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Analysis of genomic variability of hepatitis C virus
JournalofHepatology, 1991; 13([email protected]): S&S19
@ 1991 Elsevier Science Publishers B.V. AU tights reserved. 0168-8278/91/$03.50
S15
HEPAT 01019
H.T.M. Cuypers’, IN
Winkel’, CL. van der Poe12, H.W. Reesink2, P.N. L&e’, M. Houghton3 and A. Weiner3
ICentral Laboratory of The Netherlands Red Cross BIood Transj&on Service, Amsterdam, ‘Red Cross Blood Bank, Amsterdam. The Netherlandr and 3Chiron Corporation, Emeryville, CA, United Statesof America
--
-
The anti-ClOO-enzyme-linked immurrosorbent assay, the new four-antigen antibody recombinant immunoblot assay, and detection of viral RNA sequences by copy RNA-polymerase chain reaction were used to establish the course of hepatitis C virus (HCV) infection in recipients of I-ICY-infected blood products. Different patterns of infection were observed: (1) persistent HCV infection with and without chronic hepatitis, and with acute resolved hepatitis; and (2) acute resolved hepatitis with clearance of HCV. In order :o determine whether different infection- and anti-HCV recognition patterns are correlated to differences in viral nucleotide sequences, we compared sequences in the NS3 region between isolates from recipient(s) and their infective donors. Based on these comparisons we conclude that in The Netherlands two types of molecular variants circulate; one resembling the Japanese prototype isolate JWl, and the other the HCV-1 isolate from the U.S.A. The difference in sequence h,omology between the two types is approximately 24%. Comparison of sequences of donors and involved recipients determined in isolates prepared from blood samples four years after transfusion revealed that viral RNA sequences are strongly conserved (>96.8%) in the NS3 region. These data indicate that the observed differences in anti-HCV immune response patterns between recipients are more a reflection of their immune reactivity than of divergence of viral strains.
Hepatitis C virus (HCV) is believed to be the main cause of blood-borne non-A, non-B (NANB) hepatitis (1). HCV is classified as part of the flaviviridae family and contains a positive-stranded RNA molecule of approximately 10 000 nucleotides (nts) (2). Nearly the complete sequence of the viral genome has been reported (3,4). The virus encodes a single polypeptide chain, which is post-translationally processed to structural and non-structural mature proteins. It is known that RNA containing viruses are prone to spontaneous mutation at a mutation rate ranging from 10-1-10-4 base substitution per genome site per year ($6). Therefore, the amino acid sequence of viral proteins is subject to considerable change within a relatively short evolutionary period. To study this we establis7led the sequence heterogeneity in the NS3 region
(nts 4702-5233; Ref. 3) of the RNA isolate of one subject and between isolates from different individuals. Secondly, we determined whether sequences in this distinct region of the viral genome are altered during the course of infection ia a new host. Samples for these latter cornpar;sons were obtained from a prospective study to establish the incidence of post-transfusion hepatitis NANB (PTHNANB) conducted in the period 1984-1986 (7). Analysis of longitudinal anti-XV recognition patterns with 4-antigen (C33, C22c, Cl00 and 5-l-l) recombinant immunoblot assay (CRIBA) in sequential serum samples and detection of HCV-RNA by cDNA-PCR revealed different patterns of HCV infection in recipients (8). We will present some of the infection patterns of recipients and compare nucleotide and amino acid sequences within the NS3
Correspondence: H.T.M. Cuypers, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Piesmanlaan 125,1066 CX Amsterdam, The Netherlands.
H.T.M. CUYPERS et al.
S16 region between virus isolates of HCV-infected recipients and implicated donors.
Materials and Methods Historicaldata and definitions In a prospective study in 1984-86 to establish the frequency of PTH-NANB, 383 open-hart-surgery patients (recipients) received 5150 blood products that were prepared from 4906 blood donations of 4123 blood donors (7), Stored sequential serum samples of recipients were tested for HCV antibodies with HCV C-100 ELISA (Orthe, Raritan, U.S.A.) and 4-RIBA. Antibody reactivity in I-RIBA was assessed by comparison with a weak and strong IgG-positive control band (8). In storage experiments we observed that the method of collection of blood samples strongly influenced the results of cDNA-PCR (Ref. 8, Cuypers et al., unpublished data). In order to obtain reliable plasma samples for confirmation of HCV infection by cDNA-PCR, donors and recipients were invited to participate in a follow-up study in 1990. HCV isolates from HCV patients (see El, E5, Fig. 2) were prepared from blood samples of individuals visiting the hepatology out-patients clinic at the Academic Medical Centre, Amsterdam. Laboratory tests For cDNA-PCR, cell-free plasma in EDTA was snapfrozen in liquid nitrogen within 2 h of venepuncture and stored at -70°C. Presence of HCV-RNA in plasma sam-
TABLE 1 HCV infection patternof recipients
Donor
Recipient
Typeof infection Hepatitis
Don-l
ReC-4 Ree 8 Ret3
Persistent Persistent Persistent
~No hepatitis Acute resolved Chronic
Don-5
Ret-7
Cleared’
Acute resolved
Don-2
Ret-1
Persistent
Chronic
Schematic representation of infection patterns depicted in Fig. 1 and the implicated donors. Type of HCV infection is defined as: ‘persistent’, in the case of sero-conversion for I-RIBA antigen(s) during the observation period after HCV-infected bloo;l product transfusion and presenceof HCV-WA in 1990,and ‘cleared’, in the caseof
sem-conversionduringthe observationperiodand disappearance of HCV-aotibodiesin the period 1986-1990,together with cDNAPCR negativity in 1990. PTH-NANB is classified as ‘chronic’ when ALTconcentration was increased >Ztimes above the upper limit of n-al for >6 months after transfusion, and as ‘acute resolved’ when ALT normali& helow this value within six months (8). ’ cDNA-PCR positivein serum at ALT elevation.
