Journal of Virological Methods, 39 (1992) 279-290 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00
279
VIRMET 01386
Examination of the buoyant density of hepatitis C virus by the polymerase chain reaction Robert J. Cat-rick, George G. Schlauder, David A. Peterson and Isa K. Mushahwar Experimental
Biology Research,
Abbott Laboratories,
(Accepted
North Chicago, IL (USA)
30 April 1992)
Summary
Sucrose and cesium chloride density gradients were used to fractionate hepatitis C virus (HCV) infectious chimpanzee plasma. The fractionated plasma was then evaluated for HCV RNA sequences using cDNA synthesis and the polymerase chain reaction (cDNA/PCR). cDNA/PCR detectable HCV RNA was identified repeatedly in two regions. One region was at the top of the gradients with a buoyant density of < 1.03 g/cm3, the other at a density of approximately 1.18-l .21 g/cm3. Hepatitis C virus; Polymerase chain reaction; Hepatitis C viral RNA; Buoyant density
Introduction
The successful cloning of hepatitis C virus (HCV) from highly infectious chimpanzee plasma was reported by Choo et al. (1989) The nucleotide sequence of the HCV genomic RNA has been characterized as a continuous open reading frame of approximately 10 Kb in length. Further comparison with other known viral sequences showed co-linear homologies with the flaviviruses (Choo et al., 1991). The HCV genome is reported to be enclosed in a protein coat and lipid envelope with a diameter between 30 and 60 nm (Bradley et al., 1985; He et al., 1987). Native viral antigens have not been characterized. Correspondence to: Robert J. Carrick, Experimental Sheridan Road, North Chicago, IL 60064, USA.
Biology Research, Abbott
Laboratories,
1401
280
In this study, HCV infectious chimpanzee plasma was fractionated using sucrose and cesium chloride density gradients. Individual fractions and/or pools of fractions were evaluated for the presence of HCV RNA using cDNA/ PCR.
Materials and Methods Chimpanzees Inoculation of chimpanzees and acquisition of samples were performed following standards for hepatitis studies in chimpanzees (Muchmore, 1980). Chimpanzee Colonel (CH-427) was inoculated intravenously with 10 ml of the Hutchinson strain (Alter et al., 1975) passage 3 chimpanzee serum obtained 945 days post inoculation from a chronic HCV carrier chimpanzee (CH-117). Liver punch biopsies were obtained at 7-day intervals. Serum and/or plasma samples were obtained before inoculation and at 3 times weekly to monthly intervals thereafter. Chimpanzee Colonel resolved HCV infection after an acute phase of the disease. The acute phase was characterized by an elevation in alanine amniotransferase (ALT) levels, hepatocyte ultrastructural changes (Pfeifer et al., 1980), an antibody response to cl00 antigens (Dawson et al., 1991), and the detection of HCV RNA (Schlauder et al., 1991). Plasma was collected 3 times weekly during ALT elevation. Pools of plasma were prepared from the rising (pool A), plateau (pool B), and descending (pool C) portions of the ALT spike. The plasma pools were aliquoted and stored at - 70°C and subsequently titered in chimpanzees by limiting dilution. Titration of pools A, B, and C showed 1 x 104, 1 x 105, and < 1 x lo4 chimpanzee infectious doses (CID) per ml of plasma, respectively. Pool A was selected for use in this study. Biochemistry/histology ALT levels were determined from freshly drawn samples (Valenza and Muchmore, 1982). The upper limit of the normal ALT level is defined as 2.5 times the mean ALT baseline. Histologic analysis by light and electron microscopy were performed by standard methods. Density gradients Sucrose and cesium chloride preformed linear gradients and cesium chloride self-forming gradients were used. Sucrose and cesium stocks were prepared in Mill&Q filtered water (Millipore Inc.) with phosphate buffered saline, pH 7.3 (PBS). Fraction density was determined by refractive index and corrected for PBS (Rickwood, 1984). Centrifugation runs were conducted at 4°C using a Beckmen L-8 70 M ultracentrifuge and a SW-41 rotor. Fractions (0.5 ml) were collected as described below and stored at -70°C before analysis.
281
Linear preformed gradients were prepared using a gradient maker (Hoffer Scientific, San Francisco, CA) according to the manufacturer’s instructions with sucrose or cesium chloride stock solutions. The total volume of each preformed gradient was 11 ml. After gradient formation, a 1 ml aliquot of Colonel pool A or Colonel plasma obtained prior to inoculation (Colonel PreHCV) was thawed, warmed to ambient temperature, overlaid onto the gradient, and centrifuged. The conditions of centrifugation were as follows: Gradient 1 (sucrose), 17 h at 37000 rpm; gradient 2 (sucrose), 66 h at 37000 rpm; gradient 3 (cesium), 21 h at 33 000 rpm. Fractions from gradient 1 were collected by aspiration from the gradient top, fractions from gradients 2 and 3 were collected from the gradient bottom by tube puncture. Two self-forming cesium gradients were prepared, one light and one heavy (gradients 4A and 4B). The light gradient was prepared by mixing 1 ml of Colonel pool A plasma uniformly into 11 ml of 1.1500 g/cm3 cesium chloride in PBS. The heavy gradient was prepared by mixing 1 ml of Colonel pool A into 11 ml of 1.2502 g/cm3 cesium chloride in PBS. The gradients were spun at 33 000 r-pm for 72 h. Fractions were collected from the bottom of the gradient. cDNA/PCR
