Chronic hepatitis in chimpanzee carriers of hepatitis B ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 77, No. 10, pp. 6147-6151, October 1980 Medical Sciences

Chronic hepatitis in chimpanzee carriers of hepatitis B virus: Morphologic, immunologic, and viral DNA studies (hepatitis B virus serum markers/light microscopy alnd immunohistochemistry/hepatitis B virus DNA sequences/recombinant hepatitis B virus DNA probes/molecular hybridizati4

DANIEL SHOUVAL*, PRASANTA R. CHAKRABORTY*, NELSON RUIZ-OPAZO*, STEVEN BAUM*, ILYA SPIGLANDt, ELIZABETH MUCHMOREt, MICHAEL A. GERBERT, SWAN N. THUNG0, HANS POPPER", AND DAVID A. SHAFRITZ* *Departments of Medicine and Cell Biology, The Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461; tDivision of Virology, Montefiore Hospital and Medical Center, Bronx New York 10467; *Laboratory of Experimental Medicine and Surgery in Primates, New York University Medical Center, Tuxedo, New York 10987; and IStratton Laboratory for the Study of Liver Diseases and IDepartment of Pathology, The Mount Sinai School of Medicine of the City University of New York, New York, New York 10029

Contributed by Hans Popper, June 30,1980

ABSTRACT Years after infection with hepatitis B virus, chimpanzees may have manifestations of the carrier state as described in man. In addition to serologic evidence for persistent viral infection, percutaneous liver biopsy specimens showed hepatitis B virus surface antigen in the cytoplasm and hepatitis B virus core antigen in the nucleus. Four carrier animals had portal inflammatory reaction as seen in human chronic persistent hepatitis. Viral DNA was demonstrated in nucleic acid extracts of liver biopsy specimens by molecular hybridization to cloned plasmid pAO1 containing hepatitis B virus DNA sequences. Although a viral molecule of length greater than the putative virus was identified, it did not appear to represent integration of viral DNA into the host genome. The chimpanzee model may serve as a means to study the mechanism of hepatitis B viral persistence and progression to chronic liver disease.

cloned HBV DNA probe, DNA molecules of several sizes containing HBV sequences have been identified in liver specimens from all five chimpanzee carriers. Such techniques are considerably more sensitive than immunologic methods for detecting virus in the serum and have direct applicability to human liver biopsy specimens.

With development of tests for hepatitis B virus surface antigen (HBsAg) in the 1960s, it was found that some chimpanzees were carriers of this protein (1, 2) and that acute viral hepatitis could be induced in these primates by injection of serum from human hepatitis B virus (HBV) carriers (2). The histologic lesions were usually milder in chimpanzees than in man (3, 4). Recently, two distinct types of experimental hepatitis B have been described in chimpanzees, one with rapid resolution and the other with smoldering features characterized by portal inflammatory cell infiltration present for more than 1 year (5). After acute infection, some animals became long-term HBV carriers, maintaining elevated levels of HBsAg, antibody to hepatitis B core antigen (anti-HBc), and Dane particles in the serum. Therefore, it appeared important to study such chimpanzee carriers (a) to establish the histologic features in these chimpanzees, (b) to correlate serologic observations with immunohistochemical findings in the liver, and (c) to characterize the nature of viral DNA in the hepatocytes. This was considered important because human HBV carriers have a significant risk of developing hepatocellular carcinoma (6). In a human hepatocellular carcinoma line (PLC/PRF/5) in tissue culture, HBV DNA has recently been found to be integrated into the host genome (7, 8). With development of newer techniques in nucleic acid analysis and availability of recombinant cloned HBV DNA probes (9-14), this question could be addressed in small samples of liver by using very sensitive techniques. We have examined percutaneous liver biopsy specimens and serum from known chimpanzee HBV carriers that were infected experimentally or in the wild. By using a 32P-labeled