collected in 1990 was retested by using a more sensitive cDNA-PCR as described (van der Poe1 et al., unpublished data); using Trimers of the highly conserved 5’ untranslated region (UTR) of the HCV genome (9). In some cases we were also able to detect viral RNA sequences in the stored serum samples, taken during and immediately af’ter the ALT peak in the study period 1984-1986. In order to compare sequences in the NS3 region, RNA was extracted, copied into cDNA by reverse transcription, and amplified by cDNA-PCR with primer S-378 and 3-36X as described (8). If necessary, amplification with a nested primer set nts 4682-4701 and 5234-5253 (antisense) was performed. These primers were extended with a recognition site for EcoRI and BamHl, respectively, in order to subclone amplified fragments into pUC18 or pWC19. Individual molecular clones were sequenced with the dideoxy termination method with standard Ml3 primers or synthetic HCV primers, using Sequenase (USB, Cleveland, OH). Sequences were compared with the program CLUSTER (10). ples from donors and recipients,
Results Different patterns of HCV infection were observed in recipients of HCV-infected blood products in the 1984-1986 PTH study. Three consecutive donations of one donor (Don-l, Table 1) infected three recipients, all revealing a different infection pattern (see Fig. 1). Recipient R4 (Fig. 1A) was persistently infected by HCV, in view of positive RIBA results in both the study period 1984-1986 and in 1990, and presence of HCV-RNA as detected by cDNA-PCR in 1990. No ALT elevation, as sign of hepatitis, was observed during the follow-up after transfusion. In the second recipient (R8, Fig. 1B) an acute resolved hepatitis was diagnosed, indicated by ALT elevation and normalization. This persistently infected recip ient sero-converted from RIBA indeterminate to positive in the period between 1986 and 1990and did not sero-convert for Cl00 antibody. The third recipient (R3, Fig. 1C) also was persistently HCV-infected, 4-RIBA-positive in 1984-1986 and in 1990, and cDNA-PCR-positive in 1990. This patient exhibited an ALT pattern of a classical PTHhepatitis, characterized by flares of hepatitis (11). Another HCV-infected donor induced an acute resolved hepatitis in recipient R7 (Fig. 1D). However, this recipient cleared the virus, as indicated by sero-conversion from 4-RIBA positivity to negativity between 1986 and 1990 and absence of HCV-RNA sequences as determined by cDNA-PCR in 1990. We were, however, able to detect HCV-RNA in serum samples collected at ALT ele-
VARIABILITY OF HEPATITIS C VIRUS
A
Sl7 R4
Recipient:
ELISA ODlCO
0
AL?
26
SO
76
1985
loo
126
160
176 200
-
C-MO
ELISA
-
Recipient:
@
2;s
-1990
-
days after tranefuslon
300 200
2
100
0
n 60
x)0
19i6
AL1
80
260 1990
260
R3
C-100ELISA
-
ALT
R7
Recipient:
ALT
ALT
WI
60
100
160
200
260
0
days after transfuekm -
C.loO
ELISA
-
100
1986
206
daya after transfusion -
ALT
”
days after transfusion -
ELlSA ODlCO
E
6 4
C-(00
ELISA
-
ALT
Rl
Recipient:
ELISA
ALT WI
ODrCO
antibody -we antibody +ve LmmmmRmno passive antibody
6
60
100
I is0
200
260
300
days after tranefueion -
C-100 ELISA
-
AL1
Fig. 1. Pattern of HCV-infection. Anti-HCV antibody response with C-100 ELISA and ALT are depicted by lines; reactivity in 4-RIBAtest results for each individual antigen by horizontal barsat top of the graphs. A vertical line left irrthe graphs indicates the time of transfusion. The horizontal line in the graphsmark the value of the upper limit of normal for ALT andagiical density reading/cut off value(OD/CO)=l for Cl00 ELISA. Results of cDNA-PCR,of plasma samples collected in 1990 are indicated.
