Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody

Virus Research, 2 (1985) 301-315 Elsevier

301

VRR 00183

Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody Antonio Ponzetto ‘,*, Paul J. Cote ‘, Eugenie C. Ford ‘, Ronald Engle ‘, John Cicmanec *, Max Shapiro *, Robert H. Purcell 3 and John L. Gerin ‘v** ‘Division of Molecular Virology and Immunology, Georgetown University Schools of Medicine and Dentistry, RockviNe, MD 20852, 2Meloy Laboratories, Inc., 2501 Research Blvd., RockviNe, MD 20850, and “Laboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205, U.S.A. (Accepted

22 January

1985)

Summary

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection. hepadnavirus, viral hepatitis, woodchuck, radioimmunoassay

Introduction

The woodchuck hepatitis virus (WHV) (Summers et al., 1978) belongs to a group of hepatotropic DNA viruses, or hepadnaviruses (Robinson, 1980), which presently * Present address: Division of Gastroenterology, Ospedale Molinette, Corso Bramante Italy. ** To whom reprint requests should be addressed at: Division of Molecular Virology 5640 Fishers Lane, Rockville, MD 20852, U.S.A. 0168-1702/85/$03.30

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

88, 10126 Torino, and Immunology,

302 includes WHV, hepatitis B virus (HBV), ground squirrel hepatitis virus (Marion et al., 1980) and duck hepatitis B virus (Mason et al., 1980). These viruses exhibit a restricted host range and share several biological characteristics (Summers, 1981). Persistent infections with HBV and WHV are associated with chronic liver disease and hepatocellular carcinoma in the natural host (Szmuness, 1978; Beasley, 1982; Gerin, 1983; Popper et al., 1982; Snyder and Summers, 1980). WHV infection of woodchucks, therefore, represents a useful model of virus-induced liver disease and its sequelae (Germ, 1979, 1984; Popper et al., 1981). Particulate forms of the woodchuck hepatitis virus surface antigen (WHsAg) circulate in the blood of infected woodchucks. The predominant forms include subviral spherical particles and filaments composed of polypeptides also found on the surface of the WHV virion; the virion itself is further composed of an internal core particle (WHcAg) that contains the viral genome and an endogenous DNA polymerase (Summers, 1981). In order to fully characterize the woodchuck/WHV model, sensitive serological assays for antigenic markers and their antibodies are required. Radioimmunoassays (RIAs) for the detection of WHsAg and its antibody (anti-WHs) have been described elsewhere (Cote et al., 1984; Wong et al., 1982). The present study describes RIAs for the detection and characterization of WHcAg and its antibody (anti-WHc).

Materials and Methods Animals and sera Chimpanzees and woodchucks (Marmota monax) were held at the National Institute of Allergy and Infectious Diseases’ animal colony (Meloy Laboratories, Rockville, Md.). Woodchucks were caught in the wild or were born of pregnant animals caught in the wild in the Maryland, Delaware and southern Pennsylvania areas. Woodchucks trapped in Pennsylvania were also provided by a commercial supplier (Hazelton/Dutchland Laboratories, Denver, Pa.). Woodchucks indigenous to New York State were held at the Cornell University woodchuck breeding colony. Serum samples were obtained for routine testing in conjunction with experimental studies described elsewhere (Ponzetto et al., 1984a). Chimpanzee sera and sera from human subjects were used as standard reference reagents. A serum sample from a ground squirrel chronically infected with the ground squirrel hepatitis virus (GSHV) was a gift of P.L. Marion and W.S. Robinson (Marion et al., 1980) and contained a high titer of antibody to the GSHV viral core antigen (GSHcAg) (Marion et al., 1983). Serology Sera were tested for HBsAg and anti-HBs using commercial radioimmunoassays (Ausria II and Ausab, respectively; Abbott Laboratories, Chicago, Ill.). For comparison with the anti-WHc assay developed presently, all sera were assayed by the Corab assay (Abbott) at 1: 10 dilutions. HBcAg was detected by the method of Purcell et al. (1974). Sera were assayed for WHV DNA using blot-hybridization

303 procedures (Berninger et al., 1982; Mitamura et al., 1982). Serum DNA polymerase (Kaplan et al., 1973), WHsAg (Cote et al., 1984; Wong et al., 1982), and anti-WHs (Wong et al., 1982) were detected as described; WHcAg and anti-WHc were detected by methods described below. HBcAg purification