cDNA/PCR was used for the identification of HCV positive samples utilizing a modification of the methods of Weiner et al. (1990) and Kubo et al. (1989). RNA was extracted by adding 10 ~1 of the fraction pool or individual fraction to 490 ~1 of an,extraction buffer containing 50 mM Tris (pH S.O), 100 mM NaCl, 1 mM EDTA, 0.5% SDS, 1 mg/ml Proteinase K, 20 pg/ml Yeast tRNA or Poly A. The mixture was incubated at 37°C for 1 h, extracted one time with an equal volume of phenol (equilibrated in 0.1 M Tris, pH S.O), one time with phenol/chloroform/isoamylalcohol (25:24: 1) and 2 times with chloroform/isoamylalcohol (24: 1). Nucleic acids were precipitated at - 70°C for a minimum of 12 h with 2.5 volumes of ethanol after making the aqueous phase 0.2 M in sodium acetate. The nucleic acid pellet was washed with 1 ml of ice-cold ethanol and then dried under vacuum. The pellet was resuspended in 5 ~1 of diethylpyrocarbonate treated water (Maniatis et al., 1982). A total of 1 ~1 of 20 ,uM sense primer 4648, 5’GGCTATACCGGCGACTTCGA-3’ (nucleotides 4668 to 4687) (Choo et al., 1991), and 1 ,ul of 20 PM antisense primer 526A, 5’-GACATGCATGTCATGATGTA-3’ (nucleotides 5271 to 5290), was added to the resuspended pellet and incubated at 65°C for 5 min. First strand cDNA synthesis was performed in a volume of 25 ~1 using reagents from BRL’s AMV reverse transcriptase system under the following conditions: 1 mM dATP, 1 mM dGPT, 1 mM dTTP, 0.5 mM dCTP, 100 mM Tris, pH 8.3, 10 mM MgC12, 10 mM DTT, 76 mM KCl, 40 U RNasin (Promega), 5 U AMV reverse transcriptase, 37°C for 1 h. The cDNA synthesis reaction mixture was diluted with 50 ~1 of sterile MilliQ filtered water, boiled for 10 min, and cooled on ice. The DNA amplifications were carried out using the GeneAmp DNA
282
Amplification Reagent Kit (Perkin Elmer Cetus) as follows: 75 ~1 of the cDNA synthesis reaction was added to a 0.5 ml reaction tube containing 9.0 ~1 Milli-Q filtered water, 7.5 ~1 dNTP mix (1.25 mM each), 7.5 ~1 10 x reaction buffer, and 1.0 ~1 Taq polymerase. PCR was carried out for 35 cycles (1 min 94”C, 2 min 37°C 3 min 72”(Z), followed by a 7 min extension at 72°C. PCR products were analyzed by agarose gel electrophoresis as previously described (Schlauder et al., 1991). The initial cDNA/PCR evaluation was conducted on gradient fraction aliquots that were pooled into density regions for each gradient. After completing the evaluation for the pooled fractions, individual fractions of specific interest were identified and then evaluated. Colonel pool A plasma (10 ~1) served as positive control for both RNA preparation and cDNA/PCR. Fractions collected from gradient runs with Colonel pre-HCV plasma served as negative controls. RNase treatment of gradient pools
Aliquots of 200 ~1 from each pool were treated with RNase A (Boehringer Mannheim) at a final concentration of 17 pg/ml for 30 min at 37°C. RNasin (Promega) and dithiothreitol were then added to a final concentration of 1.6 U/ ~1 and 6.3 mM, respectively. Samples were stored at - 70°C. The samples were thawed and additional RNasin was added to a final concentration of 3.2 U/pi. Extraction of RNA was carried out as described previously. Results Detection of HCV RNA
Autoradiograms of PCR products after Southern transfer (Southern, 1975) were obtained as described previously (Schlauder et al., 1991). In some cases, a 620 bp band was lightly visible by ethidium bromide staining (not shown). The specificity of the major band of the expected size, 620 bp, was always confirmed by hybridization to radiolabelled probe after Southern transfer. The additional bands visible in the hybridization analyses (Figs. l-5) have been shown to be an artifact of the non-denaturing conditions under which these products were run (Schlauder et al., 1991). Sucrose and cesium preformed gradients
The first gradient evaluated was a relatively narrow, p = 1.03-l. 18 g/cm3 sucrose gradient. Fractions collected from the gradient top were pooled into 6 density regions for cDNA/PCR analysis as shown in Fig. 1. cDNA/PCR analysis of the fraction pools revealed HCV RNA concentrated at the gradient top in fraction pools 1A, lB, and 1C (p = 1.03-1.11 g/cm3) with the most
283
Gradient 1 - Preformed Sucrose Pooled Fractions
62Qbp
1.03-1.06 l-4
1A
1.07-1.09 5-8
1B
1.09-1.11 9-11
1c
1.11-1.13
12-14
1D
1.14-1.15
15-17
1E
1.17-1.18
18-20
1F
Fig. 1. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient top and pooled prior to PCR. Density expressed in g/cm3.
intense hybridization signal observed for pool 1A (p = 1.03-1.06 g/cm3), the gradient top. Light tailing of the hybridization signal was seen in fraction pools 1D and 1E (p = 1.1 l-l. 15 g/cm3). To determine if the HCV RNA present in Gradient 2 - Preformed Pooled Fractions
Sucrose
1.03-1.04
25-23
2A
1.05-1.07
22-20
28
1.09-1.12
19-17
2c
1.13-1.16
16-14
2D
1.16-1.16
13-11
2E
1.19-1.22
10-8
2F
1.23-1.26
7-5
2G
1.26-l .30
3-1
2H
Fig. 2. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom and pooled prior to PCR. Density expressed in g/cm3.