vation in the period 1984-86, using primers from the 5’
LlTR. The third HCV-infected donor (Don-2, Table 1) caused a persistent HCV infection of the recipient (Ret-1, Fig. lD), leading to a classical PTH-hepatitis pattern, with flares of hepatitis. This recipient sero-converted to 4-
RlBA positivity during the post-transfusion observation period and was still 4-RIBA-positive in 1990. In order to determine whether the different patterns are correlated to variability of viral sequences leading to multiple strains of HCV, which may be more virulent or avirulent, we compared viral sequences in the NS3 region of recipi-
H.T.M. CUYPERS et al.
Sl8 ent(s) and implicated donors between nts 4702-5233. First, we determined the degree of sequence heterogeneity in this part of the HCV-genome within one particular isolate of an individual and between isolates from different mdividuals. From these sequence analyses we can draw the following conclusions: (1) nucleotide sequence differences within individual molecular clones from one isolate appear to be minute, 0.3% difference in 2.6 kb analyzed fragments obtained from nine individual clones of isolates from two individuals. Only one in every ten differences is non-silent; (2) analysis of nucleotide sequences from different bleedings (interval 8 months) of a persistently infected individual revealed 1.6% difference from which 1.0% were silent mutations; (3) homologies on nucleotide sequence level between isolates from different individuals vary from 76.3% up to 94.0% and on deduced amino acid sequence level from 89.8% up to 97.7%, respectively, when compared with the published prototype sequence (3). The results of the comparative study are outlined in a matrix presentation in Fig. 2. In the matrix in Fig. 2, the sequences are compared with published sequences from the prototype isolate HCV-1 (3) and from the published Japanese isolate JHl (12). A remarkable phenomenon is that HCV isolates can be clustered into two groups: El, ES and Don-5 resembling the JHl isolate (91592.5% sequence homology on nucleotide level); and Don-l and Don-2 resembling the HCV-1 isolate (homology 93.7-94.0%). The difference in homology between both groups is about 24%. Sequence analysis of seven other isolates (two donors and
Jap. JHl
A
Rec_8
R
R
1
4
8
R 3
800%
98.9%
i 98.3%
I 97.6%
100%
99 A, 4% I98.9%
97.2%
98.9%’
100% ‘97.4%’ 96.8%‘
a.-%
100% &$&
Don-l
Ret-4
D
i
97.7%
: 98.3%197.4% / 100% 98.1% @X9%,98.3%: 100% :96.0%
Ret-3
’ 97.6%
96.8%’
98.1% : 100%
97.2% 97.7% 96.0% 100%
xxx%=nucleotide
homology
W
=AA homology
B D 2 Don-2
Ret-1
R 1
100%
98.3%
x?B
g&y@
96.3%
100%
U%
Img
XX%wcleotide homology ~4.A. homology
Fig. 3. Sequence comparison in the NS3 region of recipients and implicated infective donor. Matrix comparison of nucleotide and deduced amino acid sequences in the region nts 4702-5233 (3) of donor-l and three involved recipients (A) and donor-2 and recipient-l (B). Percentage homology is depicted. Underlined numbers represent polypeptide homology.
five hepatitis C patients) from The Netherlands shows that these isolates cluster into the ‘JHI group’ (data not shown). Comparison of part of the NS3 region (nts 4702-5233), as depicted in Fig. 3, between recipient(s) and implicated donor isolates showed a strong sequence conservation. Four years after transfusion of the infected blood products all sequences were still more than 96.8% homologous on nucleotide sequence level (range 96.8-98.9%).
Pat. El Pat. ES Don-5
F&. 2. Sequence comparison in the NS3 region of five independent HCV isolates. Matrix comparison of nucleotide and deduced amino acid sequence in the region nts 4702-5233 (3) as determined by the program CLUSTER (10). Percentage homology is depicted. Sequences are compared with the prototype sequence HCV-l(3) and the Japanese isolate JHl(12). Donors 1,2 and 5 participated in the I’TH-study (see Table 1). Sequences of two hepatitis-C patients (Pat.) El and ES are inserted in matrix. Underlined numbers mark the polypeptide homology. Clustering of the two types of isolates is indicated by the thick horizontal and vertical lines.
