Hepatitis C viral quasispecies in hepatitis C virus carriers with normal liver enzymes and patients with type C chronic liver disease

Hepatitis C Viral Quasispecies in Hepatitis C Virus Carriers With Normal Liver Enzymes and Patients With Type C Chronic Liver Disease MASAFUMI NAITO,1 NORIO HAYASHI,1 TOYOKI MORIBE,2 HIDEKI HAGIWARA,1 EIJI MITA, ~ YOSHIYUKI KANAZAWA,1 AKINORI KASAHARA,1 HIDEYUKI FUSAMOTO,~ AND TAKENOBU KAMADA~

Hepatitis C virus (HCV) h a s b e e n r e p o r t e d to c o n f o r m to a q u a s i s p e c i e s nature, w h i c h is m o s t e v i d e n t in hypervariable r e g i o n s of t h e p u t a t i v e e n v e l o p e 2 domain. The a i m of this study w a s to d e t e r m i n e the r e l a t i o n s h i p bet w e e n t h e n u c l e o t i d e c o m p l e x i t y a n d diversity o f hypervariable r e g i o n I a n d v a r i o u s stages o f t h e carrier states. The subjects s t u d i e d w e r e 20 HCV carriers w i t h n o r m a l a l a n i n e a m i n o t r a n s f e r a s e (ALT) levels, 50 p a t i e n t s w i t h c h r o n i c hepatitis w h o s h o w e d e l e v a t e d ALT levels, 22 w i t h cirrhosis, a n d 24 w i t h h e p a t o c e l l u l a r c a r c i n o m a . The q u a s i s p e c i e s c o m p l e x i t y w a s a n a l y z e d by m e a n s of p o l y m e r a s e c h a i n r e a c t i o n - m e d i a t e d single strand conf o r m a t i o n p o l y m o r p h i s m (PCR-SSCP). The v a l u e o f nucleotide diversity w a s c a l c u l a t e d by P C R c l o n i n g a n d s e q u e n c i n g . The n u m b e r o f SSCP b a n d s r a n g e d f r o m 1 to 7, w i t h n o significant differences in t h e m e a n n u m b e r s a m o n g t h e stages o f HCV infection. There w a s n o correlation b e t w e e n t h e a m o u n t s o f s e r u m HCV R N A a n d the n u m b e r s o f SSCP bands. N o significant difference w a s f o u n d in t h e v a l u e s o f n u c l e o t i d e diversity b e t w e e n carriers w i t h n o r m a l ALT levels (mean, 6.6 × 1 0 -2 per site) a n d p a t i e n t s w i t h c h r o n i c hepatitis (7.7 × 10 2). T h e s e findings suggest that t h e q u a s i s p e c i e s c o m p l e x i t y o f hypervariable r e g i o n 1 is i n d e p e n d e n t of t h e stage of c h r o n i c HCV infection. (HEPATOLOGY 1995;22:407-412.)

Hepatitis C virus (HCV) commonly causes persistent infection, and chronic HCV infection can lead to serious consequences, including cirrhosis and hepatocellular carcinoma (HCC). 1'2 Using competitive reverse tranAbbreviations: HCV, hepatitis C virus; HCC, hepatocellular carcinoma; RTPCR, reverse transcription-polymerase chain reaction; HVR, hypervariable region; E2, envelope 2; NSl, nonstructural protein 1; IFN, interferon; PCR-SSCP, PCR-mediated single strand conformation polymorphism; HIV, human immunodeficiency virus; ALT, alanine aminotransferase; ELISA, enzyme linked immunosorbent assay; HBsAG, hepatitis B surface antigen; nt, nucleotide positions; tRNA, transfer RNA; cDNA, complementary DNA; Sl, sense primer. From 1the First Department of Medicine, Osaka University School of Medicine, Suita, Osaka; and 2Diagnostic Science Department, Shionogi Biomedical Laboratories, Shionogi & Co. Ltd., Settsu, Osaka, Japan. Received December 8, 1994; accepted April 6, 1995. Supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan and the Research Group of Intractable Hepatitis, sponsored by the Ministry of Health and Welfare of Japan. Address reprint requests to: Norio Hayashi, MD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2202-000553.00/0

