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Serum Ferritin (SF) levels, HCV viremia and long-term response to interferon alfa therapy in patients with chronic hepatitis C
HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
297
AASLD
SERUM FERRITIN (SF) LEVELS, HCV VIREMIA AND LONGTERM RESPONSE TO INTERFERON ALFA THERAPY IN PATIENTS WITH CHRONIC HEPATITIS C. L Casteratr C Fourr~2, E Dussaix3~P Bedossa4r P Laurent-Puig~, C Altman :, G PeUetier', C Buffet'. Service d'H6patologie', de Medoeine Nueleaire', de Virologic" and d'Anatomopathologie4, CHU de Bic~tre, Le KremlinBic~tre, France.
ABSTRACTS
298 SEQUENCE ANALYSIS OF A NEW RNA VIRUS (HEPATITIS G VIRUS, HGV) REVEALS A UNIQUE VIRUS IN THE FLAVIVIRIDAE FAMILY. KE Fry, J Linnen, Z-Y Zhang-Keck, K Fun9, C Hoover, J Fernandez JP Kim. GenelabsTechnologies, Inc., Redwood City, CA. The complete nucleotide sequence of a novel blood borne virus associated with non-A-E hepatitis in humans, provisionally designated Hepatitis G Virus, has been determined. The programs BLASTN, TBLASTN, BLASTX, BLASTP, FASTA and TFASTA were used to search Genbank (ver. 88), PIR (ver. 43) and Swiss-Prot (ver. 31) databanks for sequences related or identical to the HGV sequence. No closely related sequences were found, however, limited homology was observed with the non-structural genes of HCV. Computer analysis of these sequences revealed conservation of motifs found in members of the Flaviviridae family of viruses. These include a helicase motif, two protease motifs and an RNA dependent RNA polymerase motif. These analyses confirm experimental data that indicate the virus is a positive stranded RNA virus. Primary sequence similarity analysis with other members of the Flaviviridae family revealed that the closesti related viruses to HGV are other flavilike viruses associated with hepatitis in humans and animals, HCV and the recently described GBV-A and GBV-B viruses. The nonstructural, genes of these viruses are related to HGV; the predicted RNA dependent RNA polymerasa (NS5) of HGV is 44% identical to GBV-A, 23% identical to HCV and 25% identical to GBV-B. In contrast, the primary sequence similarity of the predicted HGV structural genes shows virtually no conservation of primary sequence relative to HCV and GBV-B and only limited similarity with GBV=A. Nucleotide sequence similarity comparison of the 5' and 3' untranslated regions of HGV to the other viruses also shows that these important control regions are unique to each member of this group of viruses. In conclusion, HGV, which is found in human blood and is associated with hepatitis, represents a novel virus in the family Flaviviridae.
It has been recently reported that serum iron status and hepatic iron content correlate with response to interferon alfa (IFN-a) therapy in patients chronically infected with hepatitis C virus (HCV). In order to look for a relationship between pretreatment serum iron status and long-term response to IFN-a, we evaluated SF levels in 70 consecutive patients (M/F 43/27; median age 41yrs, range 20-70) with HCV-positive chronic active hepatitis (85,7%) or liver cirrhosis (14,3%) before standard treatment with IFN-a (3 MU three times weekly). Patients with a history of alcoholism or hemodialysis were excluded. HCV-RNA pretreatment levels (branched DNA assay) and genotype (Innolipa, Innognetics) were determined in 46 and 28 patients respectively. Forty-three patients (61,5%) normalized ALT during IFN-a treatment and were Considered as short-term responders (SR). Ten patients (14%) were long-term responders (LR). There were no significant differences with regard to sex, serum ALT pretreatment levels, source of contamination and liver histology between SR, LR and nonresponders (NR). A~e, SF and HCV-RNA levels are expressed as M+SD in the table. NR
SR
LR
Age (yrs) 50+12 35+12" 40_+11 ** Ferritin (ng/ml) 485+487 176+164" 181+132"* RNA levels (Eq/mlxl05) 116+87 43+47* 18_+21 * * Genotype (1 b/others) 813 3/11"* 1/3 • p<.01 vs NR; ** p<.05 vs NR There were no significant differences for SF and HCV-RNA levels between SR and LR. All patients with SF > 650 ng/ml were NR. SF did not correlate neither with pretreatment HCV-RNA levels, liver histology or ALT. In logistic discriminant analysis SF and pretreatment HCV-RNA levels were independant predictive factors for poor response to IFN-~. Our study shows that high pretreatment SF and HCV-RNA levels are associated with poor short and long-term responses to IFN-a in patients with chronic hepatitis C. However SF does not discriminate between shortterm and long-term responders.