Discussion
Data from this study show that host immune response to viral antigens and the course of liver disease in HCV-infected blood transfusion recipients are quite incompatible. From the data presented in this paper, roughly four different pat&emscan be deduced: (1) persistent HCV infection with chronic hepatitis (R3, Rl); (2) persistent infection without hepatitis (R4); (3) persistent infection with acute resolved hepatitis (R8); and (4) acute resolved
VARIABILITY OF HEPATITIS C VIRUS
hepatitis and clearance of the virus ( ). The course of imfection and disease seems to be more host-related than virus-related. We observed patterns (l)-(3) in recipients infected by blood products from the same individuai prepared from three consecutive donations within one year. Sequence comparison of the NS3 region from HCV isolates of these three recipients, collected 4 years after transfusion, revealed a strong sequence conservation, indicating that different anti-HCV recognition and disease patterns do not lead to difference in sequence variability in this part of the HCV genome. Heterogeneity in disease pattern and antibody response to Cl00 antigen was also observed in an established chimpanzee model of parenterally transmitted HCV (13) and in a follow-up study of recip ients of HCV-infected blood products (14). Nucleotide sequence comparison in the NS3 region reveals that HCV isolates from The Netherlands can be classified into two groups, class I isolates, resembling the prototype Chiron HCV-1 isolate, and class II, resembling the Japanese J isolate. In other presentations of this meeting, results have been presented which allow the same classification in two related groups, based on other regions of the HCV genome. Weiner et al. (15) clustered sequences by comparing the envelope-l region. Patient El (Fig. 2) was included in this comparison and also clustered in the class II
eferences 1 Choo Q-L, Kuo G, Weiner AJ, Gverby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 2 Choo Q-L, Weiner AJ, Overby LR, Kuo G, Houghton M, Bradley DW. Hepatitis C virus: the major causative agent of viral nonA, non-B hepatitis. Br Med Bull 1990; 46: 423-41. 3 Choo Q-L, Richman KH, Han JH, et al. Genetic organisation and diversity of the hepatitis C virus. Proc Nat1 Acad Sci USA 19%; 8% 245i-5. 4 Kato N, Hykata M, Ootsuyama Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Nat1 Acad Sci USA 1990; 87: 9524-8. 5 Holland J, Spindler K, Horodyski F, Grabau E, Nichol S, Van de Pal S. Rapid evolution of RNA genomes. Science 1982; 215: 1577-85. 6 Domingo E, Holland JJ. In: Domingo E, Holland JJ, Ahlquist P, eds. RNA Genetics, Vol. 3. Boca Raton, FL: CRC, 1990: 3-36. 7 Reesink HW, Leentvaar-Kuypers A, Van der Poe1 CL, et al. Non-A, non-B post-transfusion hepatitis in open heart surgery patients in The Netherlands: preliminary results of a prospective study. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 558-60. 8 Van der Poe1 CL, Cuypers HTM, Reesink HW, et al. Confirmadon of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991; 337: 317-,9. 9 Han JH, Shyama!a V, Richman KH, et al. Characterization of
s19
group by comparing envelope sequences. Comparison of CV-isolates in the e~velo~e-2~S~ region (Del&se et d., Ref. 16) and NS3 region (nts 2411-2902, Ref. 3) (Li e.t al., Ref. 1) revealed the same classification. From data of this study we conclude that most of the HCV isolates of individuals from The Netherlands can be classified as class II isolates (10 out of 12) whereas in France most (25 out of 30) belong to class I. The high-sequence conservation in the analyzed part of the NS3 region between HCVinfected recipients and implicated donor(s) demonstrates that sequence variability in this region is not remarkably changed after infection of a new host. At this moment we are performing analyses of other regions of the genome to determine whether sequences in other parts exhibit a higher variability as a result of change of immune pressure on the virus after infection of a new host.
We thank Qrtho Diagnostics for HCV-Cl00 ELISA kits, Dr. A. Polito for performing the 4-RIBA tests and Dr. W. Schaasberg and Mr. E. Bakker for assistance in preparing the graphs of the infection patterns, and for data management.
the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5’ untranslated region and poly(A) tails at the 3’ end. Proc Nat1Acad Sci USA 1991;88: 1711-5. 10 Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 1988; 73: 237-44. 11 Dienstag JL, Alter HJ. Non-A, non-B hepatitis: evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 12 Kubo Y, Takeuchi K, Boonmar S, et al. A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucieic Acids Res IY89; 17: 10367-72. 13 Bradley DW, Krawcrynski K, Ebert JW, et al. Parenterally transmitted non-A, non-B hepatitis: virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees. Gastroenterology 1990;99: 1054-60. 14 Alter JW, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engi J Med 1989;321: 1494-500. 15 Weiner A, Christopherson C, Hall JE, et al. Sequence variation in hepatitis C viral isolates. J Hepatoll991; 13 (Suppl. 4): S6-14. 16 Delisse AM, Descurieux M, Rutgers T, et al. Sequence analysis of the putative structuralgenes of hepatitis C virus from Japanese and European origin. J Hepatoll991; 13 (Suppl. 4): S20-3. 17 Li J-s, Tong S-p, Vitvitski L, Lepot D, Tr&o C. Evidence of two major genotypes of hepatitis C virus in France and close relatedness of the predominant one with the prototype virus. J Hepatol 1991; 13 (Suppl. 4): s33-7.