scription-polymerase chain reaction (RT-PCR) assay, we found that there seem to be "healthy carriers" of HCV with extremely low levels of viral replication and that higher levels of replication occur in patients with advanced liver disease. 3'4 Thus, elevation of viral replication may be related to liver injury and progression of liver disease in chronic HCV infection. The mechanisms responsible for HCV persistence are not well understood, although it is likely that both virus-induced and immunologically mediated mechanisms play important roles in them. Most RNA viruses are thought to consist of heterogeneous mixtures of different but closely related genomes resulting from high error rates in RNA replication; this is known as a quasispecies n a t u r e Y Many biological features of these viruses, including persistent infection, 7-9 are attributable to this nature. HCV, which has a hypervariable region (HVR) in the putative envelope 2 (E2)/nonstructural protein 1 (NS1), 1°-12 conforms to this model. 13 Using conventional sequence analysis, we have found the degree of nucleotide diversity of HCV HVR1 to be closely correlated with the responsiveness to interferon (IFN) therapy in chronic hepatitis C patients. 14 Also, using PCR-mediated single strand conformation polymorphism (PCR-SSCP) analysis, 15'1~ which is a method for detecting DNA sequence differences and estimating the degree of sequence complexity, we recently found that the complexity of the HVR1 quasispecies might be a factor for predicting IFN inefficacy in patients with chronic hepatitis C and that this method was suitable for evaluating the complexity in multiple samples. 17 In h u m a n immunodeficiency virus (HIV), the heterogeneity of HVRs is thought to increase gradually during infection to eventually lead to immunodeficiency disease after a long and variable incubation period. TM However, there have been no reports about the quasispecies nature in the natural history of chronic HCV infection, and it is still not known how this nature is associated with the progression of liver disease. In this study, therefore, we applied PCR-SSCP analysis to estimate the degree of complexity of the HVR1 quasispecies, which usually correlates with the degree of nucleotide diversity, and investigated the relationship between the complexity and various stages of the car-

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TABLE 1. Age, ALT Levels, HCV RNA Titers, and Genome Subtypes in Various Stages of Chronic HCV Infection Patients

No. of Patients

Carrier with normal ALT Chronic hepatitis Cirrhosis Hepatocellular carcinoma

20 50 22 24

Age* (yr)

47.2 48.3 58.3 64.6

ALT* (IU/L)

_+ 12.35 _+ 10.55 ÷ 7.2§ _+ 7.0

20.6 151.6 89.2 78.7

_+ 6.41r _+ 104.7# _+ 47.3 _+ 48.5

RNA Titer*t

6.3 7.9 7.9 7.7

_+ 1.1¶ _+ 0.8 _+ 0.7 ÷ 0.7

Genome Subtype lb/2ed2b/Mixed

17/1/0/2 40/5/3/2 18/2/0/2 22/2/0/0

* Data expressed as mean _+ SD. t HCV RNA titer was defined as lOglo (copies per milliliter of serum). $ P < .001 vs. cirrhosis; P < .0001 vs. HCC. § P < .05 vs. HCC. IIP < .0001 vs. chronic hepatitis, cirrhosis, and HCC. # P < .005 vs. cirrhosis and HCC. ¶ P < .0001 vs. chronic hepatitis and cirrhosis; P < .001 vs. HCC.

rier states including 'asymptomatic' carriers with normal alanine aminotransferase (ALT) levels. Furthermore, we compared the nucleotide diversity of HVR1 estimated by conventional sequence analysis between selected HCV carriers with normal ALT levels and chronic hepatitis C patients. PATIENTS AND METHODS