284
pools IA, IB, and 1C was in a free or protected form, additional cDNA/PCR reactions were run on RNase treated pool aliquots. Results from these assays showed that RNase treatment did not reduce the hybridization signal when compared to non-treated aliquots (not shown). In a second sucrose gradient with a broader density range, p = 1.03-1.30 g/ cm3 (gradient 2), fractions were collected from the gradient bottom to determine if a banding density had been masked by tailing of the PCR signal observed in gradient 1. Aliquots were pooled into 8 density regions as shown in Fig. 2. cDNA/PCR evaluation of the pools revealed two regions with high HCV RNA levels. One at the gradient top in pool 2A, p = 1.03-1.04 g/cm3, and a second region in pool 2F, p = 1.19-1.22 g/cm3, a density range not included in gradient 1. Aliquots of fractions 4-12 (p = 1.26-1.17 g/cm3) encompassing pool 2F were evaluated with cDNA/PCR to determine where the maximum level of HCV RNA occurred. This analysis showed peak HCV specific hybridization at the density of 1.19 g/cm3 (fraction 10, Fig. 3). A weaker signal was seen in fraction 9 (p = 1.21 g/cm3). In addition, a signal that was barely visible on the original autoradiogram was detected in fraction 11 (p = 1.18 g/cm3). To determine if a similar banding pattern to the one observed in the sucrose gradients occurred in cesium chloride, a preformed cesium gradient was prepared with the density range 1.07-1.37 g/cm3 (gradient 3). Fractions Gradient 2 - Preformed Sucrose. Individual Fractions
1.17
12
1.18
11
1.19
IO
1.21
9
1.22
8
1.23
7
1.24
6
1.26
5
1.26
4
Fig. 3. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom. PCR was run on selected individual fractions. Density expressed in g/cm3.
285
620bp
1.07-1.06
27-25
3A
1.06-1.11
24-22
38
1.12-1.14
21-19
3c
1.15-1.17
16-16
3D
1.17-1.16
15-13
3E
1.19-1.20
12-10
3F
1.22-1.25
9-7
3G
1.25-1.29 6-4
3H
1.32-1.37 3-l
31
Fig. 4. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom and pooled prior to PCR. Density expressed in g/cm’.
collected from the gradient bottom were pooled into 9 density regions as shown in Fig. 4. cDNA/PCR evaluation of the pooled fractions again showed two density regions of amplifiable HCV RNA. Light material, as previously seen at the tops of the sucrose gradients, was detected in pools 3A, 3B and 3C (p = 1.07-l. 14 g/cm3). Banding of HCV RNA was also detected within pool 3F, in the heavier density range of 1.19-1.20 g/cm3. Aliquots of fractions 8-13 @ = 1.23-l. 18 g/cm3), encompassing pool 3F, were evaluated to determine where the maximum banding density occurred within this region. This analysis (Fig. 5) showed the peak HCV specific hybridization signal at a density of 1.20 $/cm3 in fraction 11 with weaker signals detected in fractions 12 (p = 1.19 g/cm ), 10 (p = 1.20 g/cm3), and 9 (p = 1.22 g/cm3). Cesium self-forming gradients To determine if the HCV RNA being detected at the top of the sucrose and cesium gradients was an artifact of layering Colonel Pool A plasma over preformed gradients, the following study was conducted. Two self-forming cesium gradients; gradient 4A @ = 1.05-1.11 g/cm3) and gradient 4B @ = 1.12-l .34 g/cm3) (not shown) were prepared with 1 ml of Pool A plasma mixed throughout each gradient volume. After completion -of the centrifuge run,
Preformed Gradient 3 - Cesium Chloride Individual Fractions
620bp I
1.18
13
1.19
12
1.20
11
1.20
10
1.22
9
1.23
8
Fig. 5. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom. PCR was run on selected individual fractions. Density expressed in g/cm3.
fractions were collected by bottom puncture and aliquots were pooled into 14 density regions. cDNA/PCR analysis of the pooled fractions showed that for both gradients 4A and 4B, material with high HCV RNA content had migrated to the gradient tops. In gradient 4A, the lightest pool, p = 1.05 g/cm3-1 .07 g/ cm3 showed the only HCV specific hybridization signal. No banding of HCV was detected in the remainder of gradient 4A, p = 1.07-l. 11 g/cm3. In gradient 4B, the heavier self-forming gradient, banding of HCV occurred in two areas. At the gradient top, p = 1.12-l .14 g/cm3, and in the fraction pools encompassing the density region 1.15-l .21 g/cm3. Aliquots of fractions encompassing this density region were further evaluated by cDNA/PCR. Data from this analysis again showed maximum banding at p = 1.18-1.21 g/ cm3. Discussion
The data indicate that HCV viral RNA was detected at two buoyant densities, the first at the gradient top, the second within a buoyant density range of 1.18-1.21 g/cm3. Density gradient centrifugation using either sucrose preformed or cesium chloride preformed or self-forming gradients did not significantly alter these results. Tailing of the hybridization signal observed adjacent to these peak banding areas is consistent with a normal distribution of particles surrounding banding density peaks and was anticipated considering
287
the extreme sensitivity of PCR. Although the HCV RNA positive plasma component detected at the gradient tops was recovered at densities ranging between 1.03-1.14 g/cm3, the buoyant density with the maximum signal was always detected at the lightest density in a given gradient. Evaluation of the light banding density in a cesium self-forming isopycnic gradient, where HCV positive plasma was mixed uniformly throughout the gradient media prior to centrifugation, further supported this observation. The gradient top was positive for HCV RNA (p = 1.05-1.07 g/cm3) while the heavier densities (p = 1.07-l. 11 g/cm3) were negative for HCV RNA. These observations suggest an isopycnic banding density for the first peak of < 1.03 g/cm3, the density of the lightest gradient fraction evaluated. Signal tailing was observed into the heavier gradient fractions until the second peak (p = 1.18-1.21 g/cm3) was encountered. These data differ from a recent report by Miyamoto et al. (1992), who failed to detect HCV RNA in cesium chloride gradients, but reported that the HCV peak banding density was 1.08 g/cm3 in sucrose. To account for this light banding density, the possibility of a virus and lipid complex was explored. However, extraction of the gradient fractions with organic solvents destroyed the cDNA/PCR detectability of the HCV RNA suggesting virion damage with resultant exposure of the HCV RNA