299 GENETIC ORGANIZATION AND SEQUENCE VARIATION OF THE HEPATITIS G VIRUS (HGV). JM Linnen, Z-Y Zhanq-Keck, K Funo. J Waoes. C Hoover. M Piatak. J Fernandez. D Mo, SK Founq, KE Fry, JP Kim. Genelabs Technologies, Inc., Redwood City, CA 94063 The Hepatitis G Virus (HGV) is a newly discovered, parenterally transmitted RNAvirus associated with liver disease in humans. W e report here the nearly complete nucleotide sequences of viral isolates from two patients, PNF2161 and JC. Sequence analysis suggests that the HGV genome is about 9400 nt in length and encodes a polyprotein of about 3000 amino acids which includes the characteristically conserved sequence motifs of positive-strand RNA viruses. The HGV genomic organiZation is similar t o that of viruses in the Flaviviridae family with the putative structural genes found at the 5'-end of the genome followed by the putative nonstructural genes. The amino acid sequences of the PNF2161 and JC isolates are 97% identical, whereas the nucleotide sequences are about 90% identical: To further assess the sequence variation among different isolates of HGV, we used RT-PCR to obtain genome fragments from the sera of patients from widely distributed geographic regions. Multiple sequence alignments of nearly forty 700 nt long sequences demonstrated that the 5' untranslated regions are highly conserved while the regions encoding the putative structural proteins vary significantly. The identification of conserved sequences of the HGV genome has important implications for the development of both nucleic acid- and protein-based diagnostic assays.
181A
300
PROTEOLYTIC PROCESSING OF THE POLYPROTEIN OF A NEWLY DISCOVERED HUMAN RNA VIRUS ASSOCIATED WITH HEPATITIS. YF Zhan~,. Z-Y Zhan~-Keek. J Linnen. K Fun~. MY Lim. CC Huang, A. Belvaev. PO Yarbom,h. J Lifson'. and JP Kin'l. Genelabs Technologies, Inc., 505 Penobscot Dr., Redwood City, CA94063 A recently identified RNA virus, provisionallydesignated hepatitis G virus (HGV), has been linked to acute and chronic hepatitis, including posttransfusion hepatitis. Sequence analysis demonstrated that the viral genome encoded a single continuons open reading frame (OR.F) of nearly 3,000 amino acids and contained several highly conserved viral seqnence motifs common to the non-structural regions of viruses of the Flaviviridae family, although global sequence identities to individual members of the Flaviviridae were <30%. Among members of the Flaviviridae, the viral proteins are initially expressed as a single polyprotein from translation of a continuous ORF in the viral genome; proteolytie processing by vitally encoded and host cell derived proteases yields the individual viral protein necessary to complete the viral life cycle. To study the polyprutein processing in HGV, a eDNA clone of the full length HGV ORF and various portions of it were expressed using vaeeinia virus (VV). BS-C-1 cells were infected with the recombinant W and metabolically labeled with [3sS]-methionine. Cell lysates were analyzed by radioimmunoprecipitation analysis (RIPA) and SDS-PAGE, using rabbit polyclonal antisera generated against synthetic peptides or E coil expressed recombinant proteins of HGV. Three antisera, raised against the putative NS2, NS3, and NS5 regions of HGV were immunoreactive by RIPA with HGV proteins of approximately 20, 67, and 50kDa, respectively, expressed by cells infected with a recombinant VV encoding the full length HGV ORF. These results are consistent with expression and cleavage of the full length HGV polyprutein, similar to that of other members of Flaviviridae. Additional studies will confirm the precise processing site used, and clarify the involvement of viral and host cell proteases in specific cleavages. These result will help to clarify the biology of HGV infection at the cellular level, and may provide insight into the development of rationally designed antiviral compounds for treatment of HGV infection.