Patients. The subjects studied were 20 HCV carriers (7 men and 13 women) with p e r s i s t e n t l y n o r m a l ALT levels for at least 6 m o n t h s a n d 96 p a t i e n t s (63 m e n a n d 33 women) who showed elevated s e r u m ALT levels for at l e a s t 6 months. These subjects were chosen because HCV RNA was detected in t h e i r s e r a by RT-PCR a s s a y both for the 5 ' - u n t r a n s l a t e d region and the E2/NS1 region, including HVRs. 1<17 All subjects were positive for anti-HCV by second generation enzyme linked i m m u n o s o r b e n t a s s a y (ELISA). H e p a t i t i s B virus infection was r u l e d out with commercial serological tests (absence of detectable h e p a t i t i s B surface a n t i g e n [HBsAg] a n d high t i t e r of antibody to h e p a t i t i s B core a n t i g e n [HBcAg]). Liver biopsy was performed for all HCV carriers with n o r m a l ALT levels, all 50 p a t i e n t s with chronic hepatitis, and 13 p a t i e n t s with cirrhosis. The other 9 p a t i e n t s with cirrhosis and all 24 p a t i e n t s with HCC were diagnosed on the basis of l a b o r a t o r y d a t a a n d diagnostic imaging. The age of the 116 p a t i e n t s r a n g e d from 17 to 79 (mean 53.0 + 11.8 years). Thirty-four p a t i e n t s h a d histories of blood transfusion. The HCV genome subtype was d e t e r m i n e d as described previously. 19,20HCV RNA in s e r u m was quantified by competitive RT-PCR assay, and the t i t e r of HCV RNA was defined as loglo (HCV RNA copies per mL serum). 3'21 Clinical a n d virological b a c k g r o u n d s of the p a t i e n t s studied are s u m m a rized in Table 1. The m e a n t i t e r of HCV RNA of carriers with n o r m a l ALT levels was significantly lower t h a n t h a t for chronic hepatitis, cirrhosis (P < .0001), a n d HCC (P < .001). Oligonucleotides. The p r i m e r s used for the RT and the successive PCR for isolating a portion of the E2/NS1 domain including HVR1 were as follows: (1) p r i m e r s for RT and the first r o u n d PCR: S 1 (5'-TGGCTTGGGATATGATGATGAAC3', sense, nucleotide positions (nt) 1277-1299 of HC-J422) and A1 (5'-GGGGTGAAGCAaTACACTGGaCCaCA-3', antisense, n t 1831-1856 of HC-J4)/A2 (5'-GGGGTGAAGCAgTACACTGGgCCgCA-3', anti-sense, n t 1839-1864 of BK23); (2) p r i m e r s for the second round PCR: $2 (5'-TGGGATATGATGATCAACTGGTC-3', sense, based on nt 1282-1304 of HC-J4) a n d A3 (5'-GTGAAGGAATTCACTGGaCCaCACAC3', anti-sense, based on nt 1828-1853 of HC-J4)/A4 (5'-GTG-

AAGGAATTCACTGGgCCgCACAC-3', anti-sense, based on nt 1836-1861 of BK) (A1/A2 and A3/A4 were produced by mixing residues in the p a r t s indicated by small letters); and (3) p r i m e r s for the t h i r d round PCR and S S C P analysis: HS (5'-GCCTTGCCTACTATTCCATG-3', sense, n t 1405-1424 of HC-J4) and HA (5'-TTGATGTGCCAACTGCCATT-3', antisense, based on n t 1581-1600 of HC-J4). Amplification of HCV El-E2 Region by RT-PCR. The HCV E l - E 2 region was amplified by the RT-PCR a s s a y as recently described in detail. TM For extraction of RNA, 20 p L of serum sample was mixed well with 400 #L of g u a n i d i n i u m homogenization buffer. After addition of 10 #g of t r a n s f e r (t) RNA (Sigma Chemical Co., St. Louis, MO), the solution was ext r a c t e d twice with phenol/chloroform and p r e c i p i t a t e d with isopropanol. The RNA pellet was reverse t r a n s c r i b e d to comp l e m e n t a r y (c) DNA using 20 pmol of the anti-sense p r i m e r (A1/A2) a n d 100 units of Molony m u r i n e l e u k e m i a virus RT (Bethesda Research Laboratories, G a i t h e r s b u r g , MD) in a final volume of 20 #L. Five microliters of the r e s u l t i n g cDNA was a d d e d to PCR solution containing 5 pmol of the sense p r i m e r (S1) a n d 0.625 unit of Taq polymerase (Perkin E l m e r Cetus Corp., Norwalk, CT) in a final volume of 25 #L, a n d amplified in a DNA t h e r m a l cycler (Perkin E l m e r Cetus Corp.) by 40 cycles of PCR (94°C for 1 minute, 55°C for 2 minutes, 72°C for 1 minute). One microliter of the products was used for the second round PCR using nested p r i m e r s (5 pmol each of A3/A4 and $2) with the same p a r a m e t e r s and conditions as for the first round PCR. The t h i r d amplification was also carried out using S S C P p r i m e r s (5 pmol each of HA and HS) with the s a m e p a r a m e t e r s and conditions. The t h i r d PCR products were subjected to electrophoresis, a n d the predicted length (196 base pairs) was confirmed. PCR-SSCPAnalysis. We performed PCR-SSCP analysis as recently described in detail. 17 Briefly, one microliter of the 10-fold d i l u t e d - s e c o n d PCR products was a d d e d to a PCR solution comprised of 0.5 pmol of each 5'-end labeled SSCP p r i m e r and 20 mmol/L deoxynucleotide triphosphate. The amplification was carried out with 0.25 unit of Taq DNA polymerase with the s a m e p a r a m e t e r s as for the t h i r d PCR described above. One microliter of the PCR products was den a t u r e d at 95°C for 10 m i n u t e s in 49 #L of d e n a t u r a t i o n solution. The d e n a t u r e d products were chilled on ice w a t e r for 10 m i n u t e s and t h e n 2 #L was applied to a 5% polyacryla m i d e gel (acrylamide:N,N'-bisacrylamide, 49:1). After electrophoresis at 25°C for 1 hour at a c o n s t a n t power of 30 W using a Macrophor sequencing system ( P h a r m a c i a LKB Biotechnology AB., Uppsala, Sweden), the gel was dried on