to endogenous plasma RNase activity (results not shown). The possibility of lipid association remains to be further investigated since it would be unlikely that the light density represents the true banding density of the virion. The heavier buoyant density detected; p = 1.20 g/cm3 (1.19-l .22 g/cm3 range) in cesium chloride preformed; 1.18 g/cm3 (1.15-l .21 g/cm3 range) cesium chloride self-forming; and p = 1.19 g/cm3 (1.19-1.21 g/cm3 range) in sucrose gradients; is in agreement with the established buoyant densities for the flaviviruses (p = 1.19-l .20 g/cm3 sucrose) and most togaviruses (p = 1.17-l. 19 g/cm3 sucrose). The density range is heavier than that for the pestivirus (p = 1.12-1.13 g/cm3 sucrose), a proposed model for HCV (Choo et al., 1991) and significantly lighter than that for free RNA (p > 1.9 g/cm3, cesium chloride). These data are also in general agreement with a report by Abe et al. (1989) where virus like particles morphologically similar to togaviruses were visualized by electron microscopy in sucrose density gradient fractionated (p = 1.141.18 g/cm3), HCV positive, sera. Evidence suggesting that the HCV RNA being detected in both banding peaks is present in whole virus includes: (1) protection from experimental RNase activity, (2) strong correlation between HCV infectivity and the cDNA/ PCR detectability of HCV RNA in plasma inocula (manuscript in preparation). Recently, Bradley et al. (1991) reported the buoyant density of HCV in sucrose gradients with fractions collected from the gradient top and concluded that HCV infectivity in chimpanzees was associated with fractions at a buoyant density of 1.09-l. 11 g/cm3. However, fractions at the very top of the gradient were not evaluated for chimpanzee infectivity. Our data suggest significant
288
tailing in the density region p = 1.09-l. 11 g/cm3 in sucrose gradients collected from the top (Fig. 1). It is therefore likely that the HCV infectivity associated with this density region does not represent the area of peak virus concentration. Bradley et al. (1991) did not detect HCV infectivity in chimpanzees in the density range 1.18-1.21 g/cm3. This suggests that the HCV RNA detected in this region may not represent the infectious virus, but rather HCV RNA associated plasma components or incomplete and/or defective virus particles. The possibility also exists that virus was present in this density region but not in sufficient quantities to infect the chimpanzee. Work is proceeding to clarify these issues. This work was conducted in the absence of detailed information on the biology of HCV. This lack of biological characterization is due mainly to the unavailability of sufficient quantities of virus for such studies. The methods described in this paper may provide a useful means for HCV virus purification and concentration and aid in the elucidation of HCV biology. Acknowledgements
We thank Charles Kyrk, Gail Leverenz, and Chuck Amann for their competent technical assistance: and Dr. Elizabeth Muchmore and the Laboratory of Experimental Medicine and Surgery in Primates for the maintenance of chimpanzees. The work with chimpanzees was in accordance with the Institutional Code of Practice approved by the Ethical Committee (see also Muchmore, 1980). References Abe, K., Kurata, T. and Shikata, T. (1989) Non-A, non-B hepatitis: visualization of virus-like particles from chimpanzee and human sera. Arch Virol. 104, 351-355. Alter, H.J., Purcell, R.H., Holland, P.V., Finstone, S.M., Morrow, A.G. and Moritsugu, Y. (1975) Clinical and serological analysis of transfusion-associated hepatitis. Lancet ii, 838-841. Bradley, D.W., McCaustland, K.A., Cook, E.H., Schabel, C.A., Ebert, J.W. and Maynard, J.E. (1985) Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small, enveloped virus. Gastroenterology 88, 773-779. Bradley, D., McCaustland, K., Krawczynski, K., Spelbring, J., Humphrey, C. and Cook, E.H. (1991) Hepatitis C virus: Buoyant density of the factor V-III derived isolate in sucrose. J. Med. Virol. 34, 206-208. Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 244, 359-362. Choo, Q.-L., Richman, K.H., Han, J.H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, A., Barr, P.J., Weiner, A.J., Bradley, D.W., Kuo, G. and Houghton, M. (1991) Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88,24512455. Dawson, G.J., Lesniewski, R.R., Stewart, J.L., Boardway, K.M., Gutierrez, R.A., Pendy, L., Johnson, R.G., Alcalde, X., Devare, S.G., Robey, W.G. and Peterson, D.A. (1991) Detection of
289 antibodies to hepatitis C virus in U.S. blood donors. J. Clin. Microbial. 29, 1479-1486. He, L-F., Alling, D., Popkin, T., Shapiro, M., Alter, H.J. and Purcell, R.H. (1987) Determining the size of non-A, non-B, hepatitis virus by filtration. J. Infect. Dis. 156, 636-640. Kubo, Y., Takeuchi, K., Boonmar, S., Katayama, T., Choo, Q.-L., Kuo, G., Weiner, A.J., Bradley, D.W., Houghton, M., Saito, I. and Miyamura, T. (1989) A cDNA fragment of hepatitis C virus from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucl. Acids Res. 17, 10367-10372. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Miyamoto, H., Okamoto, H., Sato, K., Tanaka, T. and Mishiro, S. (1992) Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction. J. Gen. Virol. 73, 715-718. Muchmore, E. (1980) Hepatitis surveillance standards for hepatitis studies in a chimpanzee colony. Dev. Biol. Stand. 45, 13-21. Pfeifer, U., Thomssen, R., Legler, K., Bottcher, U., Gerlich, W., Weinmann, E. and Klinge, 0. (1980) Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees. Virch. Arch B Cell Pathol. 33, 233-243. Rickwood R. (Ed.) (1984) Centrifugation (2nd ed.): a practical approach. Appendix. IV, IRL Press, Washington, D.C. Schlauder, G.G., Leverenz, G.L., Amann, C.W., Lesniewski, R.R. and Peterson, D.A. (1991) Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees. J. Clin. Microbial. 29, 21752179. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Valenza, F. and Muchmore, E. (1982) The clinical chemistry of chimpanzees. I. Determination of aminotransferase baseline values for hepatitis studies. J. Med. Primatol. 11, 242-25 1. Weiner, A.J., Kuo, G., Bradley, D.W., Bonino, F., Saracco, G., Lee, C., Rosenblatt, J., Choo, Q.-L. and Houghton, M. (1990) Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 335, 1-3.