MATERIALS AND METHODS Percutaneous liver biopsy specimens (obtained with a Klatskin needle, 16T) and serum specimens were obtained from 10 chimpanzees. Five of these animals were controls and five were known chronic carriers of HBV. Clinical information and pertinent laboratory findings are listed in Table 1. Liver biopsy specimens were divided into separate portions for histologic studies (fixed in neutral formalin) and DNA analysis (frozen in liquid N2 under isopentane). In addition, an 11-year-old carrier chimpanzee studied previously (4) was reinvestigated in retrospect by obtaining additional multiple step sections from the original paraffin block of formalin-fixed tissue. Specimens were prepared for hematoxylin/eosin staining as reported (3), and Shikata's orcein stain (15) was performed with an improved technique which included 30-min oxidation with potassium permanganate and staining with orcein for 5 hr followed by counterstaining with light green. Immunohistochemical staining for HBV proteins in formalin-fixed, paraffin-embedded tissue was performed by the peroxidase-antiperoxidase technique as reported (16), using specific goat antiserum to HBsAg or human antiserum to hepatitis B core antigen (HBcAg). Controls for the specificity of antisera and immunohistochemical staining have been described (16). Normal ranges on routine tests of serum were as follows: total protein, 6.0-8.0 g/dl; albumin, 3.5-5.0 g/dl; bilirubin, 0.15-1.0 mg/dl; LDH, 100-125 international units (IU)/liter; serum glutamic-oxaloacetic transaminase, 7-40 IU/liter; serum glutamic-pyruvic transaminase, 7-40 IU/liter; and alkaline phosphatase activity, 30-115 IU/liter. These were kindly performed by A. Karmen and R. Lent (Albert Einstein College of Medicine) on the Technicon Auto-Analyzer-SMAC. Determination of HBV markers in serum was performed in duplicates on 100- to 200-,gl samples by solid-phase radioimmunoassay (125I) procedures using either commercially available kits (Abbott, AUSRIA II for HBsAg, AUSAB for anti-HBs, and CORAB for anti-HBc) or specially prepared reagents, 125I-

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Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; anti-HBs, antibody to HBsAg; HBcAg, hepatitis B core antigen; anti-HBc, antibody to HBcAg; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; IU, international units.

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Age, yr; sex

Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Clinical and serologic data on chimpanzee carriers of HBV Serum enzymes Serum HBV markers, 1251 cpm SGOT SGPT HBsAg Anti-HBs Anti-HBc HBeAg History (40) (40) (260) (260) (2046) (1041)

Anti-HBe (2682)

CH-30 12; F Known carrier, 1970 23 25 399 6,534 69 2667 4435 CH-31 13; F Infected in lab, 1970 16 10 180 1683 4944 8,034 5819 CH-116 21; F Known carrier, 1967 17 27 7,913 223 348 3296 5373 CH-125 47 26 265 123 6;M Infectedinlab, 1976 1215 9,157 1767 CH-323 21 6; M Recent acquisition 121 180 64 2265 178 14,700 Values in parentheses in column headings represent the upper (SGOT, SGPT, HBsAg, anti-HBs, HBeAg) or lower (anti-HBc and anti-HBe) limit of normal and are 2.1 SDs from means of negative controls. For HBsAg, anti-HBs, and HBeAg, 200 ul of serum was used. For anti-HBc and anti-HBe, 100 Al of serum was used and values less than the lower limit of normal represent positive results. SQ3OT, serum oxaloacetic-glutamic transaminase; SGPT, serum glutamic-pyruvic transaminase.