Dane particles from DNA-polymerase positive human sera were pelleted twice through 20% sucrose/Tris-HCI (0.01 M, pH 7.3) and the final pellets were resuspended in phosphate-buffered saline (PBS; 0.85% NaCl/O.Ol M phosphate buffer, pH 7.3) to yield a 20 X concentrate of the original sera; HBcAg was then released by adjusting the preparation to 0.3% Nonidet-P 40 (Gerin et al., 1975; Shih et al., 1980). HBcAg was also obtained from chimpanzee liver as described previously (Budkowska et al., 1977); similar methods were used in the isolation of WHcAg from woodchuck liver (see below). Purification and characterization of WHcAg

Livers were obtained from four chronic carrier woodchucks, from two woodchucks during the acute phase of a WHV infection, and from woodchucks ~thout markers of WHV infection. All liver tissues were analyzed for the presence of WHcAg by direct staining with fluorescein isothiocyanate-conjugated anti-WHc immunoglobulin (Ponzetto et al., 1984a). At autopsy, livers were perfused with sterile saline and the non-tumor tissue was cut into small cubes, and snap-frozen in liquid nitrogen. To prepare liver homogenates, the tissue was thawed, minced in chilled PBS (1: 10, w/v), homogenize in a Sorvall omni~xer (10 min, low speed; DuPont Instruments), and passed twice through a triple layer of surgical gauze. The filtrate was centrifuged for 10 min at 650 X g and the supernate was stored at - 70°C. WHcAg in liver homogenates from one chronic carrier woodchuck, and from one animal during the acute phase of experimental WHV infection, was characterized by ultracentrifugation in cesium chloride gradients. Briefly, 0.8 ml of homogenate was layered onto a discontinuous density gradient of CsCl and centrifuged (35 000 ‘pm, 18 h, 4°C Beckman SW41 rotor). The bottoms of the tubes were punctured and 0.4 ml fractions were collected; fractions were analyzed by refractometry and RIA. The peak fractions containing WHcAg were pooled and centrifuged in a second step using the above conditions. Gradient fractions with WHcAg activity by RIA were examined by electron microscopy after negative staining with 1% phosphotungstic acid. Preparation and characterization of anti- WHc reagents

Serum samples from a chronic WHV carrier woodchuck were combined and the sample was pelleted by high-speed centrifugation (40000 t-pm, 5 h, 4°C Ti 50 rotor) to remove viral particles and possible immune complexes. The immuno~obulin fraction of the serum supernate was obtained by ammonium sulfate salt precipitation (40% saturation, pH 7.0, 30 min, 4°C) centrifugation (1000 x g, 30 min), and dialysis of the pellet against PBS. The preparation was adjusted to 10 mg protein per ml and stored at -70°C. Additional samples from other animals were prepared

304 similarly. Anti-WHc immunoglobulin (5 ~1) was radioiodinated with 0.5 mCi of Na”‘I using the chloramine-T procedure of Hunter and Greenwood (1962). The specific activities of these preparations were approximately 15 pCi per pg protein. Anti-WHc in sera from chronic carrier animals was partially characterized by gel filtration over a Sephacryl 300 column (Pharmacia), by rate sedimentation in sucrose density gradients (Fudenberg and Kunkel, 1957) by sensitivity to reducing agents (Wong et al., 1982) and by protein-A affinity chromatography (Ey et al., 1978). RIA for WHcAg

The test was developed according to solid-phase RIA techniques used in the assay for HBcAg (Purcell et al., 1974). Briefly, polyvinylchloride microtiter plate wells were coated with 100 ~1 of the ultracentrifuged anti-WHc serum (1 : 1000 dilution, 6 h, 25’C) and then post-coated with 250 ~1 of 1% bovine serum in PBS (12 h, 4°C). Plates were washed with PBS, air dried, and stored at - 70°C. For WHcAg testing, 50 ~1 of sample was added to each well and incubated at 4°C for 24 h. After washing, 2 x lo5 cpm/50 ~1 of ‘251-anti-WHc was added (4 h, 37°C). The wells were then washed and analyzed for bound radioactivity. Results were expressed as the ratio of cpm for the test sample to that for a negative sample (S/N ratio); S/N ratios greater than 2.1 were considered positive for WHcAg. Tests using shorter incubation times (e.g., 1 h) were suitable for rapid screening when compared to standard assays; however the S/N values obtained were generally 50-60% lower. RIA for anti- WHc