279
VIRMET 01386
Examination of the buoyant density of hepatitis C virus by the polymerase chain reaction Robert J. Cat-rick, George G. Schlauder, David A. Peterson and Isa K. Mushahwar Experimental
Biology Research,
Abbott Laboratories,
(Accepted
North Chicago, IL (USA)
30 April 1992)
Summary
Sucrose and cesium chloride density gradients were used to fractionate hepatitis C virus (HCV) infectious chimpanzee plasma. The fractionated plasma was then evaluated for HCV RNA sequences using cDNA synthesis and the polymerase chain reaction (cDNA/PCR). cDNA/PCR detectable HCV RNA was identified repeatedly in two regions. One region was at the top of the gradients with a buoyant density of < 1.03 g/cm3, the other at a density of approximately 1.18-l .21 g/cm3. Hepatitis C virus; Polymerase chain reaction; Hepatitis C viral RNA; Buoyant density
Introduction
The successful cloning of hepatitis C virus (HCV) from highly infectious chimpanzee plasma was reported by Choo et al. (1989) The nucleotide sequence of the HCV genomic RNA has been characterized as a continuous open reading frame of approximately 10 Kb in length. Further comparison with other known viral sequences showed co-linear homologies with the flaviviruses (Choo et al., 1991). The HCV genome is reported to be enclosed in a protein coat and lipid envelope with a diameter between 30 and 60 nm (Bradley et al., 1985; He et al., 1987). Native viral antigens have not been characterized. Correspondence to: Robert J. Carrick, Experimental Sheridan Road, North Chicago, IL 60064, USA.
Biology Research, Abbott
Laboratories,
1401
280
In this study, HCV infectious chimpanzee plasma was fractionated using sucrose and cesium chloride density gradients. Individual fractions and/or pools of fractions were evaluated for the presence of HCV RNA using cDNA/ PCR.
Materials and Methods Chimpanzees Inoculation of chimpanzees and acquisition of samples were performed following standards for hepatitis studies in chimpanzees (Muchmore, 1980). Chimpanzee Colonel (CH-427) was inoculated intravenously with 10 ml of the Hutchinson strain (Alter et al., 1975) passage 3 chimpanzee serum obtained 945 days post inoculation from a chronic HCV carrier chimpanzee (CH-117). Liver punch biopsies were obtained at 7-day intervals. Serum and/or plasma samples were obtained before inoculation and at 3 times weekly to monthly intervals thereafter. Chimpanzee Colonel resolved HCV infection after an acute phase of the disease. The acute phase was characterized by an elevation in alanine amniotransferase (ALT) levels, hepatocyte ultrastructural changes (Pfeifer et al., 1980), an antibody response to cl00 antigens (Dawson et al., 1991), and the detection of HCV RNA (Schlauder et al., 1991). Plasma was collected 3 times weekly during ALT elevation. Pools of plasma were prepared from the rising (pool A), plateau (pool B), and descending (pool C) portions of the ALT spike. The plasma pools were aliquoted and stored at - 70°C and subsequently titered in chimpanzees by limiting dilution. Titration of pools A, B, and C showed 1 x 104, 1 x 105, and < 1 x lo4 chimpanzee infectious doses (CID) per ml of plasma, respectively. Pool A was selected for use in this study. Biochemistry/histology ALT levels were determined from freshly drawn samples (Valenza and Muchmore, 1982). The upper limit of the normal ALT level is defined as 2.5 times the mean ALT baseline. Histologic analysis by light and electron microscopy were performed by standard methods. Density gradients Sucrose and cesium chloride preformed linear gradients and cesium chloride self-forming gradients were used. Sucrose and cesium stocks were prepared in Mill&Q filtered water (Millipore Inc.) with phosphate buffered saline, pH 7.3 (PBS). Fraction density was determined by refractive index and corrected for PBS (Rickwood, 1984). Centrifugation runs were conducted at 4°C using a Beckmen L-8 70 M ultracentrifuge and a SW-41 rotor. Fractions (0.5 ml) were collected as described below and stored at -70°C before analysis.
281
Linear preformed gradients were prepared using a gradient maker (Hoffer Scientific, San Francisco, CA) according to the manufacturer’s instructions with sucrose or cesium chloride stock solutions. The total volume of each preformed gradient was 11 ml. After gradient formation, a 1 ml aliquot of Colonel pool A or Colonel plasma obtained prior to inoculation (Colonel PreHCV) was thawed, warmed to ambient temperature, overlaid onto the gradient, and centrifuged. The conditions of centrifugation were as follows: Gradient 1 (sucrose), 17 h at 37000 rpm; gradient 2 (sucrose), 66 h at 37000 rpm; gradient 3 (cesium), 21 h at 33 000 rpm. Fractions from gradient 1 were collected by aspiration from the gradient top, fractions from gradients 2 and 3 were collected from the gradient bottom by tube puncture. Two self-forming cesium gradients were prepared, one light and one heavy (gradients 4A and 4B). The light gradient was prepared by mixing 1 ml of Colonel pool A plasma uniformly into 11 ml of 1.1500 g/cm3 cesium chloride in PBS. The heavy gradient was prepared by mixing 1 ml of Colonel pool A into 11 ml of 1.2502 g/cm3 cesium chloride in PBS. The gradients were spun at 33 000 r-pm for 72 h. Fractions were collected from the bottom of the gradient. cDNA/PCR