@ 1991 Elsevier Science Publishers B.V. AU tights reserved. 0168-8278/91/$03.50
S15
HEPAT 01019
H.T.M. Cuypers’, IN
Winkel’, CL. van der Poe12, H.W. Reesink2, P.N. L&e’, M. Houghton3 and A. Weiner3
ICentral Laboratory of The Netherlands Red Cross BIood Transj&on Service, Amsterdam, ‘Red Cross Blood Bank, Amsterdam. The Netherlandr and 3Chiron Corporation, Emeryville, CA, United Statesof America
--
-
The anti-ClOO-enzyme-linked immurrosorbent assay, the new four-antigen antibody recombinant immunoblot assay, and detection of viral RNA sequences by copy RNA-polymerase chain reaction were used to establish the course of hepatitis C virus (HCV) infection in recipients of I-ICY-infected blood products. Different patterns of infection were observed: (1) persistent HCV infection with and without chronic hepatitis, and with acute resolved hepatitis; and (2) acute resolved hepatitis with clearance of HCV. In order :o determine whether different infection- and anti-HCV recognition patterns are correlated to differences in viral nucleotide sequences, we compared sequences in the NS3 region between isolates from recipient(s) and their infective donors. Based on these comparisons we conclude that in The Netherlands two types of molecular variants circulate; one resembling the Japanese prototype isolate JWl, and the other the HCV-1 isolate from the U.S.A. The difference in sequence h,omology between the two types is approximately 24%. Comparison of sequences of donors and involved recipients determined in isolates prepared from blood samples four years after transfusion revealed that viral RNA sequences are strongly conserved (>96.8%) in the NS3 region. These data indicate that the observed differences in anti-HCV immune response patterns between recipients are more a reflection of their immune reactivity than of divergence of viral strains.
Hepatitis C virus (HCV) is believed to be the main cause of blood-borne non-A, non-B (NANB) hepatitis (1). HCV is classified as part of the flaviviridae family and contains a positive-stranded RNA molecule of approximately 10 000 nucleotides (nts) (2). Nearly the complete sequence of the viral genome has been reported (3,4). The virus encodes a single polypeptide chain, which is post-translationally processed to structural and non-structural mature proteins. It is known that RNA containing viruses are prone to spontaneous mutation at a mutation rate ranging from 10-1-10-4 base substitution per genome site per year ($6). Therefore, the amino acid sequence of viral proteins is subject to considerable change within a relatively short evolutionary period. To study this we establis7led the sequence heterogeneity in the NS3 region
(nts 4702-5233; Ref. 3) of the RNA isolate of one subject and between isolates from different individuals. Secondly, we determined whether sequences in this distinct region of the viral genome are altered during the course of infection ia a new host. Samples for these latter cornpar;sons were obtained from a prospective study to establish the incidence of post-transfusion hepatitis NANB (PTHNANB) conducted in the period 1984-1986 (7). Analysis of longitudinal anti-XV recognition patterns with 4-antigen (C33, C22c, Cl00 and 5-l-l) recombinant immunoblot assay (CRIBA) in sequential serum samples and detection of HCV-RNA by cDNA-PCR revealed different patterns of HCV infection in recipients (8). We will present some of the infection patterns of recipients and compare nucleotide and amino acid sequences within the NS3
Correspondence: H.T.M. Cuypers, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Piesmanlaan 125,1066 CX Amsterdam, The Netherlands.
H.T.M. CUYPERS et al.
S16 region between virus isolates of HCV-infected recipients and implicated donors.