Anti-WHc coated wells were preincubated with 50 ~1 of liver homogenate from a chronic WHV carrier woodchuck. The amount of WHcAg reactive liver homogenate used was determined on the basis of the titration curve (see Fig. 2; WC31). In the present study a 1 : 10 dilution of the 10% (w/v) liver homogenate was employed. After incubation with liver homogenate for 24 h at 4°C wells were washed and the plates were either used directly or stored at - 70°C. For testing, 10 ~1 of serum and 90 ~1 of ‘2sI-anti-WHc (2 x lo5 cpm in fetal calf serum) were added to each well and incubated together for 18 h at 25°C. Wells were washed and counted and results were expressed as the percent inhibition of bound counts (cpm bound for test sample/cpm bound in the presence of normal woodchuck serum X 100%). Values greater than 50% inhibition were considered positive for anti-WHc. The anti-core titer is the reciprocal serum dilution that produced a 50% inhibition of binding of the iodinated probe.

Results RIA and characterization

of WHcAg from woodchuck

liver

Microtiter plate wells coated with serial four-fold dilutions of ultracentrifuged anti-WHc serum were inoculated with 50 ~1 of a standard WHcAg preparation and The optimal dilution of bound antigen was detected with ‘251-labeled anti-WHc. anti-WHc serum needed to construct the solid phase was between 1 : 1024 and

305 1: 4096 (Fig. 1); for subsequent studies, the anti-WHc serum was used at a 1: 1000 dilution. Serial two-fold dilutions of two WHcAg-positive liver homogenates, one from a chronic WHV carrier woodchuck and another from an animal during acute phase of WHV hepatitis, were tested in the assay. Comparison of the curves in the linear region (50% of the maximum S/N value) (Fig. 2) indicated a 32-fold difference in WHcAg concentration between the two preparations. The maximum S/N values of the two preparations were identical. An optimal dilution of liver homogenate was used in subsequent RIAs for anti-WHc. A liver homogenate from a chronic WHV carrier woodchuck was ultracentrifuged in a cesium chloride gradient; fractions were assayed for WHcAg by RIA and examined by electron microscopy. WHcAg banded at 1.31 g/cm3 CsCl (Fig. 3A) and the WHcAg peak fraction was shown by electron microscopy to contain core particles in immune aggregates formed by the reaction of cores with serum anti-WHc (Fig. 4A); according to staining patterns, both ‘empty’ and ‘full’ core particles were found in the immune complexes. WHcAg-positive fractions from Fig. 3A were recentrifuged and WHcAg again banded at 1.31 g/cm3 (Fig. 3B). WHcAg from a liver homogenate from a woodchuck during the acute phase of WHV infection (i.e., prior to the development of serum anti-WHc) had a bimodal distribution of WHcAg activity in the gradient (Fig. 3C). The first peak (1.353 g/cm3 CsCl) was enriched for electron-dense core particles (Fig. 4B) when compared to the second peak (1.31

rm -

cl

L-

1

3

5

I

9

11

n

RECIPROCAL LOG, DILUTION

Fig. 1. Conditions for the detection of WHcAg by solid-phase RIA. The wells of polyvinyl microtiter plates were coated with 0.1 ml of the appropriate dilution of ultracentrifuged anti-WHc serum. A 10% (w/v) liver homogenate from a chronic WHV carrier woodchuck was used as source of core antigen. Bound antigen was detected with ‘251-labeled anti-WHc. Fig. 2. Titration of WHcAg by solid-phase RIA. Liver homogenates from a chronic carrier woodchuck (WC 31; A) and from a woodchuck undergoing the acute phase WHV infection (WC 131; 0) were diluted as shown and 50 microliters were incubated in wells coated with 1: 1000 dilution of the standard anti-WHc serum (24 h, 4°C). After washing, each well was developed with 50 ~1 of ‘251-labeled anti-WHc (2 x lo5 cpm, 4 h, 37’C). Bound radioactivity is expressed as a sample to negative (S/N) ratio, where N represents the cpm for wells incubated with liver homogenates from WHV negative woodchucks.

5

10 FRACTION

5

15

10 FRACTION

NUMBER

15 NUMBER

FRACTION NUMBER

Fig. 3. (A) Cesium chloride gradient centrifugation of woodchuck core antigen particles from liver of a chronic carrier animal. A 0.8 ml aliquot of the liver homogenate was layered onto a discontinuous CsCl solution (density 1.15-1.40 g/cm3) and centrifuged for 18 h at 35000 rpm at 4°C in a Beckman SW41 rotor. Fractions were collected by bottom puncture and were assayed for density by refractometry (Cl) and for WHcAg activity (A) by RIA (at 1: 10 dilution). (B) The peak fraction was subjected to a second ultracentrifugation step. (C) CsCl gradient centrifugation of WHV core particles from liver of a woodchuck during the acute phase of WHV infection, prior to the appearance of serum anti-WHc. (D) Rebanding in CsCl of the 1.31 g/cm3 peak from C.

