i) Virus adsorption assay: cell inoculation (2 h at 20°C) with HCV-positive plasma (genotype lb; 2 x 10 e 6 copies/mL) was followed by 6 washes. (ii) Cells and the last wash were submitted to quantification assay: real time one step RT-PCR (Light Cycler®) using forward primer (2CH = AAC TAC TGT CTI" CAC GCA GAA), reverse primer (ITS = GCG ACC CAA CAC TAC TCG GCT), TaqMan ® probe (CRSSAc = C6-Fam-AAC CCG CTC AAT GCC TGGA-Tamra). Standard curve derived from known virus concentrations have a linear relationship over 3.5 logs from 50 to 10 e 5.2 viral copies. After inoculation of 10 e 4 HCV RNA copies on 10 e 6 cells, we detected 500 copies on ceils. The last wash remained negative. Reducing inoculum amount, bound virus number decreased. Anti-HCV E2 antibodies (J. Dubuisson, Lille, France) led to 50% reduction of HCV binding (p < 0.05, quintuplicate assay). This work reports quantitative analysis of HCV cell adsorption. This technique could be used to screen ligands involved in the first step of HCV infection and to identify molecules blocking virus binding on cells.
-1 CHARACTERISATION OF INTERFERON ALFA-2A AND PEGYLATED INTERFERON ALFA-2A IN COMBINATION WITH RIBAVlRIN, MYCOPHENOLIC ACID OR VX-497 AS INHIBITORS OF HCV REPLICON REPLICATION T.C.I. Wilkinson, C. Laxton, E. Hobbs, R. Bartenschlager, M. Graves, R. Devos, I. Najera. Roche Discovery Welwyn, UK Background: The current treatment of HCV infection relies on alpha interferon alone or in combination with ribavirin. Until recently, the lack