Materials and Methods Historicaldata and definitions In a prospective study in 1984-86 to establish the frequency of PTH-NANB, 383 open-hart-surgery patients (recipients) received 5150 blood products that were prepared from 4906 blood donations of 4123 blood donors (7), Stored sequential serum samples of recipients were tested for HCV antibodies with HCV C-100 ELISA (Orthe, Raritan, U.S.A.) and 4-RIBA. Antibody reactivity in I-RIBA was assessed by comparison with a weak and strong IgG-positive control band (8). In storage experiments we observed that the method of collection of blood samples strongly influenced the results of cDNA-PCR (Ref. 8, Cuypers et al., unpublished data). In order to obtain reliable plasma samples for confirmation of HCV infection by cDNA-PCR, donors and recipients were invited to participate in a follow-up study in 1990. HCV isolates from HCV patients (see El, E5, Fig. 2) were prepared from blood samples of individuals visiting the hepatology out-patients clinic at the Academic Medical Centre, Amsterdam. Laboratory tests For cDNA-PCR, cell-free plasma in EDTA was snapfrozen in liquid nitrogen within 2 h of venepuncture and stored at -70°C. Presence of HCV-RNA in plasma sam-
TABLE 1 HCV infection patternof recipients
Donor
Recipient
Typeof infection Hepatitis
Don-l
ReC-4 Ree 8 Ret3
Persistent Persistent Persistent
~No hepatitis Acute resolved Chronic
Don-5
Ret-7
Cleared’
Acute resolved
Don-2
Ret-1
Persistent
Chronic
Schematic representation of infection patterns depicted in Fig. 1 and the implicated donors. Type of HCV infection is defined as: ‘persistent’, in the case of sero-conversion for I-RIBA antigen(s) during the observation period after HCV-infected bloo;l product transfusion and presenceof HCV-WA in 1990,and ‘cleared’, in the caseof
sem-conversionduringthe observationperiodand disappearance of HCV-aotibodiesin the period 1986-1990,together with cDNAPCR negativity in 1990. PTH-NANB is classified as ‘chronic’ when ALTconcentration was increased >Ztimes above the upper limit of n-al for >6 months after transfusion, and as ‘acute resolved’ when ALT normali& helow this value within six months (8). ’ cDNA-PCR positivein serum at ALT elevation.
collected in 1990 was retested by using a more sensitive cDNA-PCR as described (van der Poe1 et al., unpublished data); using Trimers of the highly conserved 5’ untranslated region (UTR) of the HCV genome (9). In some cases we were also able to detect viral RNA sequences in the stored serum samples, taken during and immediately af’ter the ALT peak in the study period 1984-1986. In order to compare sequences in the NS3 region, RNA was extracted, copied into cDNA by reverse transcription, and amplified by cDNA-PCR with primer S-378 and 3-36X as described (8). If necessary, amplification with a nested primer set nts 4682-4701 and 5234-5253 (antisense) was performed. These primers were extended with a recognition site for EcoRI and BamHl, respectively, in order to subclone amplified fragments into pUC18 or pWC19. Individual molecular clones were sequenced with the dideoxy termination method with standard Ml3 primers or synthetic HCV primers, using Sequenase (USB, Cleveland, OH). Sequences were compared with the program CLUSTER (10). ples from donors and recipients,
Results Different patterns of HCV infection were observed in recipients of HCV-infected blood products in the 1984-1986 PTH study. Three consecutive donations of one donor (Don-l, Table 1) infected three recipients, all revealing a different infection pattern (see Fig. 1). Recipient R4 (Fig. 1A) was persistently infected by HCV, in view of positive RIBA results in both the study period 1984-1986 and in 1990, and presence of HCV-RNA as detected by cDNA-PCR in 1990. No ALT elevation, as sign of hepatitis, was observed during the follow-up after transfusion. In the second recipient (R8, Fig. 1B) an acute resolved hepatitis was diagnosed, indicated by ALT elevation and normalization. This persistently infected recip ient sero-converted from RIBA indeterminate to positive in the period between 1986 and 1990and did not sero-convert for Cl00 antibody. The third recipient (R3, Fig. 1C) also was persistently HCV-infected, 4-RIBA-positive in 1984-1986 and in 1990, and cDNA-PCR-positive in 1990. This patient exhibited an ALT pattern of a classical PTHhepatitis, characterized by flares of hepatitis (11). Another HCV-infected donor induced an acute resolved hepatitis in recipient R7 (Fig. 1D). However, this recipient cleared the virus, as indicated by sero-conversion from 4-RIBA positivity to negativity between 1986 and 1990 and absence of HCV-RNA sequences as determined by cDNA-PCR in 1990. We were, however, able to detect HCV-RNA in serum samples collected at ALT ele-
VARIABILITY OF HEPATITIS C VIRUS
A
Sl7 R4
Recipient:
ELISA ODlCO
0
AL?
26
SO
76
1985
loo
126
160
176 200
-
C-MO
ELISA
-
Recipient:
@
2;s
-1990
-
days after tranefuslon
300 200
2
100
0
n 60
x)0
19i6
AL1
80
260 1990
260
R3
C-100ELISA
-
ALT
R7
Recipient:
ALT
ALT
WI
60
100
160
200
260
0
days after transfuekm -
C.loO
ELISA
-
100
1986
206
daya after transfusion -
ALT
”
days after transfusion -
ELlSA ODlCO
E
6 4
C-(00
ELISA
-
ALT
Rl
Recipient:
ELISA
ALT WI
ODrCO
antibody -we antibody +ve LmmmmRmno passive antibody
6
60
100
I is0
200
260
300
days after tranefueion -
C-100 ELISA
-
AL1
Fig. 1. Pattern of HCV-infection. Anti-HCV antibody response with C-100 ELISA and ALT are depicted by lines; reactivity in 4-RIBAtest results for each individual antigen by horizontal barsat top of the graphs. A vertical line left irrthe graphs indicates the time of transfusion. The horizontal line in the graphsmark the value of the upper limit of normal for ALT andagiical density reading/cut off value(OD/CO)=l for Cl00 ELISA. Results of cDNA-PCR,of plasma samples collected in 1990 are indicated.