307

Fig. 4. Electron microscopy of purified WHcAg particles. Preparations were negatively stained with 1% phosphotungstic acid. Ma~ification: 150000X. (a) Core particles from liver of a chronically infected woodchuck banding at 1.31 g/cm3 CsCl (see Fig. 3A); note presence of bound anti-WHc antibodies. (b) Core particles from woodchuck liver banding at 1.353 g/cm3 CsCl (see Fig. 3C); anti-WHc antibodies were not evident in the preparation from this animal. The average diameter of core particles in a and b was 30 nm.

g/cm3) which contained mostly empty core particles (not shown). No particle-bound antibodies were observed in either peak fraction by electron microscopy. The second peak from this preparation again banded at 1.31 g/cm’ (Fig. 3D). Analogous to the HBV system, the radioimmunoassay detects particles from WHV-infected liver which have the mo~holo~cal and biophysical properties of WHV cores. While liver homogenates from WHV carrier woodchuck contain anti-WHc, this activity does not interfere with the detection of WHcAg by this RIA. RIA and characterization of anti- WHc in woodchucks The RIA for WHcAg was modified to develop a competitive binding assay for

TABLE

1

CORRELATION TION

OF SERUM

ANTI-WHc

ACTIVITY

WITH OTHER

WHV markers

Anti-WHc Anti-WHc

positive negative

a WHV markers are DNA polymerase, b Animal was anti-WHs positive.

MARKERS

a

Positive

Negative

88 lb

0 85

WHsAg,

anti-WHs

OF WHV INFEC-

and WHV DNA in the liver.

308 anti-WHc. The assay was specific for the detection of anti-WHc; the latter observation was based on titration analysis of anti-core positive sera and on a retrospective survey of WHV-infected colony woodchucks. All woodchuck sera negative for WHV markers were negative for anti-WHc (n = 85); in contrast, 87/88 sera positive for one or more WHV markers were also anti-WHc positive (Table 1). Only one animal with anti-WHs was negative for anti-WHc, indicating that the present RIA does not cross-react with anti-WHs. In this regard, we have also noted that anti-WHs positive sera from woodchucks vaccinated with WHsAg do not cross-react in the present RIA (unpublished data). End-point titers of anti-WHc were compared in sera from chronically-infected and recovered (anti-WHs positive) woodchucks. Higher titers of anti-WHc were found in the chronic carriers compared to recovered animals; we also noted that animals convalescent from recent hepatitis that had not seroconverted to anti-WHs exhibited titers midway between those of chronic carriers and recovered animals (Fig. 5; see also Ponzetto et al., 1984a). Differences among plateau values for maximum inhibition by various test sera were also observed (Fig. 5). The anti-WHc activities of sera from four chronic carriers (WC No. 8, 17, 21, 69) were resistant to treatment with reducing agents (dithiothreitol, 2-mercaptoethanol), which, in other systems, disrupt IgM but not IgG. Anti-WHc from two of the above woodchucks was found in both the 7s and 19s fractions of sucrose density gradients,

Fig. 5. Titration of three anti-WHc positive sera by solid-phase competitive inhibition RIA. Serum from chronic WHV carrier (WC 8; l), serum from convalescent, anti-WHs positive woodchuck at week 14 following experimental infection with WHV (WC 13; 0). serum from wild-caught, anti-WHs positive woodchuck (WC 51; n).