cDNA/PCR was used for the identification of HCV positive samples utilizing a modification of the methods of Weiner et al. (1990) and Kubo et al. (1989). RNA was extracted by adding 10 ~1 of the fraction pool or individual fraction to 490 ~1 of an,extraction buffer containing 50 mM Tris (pH S.O), 100 mM NaCl, 1 mM EDTA, 0.5% SDS, 1 mg/ml Proteinase K, 20 pg/ml Yeast tRNA or Poly A. The mixture was incubated at 37°C for 1 h, extracted one time with an equal volume of phenol (equilibrated in 0.1 M Tris, pH S.O), one time with phenol/chloroform/isoamylalcohol (25:24: 1) and 2 times with chloroform/isoamylalcohol (24: 1). Nucleic acids were precipitated at - 70°C for a minimum of 12 h with 2.5 volumes of ethanol after making the aqueous phase 0.2 M in sodium acetate. The nucleic acid pellet was washed with 1 ml of ice-cold ethanol and then dried under vacuum. The pellet was resuspended in 5 ~1 of diethylpyrocarbonate treated water (Maniatis et al., 1982). A total of 1 ~1 of 20 ,uM sense primer 4648, 5’GGCTATACCGGCGACTTCGA-3’ (nucleotides 4668 to 4687) (Choo et al., 1991), and 1 ,ul of 20 PM antisense primer 526A, 5’-GACATGCATGTCATGATGTA-3’ (nucleotides 5271 to 5290), was added to the resuspended pellet and incubated at 65°C for 5 min. First strand cDNA synthesis was performed in a volume of 25 ~1 using reagents from BRL’s AMV reverse transcriptase system under the following conditions: 1 mM dATP, 1 mM dGPT, 1 mM dTTP, 0.5 mM dCTP, 100 mM Tris, pH 8.3, 10 mM MgC12, 10 mM DTT, 76 mM KCl, 40 U RNasin (Promega), 5 U AMV reverse transcriptase, 37°C for 1 h. The cDNA synthesis reaction mixture was diluted with 50 ~1 of sterile MilliQ filtered water, boiled for 10 min, and cooled on ice. The DNA amplifications were carried out using the GeneAmp DNA
282
Amplification Reagent Kit (Perkin Elmer Cetus) as follows: 75 ~1 of the cDNA synthesis reaction was added to a 0.5 ml reaction tube containing 9.0 ~1 Milli-Q filtered water, 7.5 ~1 dNTP mix (1.25 mM each), 7.5 ~1 10 x reaction buffer, and 1.0 ~1 Taq polymerase. PCR was carried out for 35 cycles (1 min 94”C, 2 min 37°C 3 min 72”(Z), followed by a 7 min extension at 72°C. PCR products were analyzed by agarose gel electrophoresis as previously described (Schlauder et al., 1991). The initial cDNA/PCR evaluation was conducted on gradient fraction aliquots that were pooled into density regions for each gradient. After completing the evaluation for the pooled fractions, individual fractions of specific interest were identified and then evaluated. Colonel pool A plasma (10 ~1) served as positive control for both RNA preparation and cDNA/PCR. Fractions collected from gradient runs with Colonel pre-HCV plasma served as negative controls. RNase treatment of gradient pools
Aliquots of 200 ~1 from each pool were treated with RNase A (Boehringer Mannheim) at a final concentration of 17 pg/ml for 30 min at 37°C. RNasin (Promega) and dithiothreitol were then added to a final concentration of 1.6 U/ ~1 and 6.3 mM, respectively. Samples were stored at - 70°C. The samples were thawed and additional RNasin was added to a final concentration of 3.2 U/pi. Extraction of RNA was carried out as described previously. Results Detection of HCV RNA
Autoradiograms of PCR products after Southern transfer (Southern, 1975) were obtained as described previously (Schlauder et al., 1991). In some cases, a 620 bp band was lightly visible by ethidium bromide staining (not shown). The specificity of the major band of the expected size, 620 bp, was always confirmed by hybridization to radiolabelled probe after Southern transfer. The additional bands visible in the hybridization analyses (Figs. l-5) have been shown to be an artifact of the non-denaturing conditions under which these products were run (Schlauder et al., 1991). Sucrose and cesium preformed gradients
The first gradient evaluated was a relatively narrow, p = 1.03-l. 18 g/cm3 sucrose gradient. Fractions collected from the gradient top were pooled into 6 density regions for cDNA/PCR analysis as shown in Fig. 1. cDNA/PCR analysis of the fraction pools revealed HCV RNA concentrated at the gradient top in fraction pools 1A, lB, and 1C (p = 1.03-1.11 g/cm3) with the most
283
Gradient 1 - Preformed Sucrose Pooled Fractions
62Qbp
1.03-1.06 l-4
1A
1.07-1.09 5-8
1B
1.09-1.11 9-11
1c
1.11-1.13
12-14
1D
1.14-1.15
15-17
1E
1.17-1.18
18-20
1F
Fig. 1. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient top and pooled prior to PCR. Density expressed in g/cm3.
intense hybridization signal observed for pool 1A (p = 1.03-1.06 g/cm3), the gradient top. Light tailing of the hybridization signal was seen in fraction pools 1D and 1E (p = 1.1 l-l. 15 g/cm3). To determine if the HCV RNA present in Gradient 2 - Preformed Pooled Fractions
Sucrose
1.03-1.04
25-23
2A
1.05-1.07
22-20
28
1.09-1.12
19-17
2c
1.13-1.16
16-14
2D
1.16-1.16
13-11
2E
1.19-1.22
10-8
2F
1.23-1.26
7-5
2G
1.26-l .30
3-1
2H
Fig. 2. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom and pooled prior to PCR. Density expressed in g/cm3.
284
pools IA, IB, and 1C was in a free or protected form, additional cDNA/PCR reactions were run on RNase treated pool aliquots. Results from these assays showed that RNase treatment did not reduce the hybridization signal when compared to non-treated aliquots (not shown). In a second sucrose gradient with a broader density range, p = 1.03-1.30 g/ cm3 (gradient 2), fractions were collected from the gradient bottom to determine if a banding density had been masked by tailing of the PCR signal observed in gradient 1. Aliquots were pooled into 8 density regions as shown in Fig. 2. cDNA/PCR evaluation of the pools revealed two regions with high HCV RNA levels. One at the gradient top in pool 2A, p = 1.03-1.04 g/cm3, and a second region in pool 2F, p = 1.19-1.22 g/cm3, a density range not included in gradient 1. Aliquots of fractions 4-12 (p = 1.26-1.17 g/cm3) encompassing pool 2F were evaluated with cDNA/PCR to determine where the maximum level of HCV RNA occurred. This analysis showed peak HCV specific hybridization at the density of 1.19 g/cm3 (fraction 10, Fig. 3). A weaker signal was seen in fraction 9 (p = 1.21 g/cm3). In addition, a signal that was barely visible on the original autoradiogram was detected in fraction 11 (p = 1.18 g/cm3). To determine if a similar banding pattern to the one observed in the sucrose gradients occurred in cesium chloride, a preformed cesium gradient was prepared with the density range 1.07-1.37 g/cm3 (gradient 3). Fractions Gradient 2 - Preformed Sucrose. Individual Fractions
1.17
12
1.18
11
1.19
IO
1.21
9
1.22
8
1.23
7
1.24
6
1.26
5
1.26
4
Fig. 3. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom. PCR was run on selected individual fractions. Density expressed in g/cm3.