labeled hepatitis Be antigen (125I-HBeAg), and appropriate antibodies (17), kindly provided by I. K. Mushahwar and L. R. Overby (Diagnostic Division, Abbott). Total nucleic acid was extracted from frozen liver biopsy specimens (--30 mg) by homogenization in 5 vol of a 1:1 (vol/ vol) mixture of phenol and 1.0% NaDodSO4/50 mM Tris-HCl, pH 7.5/150 mM NaCl/2 mM EDTA, followed by two additional phenol extractions and ethanol precipitation. Total nucleic acids were resuspended in 50 mM Tris-HCl, pH 7.4/2 mM EDTA, 0.2 M Na acetate (pH 5.5) was added, and nucleic acids were reprecipitated with 3 vol of ethanol at -20°C. The final material was resuspended in 50 mM Tris-HCl, pH 7.4/100 mM NaCI/2 mM EDTA, digested with pancreatic RNase (Worthington, recrystallized) at 25-50,ug/ml, and treated with proteinase K (50-100 ,g/ml) at 370C for 1.5 hr. This was followed by two phenol extractions and ethanol precipitation. The final DNA pellet was washed with 70% ethanol, resuspended in appropriate buffer, and either applied directly to a 5-mm-thick 0.8% agarose slab gel or digested three times with restriction enzymes (1 unit of enzyme per ,ug of DNA for each digestion) for 12-18 hr. The final material was then diluted in electrophoresis buffer (10.8 g of Tris base, 5.5 g of boric acid, and 1.0 g of Na2EDTA per liter) and applied to the agarose gel. Gel electrophoresis with 125I-labeled restriction fragments of plasmid pBR322 as standards was performed in a submerged gel at 50 V for 6-7 hr. DNA was depurinated and transferred to diazobenzyloxymethyl-paper (Schleicher & Schuell) according to the procedure of Alwine et al. (18). Hybridization was performed with plasmid pA01 HBV DNA kindly supplied by J. Summers (The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA) and labeled with 32P to a specific activity of 1-2 X 108 cpm/,ug by the method of Rigby et al. (19); the diazobenzyloxymethyl-paper was then washed, dried, and radioautographed as described (18), using Picker Max B intensifying screens at -70°C. RESULTS Five control chimpanzees with sera negative for HBsAg had essentially negative histologic and immunologic findings in their livers. With the exception of CH-323, all carrier chimpanzees were HBsAg seropositive for at least 4-14 years (Table 1). Results of hepatic "function" tests (including total protein, albumin, bilirubin, lactate dehydrogenase, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase and alkaline phosphatase activities) were normal, except for a mild increase in serum glutamic-pyruvic transaminase in CH-125 and CH-323. Carrier status for HBV was demonstrated by increased serum levels of HBsAg and anti-HBc and absence or low levels of antibody to HBsAg (anti-HBs). HBeAg was present in all carriers and antibody to HBeAg (anti-HBe) was found in CH-125 and CH-323. On light microscopy, four of these chimpanzees (CH-30,

CH-31, CH-125, and CH-323) and the chimpanzee previously reported (4) to be negative showed normal hepatocytes in the lobular parenchyma and mild focal activation of periodic acid/Schiff-negative sinusoidal cells. Although many portal tracts were normal, some (usually about 0.1 mm in diameter) showed dense infiltration with mononuclear cells, mainly lymphocytes intermixed with a few histiocytes and an occasional periodic acid/Schiff-positive macrophage (Fig. 1). This inflammatory exudate surrounded bile ducts with essentially normal epithelium, between which scattered lymphocytes were noted. The limiting plate of the parenchyma was in general intact. In a few areas, hepatocytes in the periportal zone had disappeared and were replaced by lymphocytes intermixed with histiocytes. This finding was most conspicuous in CH-125. However, inflammatory exudate did not extend further into the parenchyma. In some areas, neighboring portal tracts appeared linked by exudate, probably as a result of their normal bifurcation. However, the connective tissue stroma was not altered, except where hepatocytes had disappeared. The portal inflammatory reaction was most prominent in CH-323 (Fig. 1). In one specimen (CH-31), many orcein-stained hepatocytes were scattered throughout the parenchyma (Fig. 2). In three others (CH-30, CH-125, and CH-323) and in the reinvestigated animal, few orcein-positive hepatocytes were seen. The orcein-positive material was localized in the cytoplasm, was primarily perinuclear, and had a reticular appearance. Occasionally, it was aggregated in half-moon form along the cell

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FIG. 1. Percutaneous liver biopsy specimen from chimpanzee carrier of HBV, showing normal hepatocytes, mild focal activation of periodic acid/Schiff-negative sinusoidal cells, and dense infiltration of portal tract by mononuclear cells, predominantly lymphocytes. The limiting plate of the parenchyma is predominantly intact as seen in chronic persistent hepatitis. (Hematoxylin and eosin; X100.)

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Proc. Natl. Acad. Sc. USA 77 (1980)

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Table 2. Morphologic and immunologic findings in liver biopsies from chimpanzee carriers of HBV

Animal

Histologic findings*

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Immunoperoxidase reactiont HBsAg HBcAg

CH-30 Moderate + ++ CH-31 Mild ++ ++ ++ CH-116 Normal + + CH-125 Marked + ++ ++ CH-323 Marked + ++ * Abnormalities consistent with chronic persistent hepatitis. t Number of reactive cells estimated on a scale from - to ++++.