HEPATOLOGYVol. 22, No. 2, 1995

NAITO ET AL

409

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FIG. 1. Number of SSCP bands in various stages of chronic HCV infection• Closed circles (O), open circles (©), and open squares ([~) denote patients with genome subtype lb, 2a, and 2b, respectively. × denotes patients with mixed subtype. No significant differences were found in the mean numbers of SSCP bands among each stage of chronic HCV infection.

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a glass board a n d t h e n exposed to Fuji X-ray film (Fuji Film Co., Tokyo, J a p a n ) for 20 hours at room temperature. The n u m b e r of noted b a n d s was counted. Cloning and Sequencing. The second PCR product was purifled a n d cleaved with restriction endonucleases Bcl I a n d EcoRI (Toyobo Co., Ltd., Osaka, Japan). The digested fragm e n t s were cloned into EcoRUBamHI (Toyobo Co., Ltd.)-digested plasmid vector pUC 19. Eight to 12 i n d e p e n d e n t rec o m b i n a n t clones were selected from each sample. Both plus and m i n u s s t r a n d s of their clones were sequenced by the dideoxy chain t e r m i n a t i o n method u s i n g the Taq Dye P r i m e r Cycle Sequencing Kit (Applied Biosystems, Inc., Foster, CA) on either the Applied Biosystem Model 373A or Model 370A DNA Sequencing System (Applied Biosystems, Inc.). The sequencing was carried out according to the m a n u f a c t u r e r ' s instructions. Nucleotide diversity 24 in each set of sequences was calcu-

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RESULTS SSCP Analysis. The complexity of HVR1 quasispecies in the s e r u m of 116 p a t i e n t s with chronic H C V infection was a n a l y z e d by m e a n s of S S C P a n d exp r e s s e d as the n u m b e r of observed bands. The n u m b e r of S S C P b a n d s at e v e r y stage of chronic H C V infection, r a n g i n g from 1 to 7, was plotted in Fig. 1. T h e r e were

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410

NAITO ET AL

HEPATOLOGY A u g u s t 1995

TABLE 2. Comparison of Clinical and Virological Characteristics B e t w e e n HCV Carriers With N o r m a l A L T L e v e l s and Chronic Hepatitis Patients With E l e v a t e d A L T L e v e l s No. of

Patients

Patients

Age* (yr)

ALT* (IU/L)

RNA Titer*'t

Carrier with normal ALT Chronic hepatitis

6 8

41.8 ± 9.1 49.4 _+ 9.2

25.3 _+ 5.35 147.8 i 116.8

6.3 _+ 1.3§ 8.1 _+ 0.6

Genome Subtype

No. of SSCP

lb/2a/2b/Mixed

Bands*

ND*

5/1/0/0 5/3/0/0

2.3 _+ 0.8 2.0 ± 0.9

6.6 ± 4.5 7.7 _+ 7.1

Abbreviation: ND, nucleotide d i v e r s i t y ( × 1 0 2 p e r site) * D a t a e x p r e s s e d as m e a n _+ SD. T i t e r of H C V R N A w a s defined as log10 (copies p e r milliliter of s e r u m ) . $ P < .05 vs. chronic h e p a t i t i s . § P < .005 vs. chronic h e p a t i t i s .

no significant differences in the mean numbers of SSCP bands among the stages of HCV infection; 2.7 _ 1.2 for carriers with normal ALT levels, 2.7 ± 1.2 for patients with chronic hepatitis, 3.0 ± 1.5 for patients with cirrhosis, and 2.4 ± 1.5 for patients with HCC. Also, no significant differences were found among the stages in the patients infected with subtype lb, which is the major subtype in Japan. There was no correlation (r = .06) between the amounts of serum HCV RNA and the numbers of SSCP bands (Fig. 2). In patients with a small amount of serum HCV RNA in whom the RNA titers were less t h a n 6.0, the numbers of SSCP bands ranged from 1 to 4. In patients in whom the HCV RNA titers were more t h a n 6.5, the range was wider, from 1 to 7. Also, no significant correlation (r = .07) was found between age and the numbers of SSCP bands. Furthermore, in 34 patients with histories of blood transfusion, no significant correlation (r = .27) was found between the numbers of SSCP bands and the time elapsed after blood transfusion. Comparison of HCV Carriers with Normal ALT and