285
620bp
1.07-1.06
27-25
3A
1.06-1.11
24-22
38
1.12-1.14
21-19
3c
1.15-1.17
16-16
3D
1.17-1.16
15-13
3E
1.19-1.20
12-10
3F
1.22-1.25
9-7
3G
1.25-1.29 6-4
3H
1.32-1.37 3-l
31
Fig. 4. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom and pooled prior to PCR. Density expressed in g/cm’.
collected from the gradient bottom were pooled into 9 density regions as shown in Fig. 4. cDNA/PCR evaluation of the pooled fractions again showed two density regions of amplifiable HCV RNA. Light material, as previously seen at the tops of the sucrose gradients, was detected in pools 3A, 3B and 3C (p = 1.07-l. 14 g/cm3). Banding of HCV RNA was also detected within pool 3F, in the heavier density range of 1.19-1.20 g/cm3. Aliquots of fractions 8-13 @ = 1.23-l. 18 g/cm3), encompassing pool 3F, were evaluated to determine where the maximum banding density occurred within this region. This analysis (Fig. 5) showed the peak HCV specific hybridization signal at a density of 1.20 $/cm3 in fraction 11 with weaker signals detected in fractions 12 (p = 1.19 g/cm ), 10 (p = 1.20 g/cm3), and 9 (p = 1.22 g/cm3). Cesium self-forming gradients To determine if the HCV RNA being detected at the top of the sucrose and cesium gradients was an artifact of layering Colonel Pool A plasma over preformed gradients, the following study was conducted. Two self-forming cesium gradients; gradient 4A @ = 1.05-1.11 g/cm3) and gradient 4B @ = 1.12-l .34 g/cm3) (not shown) were prepared with 1 ml of Pool A plasma mixed throughout each gradient volume. After completion -of the centrifuge run,
Preformed Gradient 3 - Cesium Chloride Individual Fractions
620bp I
1.18
13
1.19
12
1.20
11
1.20
10
1.22
9
1.23
8
Fig. 5. Agarose gel analysis. Gel was run from left to right. Fractions were collected from the gradient bottom. PCR was run on selected individual fractions. Density expressed in g/cm3.
fractions were collected by bottom puncture and aliquots were pooled into 14 density regions. cDNA/PCR analysis of the pooled fractions showed that for both gradients 4A and 4B, material with high HCV RNA content had migrated to the gradient tops. In gradient 4A, the lightest pool, p = 1.05 g/cm3-1 .07 g/ cm3 showed the only HCV specific hybridization signal. No banding of HCV was detected in the remainder of gradient 4A, p = 1.07-l. 11 g/cm3. In gradient 4B, the heavier self-forming gradient, banding of HCV occurred in two areas. At the gradient top, p = 1.12-l .14 g/cm3, and in the fraction pools encompassing the density region 1.15-l .21 g/cm3. Aliquots of fractions encompassing this density region were further evaluated by cDNA/PCR. Data from this analysis again showed maximum banding at p = 1.18-1.21 g/ cm3. Discussion
The data indicate that HCV viral RNA was detected at two buoyant densities, the first at the gradient top, the second within a buoyant density range of 1.18-1.21 g/cm3. Density gradient centrifugation using either sucrose preformed or cesium chloride preformed or self-forming gradients did not significantly alter these results. Tailing of the hybridization signal observed adjacent to these peak banding areas is consistent with a normal distribution of particles surrounding banding density peaks and was anticipated considering
287
the extreme sensitivity of PCR. Although the HCV RNA positive plasma component detected at the gradient tops was recovered at densities ranging between 1.03-1.14 g/cm3, the buoyant density with the maximum signal was always detected at the lightest density in a given gradient. Evaluation of the light banding density in a cesium self-forming isopycnic gradient, where HCV positive plasma was mixed uniformly throughout the gradient media prior to centrifugation, further supported this observation. The gradient top was positive for HCV RNA (p = 1.05-1.07 g/cm3) while the heavier densities (p = 1.07-l. 11 g/cm3) were negative for HCV RNA. These observations suggest an isopycnic banding density for the first peak of < 1.03 g/cm3, the density of the lightest gradient fraction evaluated. Signal tailing was observed into the heavier gradient fractions until the second peak (p = 1.18-1.21 g/cm3) was encountered. These data differ from a recent report by Miyamoto et al. (1992), who failed to detect HCV RNA in cesium chloride gradients, but reported that the HCV peak banding density was 1.08 g/cm3 in sucrose. To account for this light banding density, the possibility of a virus and lipid complex was explored. However, extraction of the gradient fractions with organic solvents destroyed the cDNA/PCR detectability of the HCV RNA suggesting virion damage with resultant exposure of the HCV RNA to endogenous plasma RNase activity (results not shown). The possibility of lipid association remains to be further investigated since it would be unlikely that the light density represents the true banding density of the virion. The heavier buoyant density detected; p = 1.20 g/cm3 (1.19-l .22 g/cm3 range) in cesium chloride preformed; 1.18 g/cm3 (1.15-l .21 g/cm3 range) cesium chloride self-forming; and p = 1.19 g/cm3 (1.19-1.21 g/cm3 range) in sucrose gradients; is in agreement with the established buoyant densities for the flaviviruses (p = 1.19-l .20 g/cm3 sucrose) and most togaviruses (p = 1.17-l. 19 g/cm3 sucrose). The density range is heavier than that for the pestivirus (p = 1.12-1.13 g/cm3 sucrose), a proposed model for HCV (Choo et al., 1991) and significantly lighter than that for free RNA (p > 1.9 g/cm3, cesium chloride). These data are also in general agreement