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46. FIG. 2. Liver biopsy specimen from a chimpanzee HBV carrier, showing orcein-positive material (arrows) in the cytoplasm of scattered hepatocytes. (Shikata's orcein; X100.)

membrane. The nuclei of orcein-positive cells were not altered. In the animal in which orcein-positive hepatocytes were most numerous (CH-31), the portal inflammatory reaction was least conspicuous. Immunoperoxidase studies showed focal or diffuse cytoplasmic staining for HBsAg in a somewhat higher number of cells than did the orcein stain (Fig. 3 Left). In three chimpanzees, particularly in CH-125 and the reinvestigated animal, scattered nuclei stained for HBcAg (Fig. 3 Right). Viral core antigen was never seen in cells containing surface antigen. In chimpanzee CH-1 16, whose serum contained HBV markers but failed to show biochemical alterations, the liver did not have significant histologic abnormalities. Orcein-positive cells were absent, but rare hepatocytes were weakly positive for HBcAg in the, nucleus or HBsAg in the cytoplasm (data summarized in Table 2). This chimpanzee had been reported previously to give weakly positive immunofluorescence for HBsAg or HBcAg in 1-5% of hepatocytes (20). None of the chimpanzee carriers

had increased serum a-fetoprotein, as determined by radioimmunoassay. In contrast to controls, hybridization analysis of fragments produced upon digestion of total liver DNA with EcoRI and HindIII showed several specific HBV bands in all five chimpanzee carriers (although CH-116 produced the weakest hybridization signal). HBV DNA contains one internal cleavage site for EcoRI and no internal site for HindIII (7, 8). Therefore, a comparison of results obtained with these enzymes can determine whether HBV sequences are episomal (presumably contained in replicating complexes or virions) or integrated into the host genome. Similar banding patterns were obtained upon digestion of liver DNA from CH-30 and CH-31 with EcoRl and HindIII (Fig. 4). The major bands containing HBV sequences had lengths of 3t400 and -4100 base pairs, respectively. The 3400-base-pair molecule presumably represents fully double-stranded Dane particle DNA. Characterization of the 4100-base-pair molecule will require further restriction enzyme analysis. However, because this molecule was observed with EcoRl and HindIII digestions, it does not appear to be integrated into the host genome. The 4100-base-pair band was also seen with undigested chimpanzee liver DNA (data not shown). Other minor bands in the 3000-base-pair region may represent linear versus various circular forms of HBV DNA (21).

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FIG. 3. Immunohistochemical identification of HBsAg (Left) and HBcAg (Right) in liver biopsy specimen of chimpanzee HBV carrier by the peroxidase-antiperoxidase technique. (Left) HBsAg is identified as dark-brown reaction product in the cytoplasm of 10%o of hepatocytes. (anti-HBs; X400.) (Right) HBcAg is seen as dark-brown reaction product in the nucleus of 10%o of hepatocytes. (anti-HBc; X400.)

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FIG. 4. Identification of HBV DNA sequences in nucleic acid extracts of percutaneous liver biopsy specimens from two chimpanzee

HBV carriers. Samples (20,Mg) of total liver DNA were digested overnight with either EcoRl or HindIII, electrophoresed in a 0.8%/1 agarose slab gel at 50 V for 7 hr, transferred to diazobenzyloxymethyl-paper, hybridized'to 32p-labeled pAOl HBV DNA (specific activity, 2 X i08 cpmIg), washed, dried, and autoradiographed (15). The apparent length (base pairs) of the various bands was calculated from the migration of standard restriction fragments of plasmid pBR322. Lanes A and C, CH-30; lanes B and D, CH-31.