vation in the period 1984-86, using primers from the 5’
LlTR. The third HCV-infected donor (Don-2, Table 1) caused a persistent HCV infection of the recipient (Ret-1, Fig. lD), leading to a classical PTH-hepatitis pattern, with flares of hepatitis. This recipient sero-converted to 4-
RlBA positivity during the post-transfusion observation period and was still 4-RIBA-positive in 1990. In order to determine whether the different patterns are correlated to variability of viral sequences leading to multiple strains of HCV, which may be more virulent or avirulent, we compared viral sequences in the NS3 region of recipi-
H.T.M. CUYPERS et al.
Sl8 ent(s) and implicated donors between nts 4702-5233. First, we determined the degree of sequence heterogeneity in this part of the HCV-genome within one particular isolate of an individual and between isolates from different mdividuals. From these sequence analyses we can draw the following conclusions: (1) nucleotide sequence differences within individual molecular clones from one isolate appear to be minute, 0.3% difference in 2.6 kb analyzed fragments obtained from nine individual clones of isolates from two individuals. Only one in every ten differences is non-silent; (2) analysis of nucleotide sequences from different bleedings (interval 8 months) of a persistently infected individual revealed 1.6% difference from which 1.0% were silent mutations; (3) homologies on nucleotide sequence level between isolates from different individuals vary from 76.3% up to 94.0% and on deduced amino acid sequence level from 89.8% up to 97.7%, respectively, when compared with the published prototype sequence (3). The results of the comparative study are outlined in a matrix presentation in Fig. 2. In the matrix in Fig. 2, the sequences are compared with published sequences from the prototype isolate HCV-1 (3) and from the published Japanese isolate JHl (12). A remarkable phenomenon is that HCV isolates can be clustered into two groups: El, ES and Don-5 resembling the JHl isolate (91592.5% sequence homology on nucleotide level); and Don-l and Don-2 resembling the HCV-1 isolate (homology 93.7-94.0%). The difference in homology between both groups is about 24%. Sequence analysis of seven other isolates (two donors and
Jap. JHl
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R
R
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4
8
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i 98.3%
I 97.6%
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99 A, 4% I98.9%
97.2%
98.9%’
100% ‘97.4%’ 96.8%‘
a.-%
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D
i
97.7%
: 98.3%197.4% / 100% 98.1% @X9%,98.3%: 100% :96.0%
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’ 97.6%
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97.2% 97.7% 96.0% 100%
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Fig. 3. Sequence comparison in the NS3 region of recipients and implicated infective donor. Matrix comparison of nucleotide and deduced amino acid sequences in the region nts 4702-5233 (3) of donor-l and three involved recipients (A) and donor-2 and recipient-l (B). Percentage homology is depicted. Underlined numbers represent polypeptide homology.
five hepatitis C patients) from The Netherlands shows that these isolates cluster into the ‘JHI group’ (data not shown). Comparison of part of the NS3 region (nts 4702-5233), as depicted in Fig. 3, between recipient(s) and implicated donor isolates showed a strong sequence conservation. Four years after transfusion of the infected blood products all sequences were still more than 96.8% homologous on nucleotide sequence level (range 96.8-98.9%).
Pat. El Pat. ES Don-5
F&. 2. Sequence comparison in the NS3 region of five independent HCV isolates. Matrix comparison of nucleotide and deduced amino acid sequence in the region nts 4702-5233 (3) as determined by the program CLUSTER (10). Percentage homology is depicted. Sequences are compared with the prototype sequence HCV-l(3) and the Japanese isolate JHl(12). Donors 1,2 and 5 participated in the I’TH-study (see Table 1). Sequences of two hepatitis-C patients (Pat.) El and ES are inserted in matrix. Underlined numbers mark the polypeptide homology. Clustering of the two types of isolates is indicated by the thick horizontal and vertical lines.