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FIG. 3. P r e d i c t e d a m i n o acid s e q u e n c e s of HVR1 from H C V c a r r i e r s w i t h n o r m a l A L T levels. E i g h t to 12 a m i n o acid s e q u e n c e s from e a c h p a t i e n t are s h o w n usi n g one-letter coding a n d t h e s e s e t s of s e q u e n c e s a r e a r r a n g e d f r o m p a t i e n t No. 1 to 6. D a s h e s (- - -) indicate s e q u e n c e i d e n t i t y to t h e top s e q u e n c e s , a s t e r i s k s (*) denote deletions. T h e v a l u e s of nucleotide d i v e r s i t y (N.D.) in HVR1 a r e s h o w n ( x l 0 2 per site) from c a l c u l a t i o n as t h e a v e r a g e n u m b e r of nucleotide differences per site b e t w e e n two r a n d o m l y chosen sequences.

HEPATOLOGYVol. 22, No. 2, 1995

tions of this quasispecies model, owing to the adaptation advantage that arises from rapid selection among the mutants, have been found for several viruses. Envelope glycoproteins have been reported to be important targets for the immune response of the host in some viruses, such as pestiviruses, which are close relatives of HCV. 2~'26 In HIV, the targets of virus-neutralizing antibody and cytotoxic T lymphocytes have been reported to be located in the third variable domain of glycoprotein 120, 27.29 and HVRs are thought to change their amino acid residues for better adaptation to the surroundings. In the case of HCV, escape variants of E2 HVR advantageous to the immune system of the host have been suggested to survive and play an important role in persistent infection. 3° There may exist the pressure of positive selection for changing the antigenicity of HVR for better fitness to the environmental conditions during chronic infection. The change of HVR antigenicity in the HCV genome is sequential and the immune system of the host follows the change. 3°'31 The heterogeneity of HVR m a y increase with the progression of liver disease, because the evolution of the HIV quasispecies leads to immunodeficiency disease. TM In this study, however, broad distribution both in the complexity of HVR1 determined by PCR-SSCP analysis and in the nucleotide diversity of HVR1 estimated by conventional sequence analysis was found even in HCV carriers with normal ALT levels whose mean titer of HCV RNA was significantly lower than those of patients with chronic hepatitis, cirrhosis, and HCC. The SSCP method was originally established to detect even one point mutation in a region of interest. 15'16 In this technique, the separated strands show particular mobilities depending on their sequence-specific three-dimensional conformations. Therefore, heterogeneous mixtures of m u t a n t genomes, such as a viral quasispecies, can be separated into different bands by this analysis and it is expected that the number of the bands is proportional to the degree of the sequence complexity. On the basis of the previous investigation on the influence of sensitivity-affecting factors, especially of the temperature of the gel, we selected 25°C for the temperature of electrophoresis of our system. Under this condition, clones of more than 10% nucleotide difference could be differentiated, but not two clones with only one or two differences in the nucleotide sequence. However, we compared the values of nucleoo tide diversity by conventional sequence analysis with the number of SSCP bands in several serum samples from patients and found a statistically significant correlation between the two variables (data not shown). Therefore, our PCR-SSCP system may be able to evaluate the approximate degree of the complexity. What we showed i n t h e present study using SSCP analysis was the relationship between the estimated major quasispecies complexity and the various stages of HCV carrier states. Our data suggested that there was no significant difference in the complexity of HVR1 among

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HCV carriers with normal ALT levels, patients with chronic hepatitis, cirrhosis, and HCC. In summary, broad distribution in the complexity and diversity of HVR1 was found even in the early stage of chronic HCV infection in spite of the small quantity in serum HCV RNA. The complexity of HVR1 ranged widely in each stage of infection. Furthermore, for the patients receiving blood transfusion, there was no significant correlation between the complexity of HVR1 and the time elapsed after blood transfusion, and some patients showed high complexity of HVR1 a few years after blood transfusion. Therefore, we conclude that the quasispecies complexity of HVR1 has no correlation with the stage of chronic HCV infection. REFERENCES

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