297
AASLD
SERUM FERRITIN (SF) LEVELS, HCV VIREMIA AND LONGTERM RESPONSE TO INTERFERON ALFA THERAPY IN PATIENTS WITH CHRONIC HEPATITIS C. L Casteratr C Fourr~2, E Dussaix3~P Bedossa4r P Laurent-Puig~, C Altman :, G PeUetier', C Buffet'. Service d'H6patologie', de Medoeine Nueleaire', de Virologic" and d'Anatomopathologie4, CHU de Bic~tre, Le KremlinBic~tre, France.
ABSTRACTS
298 SEQUENCE ANALYSIS OF A NEW RNA VIRUS (HEPATITIS G VIRUS, HGV) REVEALS A UNIQUE VIRUS IN THE FLAVIVIRIDAE FAMILY. KE Fry, J Linnen, Z-Y Zhang-Keck, K Fun9, C Hoover, J Fernandez JP Kim. GenelabsTechnologies, Inc., Redwood City, CA. The complete nucleotide sequence of a novel blood borne virus associated with non-A-E hepatitis in humans, provisionally designated Hepatitis G Virus, has been determined. The programs BLASTN, TBLASTN, BLASTX, BLASTP, FASTA and TFASTA were used to search Genbank (ver. 88), PIR (ver. 43) and Swiss-Prot (ver. 31) databanks for sequences related or identical to the HGV sequence. No closely related sequences were found, however, limited homology was observed with the non-structural genes of HCV. Computer analysis of these sequences revealed conservation of motifs found in members of the Flaviviridae family of viruses. These include a helicase motif, two protease motifs and an RNA dependent RNA polymerase motif. These analyses confirm experimental data that indicate the virus is a positive stranded RNA virus. Primary sequence similarity analysis with other members of the Flaviviridae family revealed that the closesti related viruses to HGV are other flavilike viruses associated with hepatitis in humans and animals, HCV and the recently described GBV-A and GBV-B viruses. The nonstructural, genes of these viruses are related to HGV; the predicted RNA dependent RNA polymerasa (NS5) of HGV is 44% identical to GBV-A, 23% identical to HCV and 25% identical to GBV-B. In contrast, the primary sequence similarity of the predicted HGV structural genes shows virtually no conservation of primary sequence relative to HCV and GBV-B and only limited similarity with GBV=A. Nucleotide sequence similarity comparison of the 5' and 3' untranslated regions of HGV to the other viruses also shows that these important control regions are unique to each member of this group of viruses. In conclusion, HGV, which is found in human blood and is associated with hepatitis, represents a novel virus in the family Flaviviridae.
It has been recently reported that serum iron status and hepatic iron content correlate with response to interferon alfa (IFN-a) therapy in patients chronically infected with hepatitis C virus (HCV). In order to look for a relationship between pretreatment serum iron status and long-term response to IFN-a, we evaluated SF levels in 70 consecutive patients (M/F 43/27; median age 41yrs, range 20-70) with HCV-positive chronic active hepatitis (85,7%) or liver cirrhosis (14,3%) before standard treatment with IFN-a (3 MU three times weekly). Patients with a history of alcoholism or hemodialysis were excluded. HCV-RNA pretreatment levels (branched DNA assay) and genotype (Innolipa, Innognetics) were determined in 46 and 28 patients respectively. Forty-three patients (61,5%) normalized ALT during IFN-a treatment and were Considered as short-term responders (SR). Ten patients (14%) were long-term responders (LR). There were no significant differences with regard to sex, serum ALT pretreatment levels, source of contamination and liver histology between SR, LR and nonresponders (NR). A~e, SF and HCV-RNA levels are expressed as M+SD in the table. NR
SR
LR
Age (yrs) 50+12 35+12" 40_+11 ** Ferritin (ng/ml) 485+487 176+164" 181+132"* RNA levels (Eq/mlxl05) 116+87 43+47* 18_+21 * * Genotype (1 b/others) 813 3/11"* 1/3 • p<.01 vs NR; ** p<.05 vs NR There were no significant differences for SF and HCV-RNA levels between SR and LR. All patients with SF > 650 ng/ml were NR. SF did not correlate neither with pretreatment HCV-RNA levels, liver histology or ALT. In logistic discriminant analysis SF and pretreatment HCV-RNA levels were independant predictive factors for poor response to IFN-~. Our study shows that high pretreatment SF and HCV-RNA levels are associated with poor short and long-term responses to IFN-a in patients with chronic hepatitis C. However SF does not discriminate between shortterm and long-term responders.