DISCUSSION The carrier state for HBV in man is usually characterized by (i0 absence of clinical manifestations, (ii) minor, if any,, laboratory abnormalities or histologic findings in the liver, and (Mi) presence of HBsAg and anti-HBc in the serum. An uncommon variant of the carrier state, in which transmissibility is high and HBcAg is found in hepatocyte nuclei, has been described in individuals under immunosuppression therapy but is observed rarely in healthy carriers (22). It has been estimated that there are approximately 180 million HBV carriers worldwide (23). The carrier state in the chimpanzee seems to correspond to the more common carrier type in man.Most of the chimpanzees examined here were relatively old in contrast to most animals used in other tra;nsmison experiments (5, 24). In contrast-to common experience in man (25), male predominance of the carrier state was not observed in the small group of chimpanzees studied. Four of the five animals revealed histologic lesions consistent with chronic persistent hepatitis and two showed mild

Proc. Natl. Acad. Sci. USA 77 (1980)

increases in serum glutamic-pyruvic transaminaze. However, some portal tracts were normal. Human carriers of HBV may show chronic persistent hepatitis. Whether the liver from human carriers without inflammatory changes would also demonstrate chronic portal inflammation in serial sections remains to be determined. Chronic active (or "aggressive") hepatitis was not observed in the specimens examined, except for borderline findings in chimpanzee CH-125. In two animals chronic persistent hepatitis clearly followed the onset of the hepatitis B carrier state. After the carrier state had already been recognized, chimpanzees CH-30 and CH-31 had only nonspecific liver histologic findings prior to the present study (20). Chronic persistent hepatitis has also been reported in chimpanzees after infection with hepatitis non-A/non-B (4). All five carrier chimpanzees had high serum levels of HBsAg, anti-HBc, and HBeAg; the last is found infrequently in human HBV carriers (25). HBsAg was found in the cytoplasm in a varying number of hepatocytes in all animals and HBcAg was observed in hepatocellular nuclei in three animals. The orcein reaction was positive in four chimpanzees. These findings correspond to the observations in human carriers. Viral DNA was found in the nucleic acid extract of percutaneous liver biopsy specimens from all five carriers. This adds a new dimension to our ability to study the nature of HBV infection and its persistence. Hybridization of viral DNA on nitrocellulose filters with 32P-labeled plasmid HBV probe permits detection of as little as 1 pg of HBV DNA sequence. This is approximately 103 times more sensitive than any known serologic test for HBV, including radioimmunoassay. Therefore, when serum markers for HBV are absent, hybridization analysis can be used to identify viral DNA in the liver. This might be particularly helpful in evaluating patients with unexplained chronic hepatitis or cirrhosis. Because no data are available at present, characterization of viral DNA molecules for size, copy number per cell, and state of integration would be particularly valuable in following all stages in HBV infection sequentially from onset of acute hepatitis to chronic liver disease. The present study suggests that much of this information might be obtainable in the chimpanzee model. Under circumstances in which viral DNA is present in cells but viral structural proteins are not expressed, hybridization analysis will permit investigation of many aspects of viral gene regulation, viral replication, and viral oncogenicity. In the present study, we found viral DNA in two major molecular size classes, tS3400 and t4100 base pairs. Although the specific function of these DNAs has not been established, neither appears to be integrated into the host genome in this model. Further studies will be necessary to rule out this possibility fully. This is in contrast to rapidly sedimenting HBV DNA sequences reported in patients with chronic active hepatitis (26) as well as other hypotheses (27). Until more information is obtained, the present studies suggest that integration of HBV DNA may

occur only in conjunction with malignant transformation. The authors thank Dr. J. Summers (The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA) for providing recombinant pA01 HBV-plasmid, Drs. L. R. Overby and I. K. Mushahwar (Research Division, Abbott Laboratories, Chicago, IL) for providing special antigens and antibodies for HBV marker determination, and Mr. Max Shapiro (Laboratory of Experimental Medicine and Surgery in Primates, Tuxedo, NY) for coordinating the collection of liver biopsy specimens. The research was supported in part by National Institutes of Health Grant AM 17702 and National Institute of Environmental Health Sciences Grant 2 P30 E500928 06, American Cancer Society Institutional Grant IN-28U, and the Sara Chait Memorial Foundation. D.S., the recipient of a National Institutes of Health John E. Fogarty International Fellowship, is on leave from Hadassah

Medical Sciences: Shouval et al. University Hospital, Jerusalem, Israel. These studies represent a portion of a thesis by N.R.-O. to be submitted to the Sue Golding Graduate Division, Albert Einstein College of Medicine, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. M.A.G. is the recipient of Research Career Development Award Al 0035-05 from the U.S. Public Health Service.