with a report by Abe et al. (1989) where virus like particles morphologically similar to togaviruses were visualized by electron microscopy in sucrose density gradient fractionated (p = 1.141.18 g/cm3), HCV positive, sera. Evidence suggesting that the HCV RNA being detected in both banding peaks is present in whole virus includes: (1) protection from experimental RNase activity, (2) strong correlation between HCV infectivity and the cDNA/ PCR detectability of HCV RNA in plasma inocula (manuscript in preparation). Recently, Bradley et al. (1991) reported the buoyant density of HCV in sucrose gradients with fractions collected from the gradient top and concluded that HCV infectivity in chimpanzees was associated with fractions at a buoyant density of 1.09-l. 11 g/cm3. However, fractions at the very top of the gradient were not evaluated for chimpanzee infectivity. Our data suggest significant
288
tailing in the density region p = 1.09-l. 11 g/cm3 in sucrose gradients collected from the top (Fig. 1). It is therefore likely that the HCV infectivity associated with this density region does not represent the area of peak virus concentration. Bradley et al. (1991) did not detect HCV infectivity in chimpanzees in the density range 1.18-1.21 g/cm3. This suggests that the HCV RNA detected in this region may not represent the infectious virus, but rather HCV RNA associated plasma components or incomplete and/or defective virus particles. The possibility also exists that virus was present in this density region but not in sufficient quantities to infect the chimpanzee. Work is proceeding to clarify these issues. This work was conducted in the absence of detailed information on the biology of HCV. This lack of biological characterization is due mainly to the unavailability of sufficient quantities of virus for such studies. The methods described in this paper may provide a useful means for HCV virus purification and concentration and aid in the elucidation of HCV biology. Acknowledgements
We thank Charles Kyrk, Gail Leverenz, and Chuck Amann for their competent technical assistance: and Dr. Elizabeth Muchmore and the Laboratory of Experimental Medicine and Surgery in Primates for the maintenance of chimpanzees. The work with chimpanzees was in accordance with the Institutional Code of Practice approved by the Ethical Committee (see also Muchmore, 1980). References Abe, K., Kurata, T. and Shikata, T. (1989) Non-A, non-B hepatitis: visualization of virus-like particles from chimpanzee and human sera. Arch Virol. 104, 351-355. Alter, H.J., Purcell, R.H., Holland, P.V., Finstone, S.M., Morrow, A.G. and Moritsugu, Y. (1975) Clinical and serological analysis of transfusion-associated hepatitis. Lancet ii, 838-841. Bradley, D.W., McCaustland, K.A., Cook, E.H., Schabel, C.A., Ebert, J.W. and Maynard, J.E. (1985) Posttransfusion non-A, non-B hepatitis in chimpanzees. Physicochemical evidence that the tubule-forming agent is a small, enveloped virus. Gastroenterology 88, 773-779. Bradley, D., McCaustland, K., Krawczynski, K., Spelbring, J., Humphrey, C. and Cook, E.H. (1991) Hepatitis C virus: Buoyant density of the factor V-III derived isolate in sucrose. J. Med. Virol. 34, 206-208. Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 244, 359-362. Choo, Q.-L., Richman, K.H., Han, J.H., Berger, K., Lee, C., Dong, C., Gallegos, C., Coit, D., Medina-Selby, A., Barr, P.J., Weiner, A.J., Bradley, D.W., Kuo, G. and Houghton, M. (1991) Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88,24512455. Dawson, G.J., Lesniewski, R.R., Stewart, J.L., Boardway, K.M., Gutierrez, R.A., Pendy, L., Johnson, R.G., Alcalde, X., Devare, S.G., Robey, W.G. and Peterson, D.A. (1991) Detection of
289 antibodies to hepatitis C virus in U.S. blood donors. J. Clin. Microbial. 29, 1479-1486. He, L-F., Alling, D., Popkin, T., Shapiro, M., Alter, H.J. and Purcell, R.H. (1987) Determining the size of non-A, non-B, hepatitis virus by filtration. J. Infect. Dis. 156, 636-640. Kubo, Y., Takeuchi, K., Boonmar, S., Katayama, T., Choo, Q.-L., Kuo, G., Weiner, A.J., Bradley, D.W., Houghton, M., Saito, I. and Miyamura, T. (1989) A cDNA fragment of hepatitis C virus from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan. Nucl. Acids Res. 17, 10367-10372. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Miyamoto, H., Okamoto, H., Sato, K., Tanaka, T. and Mishiro, S. (1992) Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction. J. Gen. Virol. 73, 715-718. Muchmore, E. (1980) Hepatitis surveillance standards for hepatitis studies in a chimpanzee colony. Dev. Biol. Stand. 45, 13-21. Pfeifer, U., Thomssen, R., Legler, K., Bottcher, U., Gerlich, W., Weinmann, E. and Klinge, 0. (1980) Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees. Virch. Arch B Cell Pathol. 33, 233-243. Rickwood R. (Ed.) (1984) Centrifugation (2nd ed.): a practical approach. Appendix. IV, IRL Press, Washington, D.C. Schlauder, G.G., Leverenz, G.L., Amann, C.W., Lesniewski, R.R. and Peterson, D.A. (1991) Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees. J. Clin. Microbial. 29, 21752179. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. Valenza, F. and Muchmore, E. (1982) The clinical chemistry of chimpanzees. I. Determination of aminotransferase baseline values for hepatitis studies. J. Med. Primatol. 11, 242-25 1. Weiner, A.J., Kuo, G., Bradley, D.W., Bonino, F., Saracco, G., Lee, C., Rosenblatt, J., Choo, Q.-L. and Houghton, M. (1990) Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 335, 1-3.