299 GENETIC ORGANIZATION AND SEQUENCE VARIATION OF THE HEPATITIS G VIRUS (HGV). JM Linnen, Z-Y Zhanq-Keck, K Funo. J Waoes. C Hoover. M Piatak. J Fernandez. D Mo, SK Founq, KE Fry, JP Kim. Genelabs Technologies, Inc., Redwood City, CA 94063 The Hepatitis G Virus (HGV) is a newly discovered, parenterally transmitted RNAvirus associated with liver disease in humans. W e report here the nearly complete nucleotide sequences of viral isolates from two patients, PNF2161 and JC. Sequence analysis suggests that the HGV genome is about 9400 nt in length and encodes a polyprotein of about 3000 amino acids which includes the characteristically conserved sequence motifs of positive-strand RNA viruses. The HGV genomic organiZation is similar t o that of viruses in the Flaviviridae family with the putative structural genes found at the 5'-end of the genome followed by the putative nonstructural genes. The amino acid sequences of the PNF2161 and JC isolates are 97% identical, whereas the nucleotide sequences are about 90% identical: To further assess the sequence variation among different isolates of HGV, we used RT-PCR to obtain genome fragments from the sera of patients from widely distributed geographic regions. Multiple sequence alignments of nearly forty 700 nt long sequences demonstrated that the 5' untranslated regions are highly conserved while the regions encoding the putative structural proteins vary significantly. The identification of conserved sequences of the HGV genome has important implications for the development of both nucleic acid- and protein-based diagnostic assays.
181A
300
PROTEOLYTIC PROCESSING OF THE POLYPROTEIN OF A NEWLY DISCOVERED HUMAN RNA VIRUS ASSOCIATED WITH HEPATITIS. YF Zhan~,. Z-Y Zhan~-Keek. J Linnen. K Fun~. MY Lim. CC Huang, A. Belvaev. PO Yarbom,h. J Lifson'. and JP Kin'l. Genelabs Technologies, Inc., 505 Penobscot Dr., Redwood City, CA94063 A recently identified RNA virus, provisionallydesignated hepatitis G virus (HGV), has been linked to acute and chronic hepatitis, including posttransfusion hepatitis. Sequence analysis demonstrated that the viral genome encoded a single continuons open reading frame (OR.F) of nearly 3,000 amino acids and contained several highly conserved viral seqnence motifs common to the non-structural regions of viruses of the Flaviviridae family, although global sequence identities to individual members of the Flaviviridae were <30%. Among members of the Flaviviridae, the viral proteins are initially expressed as a single polyprotein from translation of a continuous ORF in the viral genome; proteolytie processing by vitally encoded and host cell derived proteases yields the individual viral protein necessary to complete the viral life cycle. To study the polyprutein processing in HGV, a eDNA clone of the full length HGV ORF and various portions of it were expressed using vaeeinia virus (VV). BS-C-1 cells were infected with the recombinant W and metabolically labeled with [3sS]-methionine. Cell lysates were analyzed by radioimmunoprecipitation analysis (RIPA) and SDS-PAGE, using rabbit polyclonal antisera generated against synthetic peptides or E coil expressed recombinant proteins of HGV. Three antisera, raised against the putative NS2, NS3, and NS5 regions of HGV were immunoreactive by RIPA with HGV proteins of approximately 20, 67, and 50kDa, respectively, expressed by cells infected with a recombinant VV encoding the full length HGV ORF. These results are consistent with expression and cleavage of the full length HGV polyprutein, similar to that of other members of Flaviviridae. Additional studies will confirm the precise processing site used, and clarify the involvement of viral and host cell proteases in specific cleavages. These result will help to clarify the biology of HGV infection at the cellular level, and may provide insight into the development of rationally designed antiviral compounds for treatment of HGV infection.