1. Blumberg, B. S., Sutnick, A. I. & London, W. T. (1968) Bull. N.Y. Acad. Med. 44, 1566-1586. 2. Barker, L. F., Chisari, F. V., McGrath, P. P., Dalgard, D. W., Kirschstein, R. L., Almeida, J. D., Edgington, T. S., Sharp, D. G. & Peterson, M. P. (1973) J. Infect. Dis. 127,648-662. 3. Dienstag, J. L., Popper, H. & Purcell, R. H. (1976) Am. J. Pathol. 83, 131-148. 4. Popper, H., Dienstag, J. L., Feinstone, S. M., Alter, H. J. & Purcell, R. H. (1980) Virchows Arch. A Pathol. Anat. Histol. 387,91106. 5. Shikata, T., Karasawa, T. & Abe, K. (1980) Am. J. Pathol. 99, 343-368. 6. Kew, M. C. (1978) in Viral Hepatitis, eds. Vyas, G. N., Cohen, S. N. & Schmid, R..(Franklin Institute Press, Philadelphia), pp. 439-450. 7. Marion, P. L., Salazar, F. H., Alexander, J. J. & Robinson, W. S. (1980) J. Virol. 33, 795-806. 8. Chakraborty, P. R., Ruiz-Opazo, N., Shouval, D. & Shafritz, D. A. (1980) Nature (London), 286,531-533. 9. Burrell, C. J., Mackay, P., Greenway, P. J., Hofschneider, P. H. & Murray, K. (1979) Nature (London) 279,43-47. 10. Charnay, P., Pourcel, C., Louise, A., Fritsch, A. & Tiollais, P. (1979) Proc. Natl. Acad. Sci. USA 76,2222-2226.

Proc. Natl. Acad. Sci. USA 77 (1980)

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11. Sninsky, J. J., Siddiqui, A., Robinson, W. & Cohen, S. N. (1979) Nature (London) 279,346-348. 12. Valenzuela, P., Gray, P., Quiroga, M., Zaldivar, J., Goodman, H. M. & Rutter, W. J. (1979) Nature (London) 280, 815-819. 13. Pasek, M., Goto, T., Gilbert, W., Zink, B., Schaller, H., Mackay, P., Leadbetter, G. & Murray, K. (1979) Nature (London) 282, 575-579. 14. Cummings, I. W., Browne, J. K., Salser, W. A., Tyler, G. V., Snyder, R. L., Smolec, J. M. & Summers, J. (1980) Proc. Natl. Acad. Sci. USA 77, 1842-1846. 15. Shikata, T., Uzawa, T., Yoshiwara, N., Akatsuka, T. & Yamazaki, S. (1974) Jpn. J. Exp. Med. 44,25-36. 16. Thung, S. N., Gerber, M. A., Sarno, E. & Popper, H. (1979) Lab. Invest. 41, 101-105. 17. Mushahwar, I. K., Overby, L. R., Frosner, G., Deinhardt, F. & Ling, C. M. (1978) J. Med. Virol. 2,77-87. 18. Alwine, J. C., Kemp, D. J. & Stark, G. R. (1977) Proc. Natl. Acad. Sci. USA 74,5350-5354. 19. Rigby, P. W., Dickmann, M. A., Rhodes, C. & Berg, P. (1977) J. Mol. Biol. 113,237-251. 20. Krawczynski, K., Prince, A. M. & Nowoslawski, A. (1979) J. Med. Primatol. 8, 222-232. 21. Brown, P. 0. & Cozzarelli, N. R. (1979) Science 206, 10811083. 22. Gudat, F., Bianchi, L., Sonnabend, W., Thiel, G., Aenishaenslin, W. & Stalder, G. A. (1975) Lab. Invest. 32, 1-9. 23. Szmuness, W. (1978) Prog. Med. Virol. 24, 40-69. 24. Hoofnagle, J. H., Michalak, T., Nowoslawski, A., Gerety, R. J. & Barker, L. F. (1978) Gastroenterology 74, 182-187. 25. Szmuness, W. (1975) Am. J. Pathol. 81, 629-650. 26. Lutwick, L. I. & Robinson, W. S. (1977) J. Virol. 21, 96-104. 27. Hirschman, S. Z. (1975) Lancet ii, 436-438.