Discussion
Data from this study show that host immune response to viral antigens and the course of liver disease in HCV-infected blood transfusion recipients are quite incompatible. From the data presented in this paper, roughly four different pat&emscan be deduced: (1) persistent HCV infection with chronic hepatitis (R3, Rl); (2) persistent infection without hepatitis (R4); (3) persistent infection with acute resolved hepatitis (R8); and (4) acute resolved
VARIABILITY OF HEPATITIS C VIRUS
hepatitis and clearance of the virus ( ). The course of imfection and disease seems to be more host-related than virus-related. We observed patterns (l)-(3) in recipients infected by blood products from the same individuai prepared from three consecutive donations within one year. Sequence comparison of the NS3 region from HCV isolates of these three recipients, collected 4 years after transfusion, revealed a strong sequence conservation, indicating that different anti-HCV recognition and disease patterns do not lead to difference in sequence variability in this part of the HCV genome. Heterogeneity in disease pattern and antibody response to Cl00 antigen was also observed in an established chimpanzee model of parenterally transmitted HCV (13) and in a follow-up study of recip ients of HCV-infected blood products (14). Nucleotide sequence comparison in the NS3 region reveals that HCV isolates from The Netherlands can be classified into two groups, class I isolates, resembling the prototype Chiron HCV-1 isolate, and class II, resembling the Japanese J isolate. In other presentations of this meeting, results have been presented which allow the same classification in two related groups, based on other regions of the HCV genome. Weiner et al. (15) clustered sequences by comparing the envelope-l region. Patient El (Fig. 2) was included in this comparison and also clustered in the class II
eferences 1 Choo Q-L, Kuo G, Weiner AJ, Gverby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 2 Choo Q-L, Weiner AJ, Overby LR, Kuo G, Houghton M, Bradley DW. Hepatitis C virus: the major causative agent of viral nonA, non-B hepatitis. Br Med Bull 1990; 46: 423-41. 3 Choo Q-L, Richman KH, Han JH, et al. Genetic organisation and diversity of the hepatitis C virus. Proc Nat1 Acad Sci USA 19%; 8% 245i-5. 4 Kato N, Hykata M, Ootsuyama Y, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Nat1 Acad Sci USA 1990; 87: 9524-8. 5 Holland J, Spindler K, Horodyski F, Grabau E, Nichol S, Van de Pal S. Rapid evolution of RNA genomes. Science 1982; 215: 1577-85. 6 Domingo E, Holland JJ. In: Domingo E, Holland JJ, Ahlquist P, eds. RNA Genetics, Vol. 3. Boca Raton, FL: CRC, 1990: 3-36. 7 Reesink HW, Leentvaar-Kuypers A, Van der Poe1 CL, et al. Non-A, non-B post-transfusion hepatitis in open heart surgery patients in The Netherlands: preliminary results of a prospective study. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 558-60. 8 Van der Poe1 CL, Cuypers HTM, Reesink HW, et al. Confirmadon of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991; 337: 317-,9. 9 Han JH, Shyama!a V, Richman KH, et al. Characterization of
s19
group by comparing envelope sequences. Comparison of CV-isolates in the e~velo~e-2~S~ region (Del&se et d., Ref. 16) and NS3 region (nts 2411-2902, Ref. 3) (Li e.t al., Ref. 1) revealed the same classification. From data of this study we conclude that most of the HCV isolates of individuals from The Netherlands can be classified as class II isolates (10 out of 12) whereas in France most (25 out of 30) belong to class I. The high-sequence conservation in the analyzed part of the NS3 region between HCVinfected recipients and implicated donor(s) demonstrates that sequence variability in this region is not remarkably changed after infection of a new host. At this moment we are performing analyses of other regions of the genome to determine whether sequences in other parts exhibit a higher variability as a result of change of immune pressure on the virus after infection of a new host.
We thank Qrtho Diagnostics for HCV-Cl00 ELISA kits, Dr. A. Polito for performing the 4-RIBA tests and Dr. W. Schaasberg and Mr. E. Bakker for assistance in preparing the graphs of the infection patterns, and for data management.
the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5’ untranslated region and poly(A) tails at the 3’ end. Proc Nat1Acad Sci USA 1991;88: 1711-5. 10 Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 1988; 73: 237-44. 11 Dienstag JL, Alter HJ. Non-A, non-B hepatitis: evolving epidemiologic and clinical perspective. Semin Liver Dis 1986; 6: 67-81. 12 Kubo Y, Takeuchi K, Boonmar S, et al. A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucieic Acids Res IY89; 17: 10367-72. 13 Bradley DW, Krawcrynski K, Ebert JW, et al. Parenterally transmitted non-A, non-B hepatitis: virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees. Gastroenterology 1990;99: 1054-60. 14 Alter JW, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engi J Med 1989;321: 1494-500. 15 Weiner A, Christopherson C, Hall JE, et al. Sequence variation in hepatitis C viral isolates. J Hepatoll991; 13 (Suppl. 4): S6-14. 16 Delisse AM, Descurieux M, Rutgers T, et al. Sequence analysis of the putative structuralgenes of hepatitis C virus from Japanese and European origin. J Hepatoll991; 13 (Suppl. 4): S20-3. 17 Li J-s, Tong S-p, Vitvitski L, Lepot D, Tr&o C. Evidence of two major genotypes of hepatitis C virus in France and close relatedness of the predominant one with the prototype virus. J Hepatol 1991; 13 (Suppl. 4): s33-7.