309 indicating the presence of both IgM and IgG anti-WHc antibodies. In addition, anti-WHc activity in serum from one chronic carrier (WC No. 35) was found in the IgG fraction eluted from a Sephacryl 300 column. Anti-WHc activity was also isolated by fractionation of centrifuged (40000 rpm, 5 h) chronic carrier serum over Staphylococcus aureus protein A columns (Ey et al., 1978); the activity was present in two putative IgG peaks, one eluting between 5.0 and 4.0, and the other eluting at pH 3.2. Taken together, the above observations suggest that the response to WHV involves the production of anti-WHc IgM and possibly two different isotypes of anti-WHc IgG. Core and anti-core reactivities in heterologous and combination RIAs Anti-WHc positive woodchuck sera were tested for anti-HBc activity in the CORAB Assay (Abbott Labs). Woodchuck anti-WHc was not detected in this test, although serum from one animal was marginally positive (Table 2). Human anti-HBc was not detected in the present assay for anti-WHc (Table 2). Cross-reacting anti-WHc activity was detected in serum from one ground squirrel chronically infected with GSHV, but only at less than 1 : 25 serum dilution (data not shown). Six WHcAg-positive liver preparations and 10 HBcAg-positive preparations were analyzed by RIAs. In these assays, homologous and heterologous antibody reagents were used in different combinations as catch antibody and probe. When tested in completely heterologous assays, no cross-reactivities were observed between WHcAg and HBcAg; this was consistent for several different anti-WHc and anti-HBc sera used as catch and probe antibodies (Table 3). We were, however, able to demonstrate partial cross-reactivity for one woodchuck and one human core preparation with combination RIAs. In these assays, anti-HBc and anti-WHc were used alternately as catch antibody and probe. The anti-HBc reagent recognized WHcAg when used as an immunoadsorbent or probe in combination with the anti-WHc reagent; the anti-WHc reagent recognized HBcAg when used as an immunoadsorbent, but not when used as an iodinated probe (Table 4).

TABLE LACK Anti-core

2 OF CROSS-REACTIVITY (+)

Woodchuck= Human d

sera

BETWEEN Anti-WHc 63 0

RIAs FOR ANTI-WHc RIAa

AND ANTI-HBc Anti-HBc

RIA b

1 23

a Standard anti-WHc RIA at 1: 10 dilution. b CORAB assay (Abbott) at 1 : 10 dilution. ’ Represents 18 chronic WHV carrier woodchucks with anti-WHc titers between lo4 and 105.3 and 46 convalescent woodchucks with anti-WHc titers between 10’ and 104; the one CORAB positive woodchuck was a chronic WHV carrier with an anti-WHc titer of 105; the N/S value was 2.1 (borderline). d Samples are from chronic type B hepatitis patients with anti-HBc titers between lo3 and lo4 by CORAB.

310 TABLE LACK

3 OF CROSS-REACTIVITY HBcAg C

84

AND

HBcAg

IN HETEROLOGOUS

WHcAg E

25

Woodchuck 8 _ 17 21 _ 35 _

WHcAg

a D

Human anti-HBc ’ A 80 19 B

BETWEEN

F

G

H

I

7

J

b 15

6

80

44

25

8

142

-

-

10

71

21

52

13

112

-

-

d _ _ _

_ _

_ _

_ _ _

_ _

_ _ _ _

26 31 35 29

anti-WHc _ _

RIAs

7.6 7.4 5.2 7.8

31

40

62

_

_

_

28 36 37 39

26 32 36 30

ND ND ND 63

Values are S/N ratios; ratios greater than 2.1 are considered positive; average S/N ratio for negative samples (-) was 1.02; range 0.6-1.6. HBcAg preparations C through J were obtained from different Dane particle concentrates (see Materials and Methods). WHcAg preparations 7, 15, 31, 40 and 62 were obtained from liver homogenates; numbers represent the NIAID woodchuck identification number; woodchuck No. 15 liver was obtained during the late phase of a naturally occurring acute hepatitis; the remaining animals were chronic carriers of WHV. Two sources of human anti-HBc used in the sandwich RIAs for core antigens (A and B). Four sources of woodchuck anti-WHc used in the sandwich RIAs for core antigens (WC Nos. 8,17,21, 35).

TABLE

4

PARTIAL

CROSS-REACTIVITIES

Solid-phase antibody

Anti-WHc Anti-HBc Values are S/N

‘251-labeled

WHcAg

probe for bound

AND HBcAg

IN MIXED

RIAs

antigen HBcAg

WHcAg ‘251-anti-WHc

‘251-anti-HBc

‘251-anti-WHc

‘251-anti-HBc

33.3 10.8

11.6 1.1

1.0 1.7

17.2 52.1

ratios;

ratios greater

than 2.1 are considered

positive.

Discussion In its natural host, the woodchuck hepatitis virus causes acute liver disease (hepatitis) and a broad spectrum of chronic sequelae (chronic hepatitis and hepatocellular carcinoma) (Summers et al., 1978; Gerin, 1979; Popper et al., 1981; Snyder and Summers, 1980; Ponzetto et al., 1984a; Frommel et al., 1984). This range of liver disease mimics that caused by the hepatitis B virus in man. In the HBV system, the status of HBcAg and anti-HBc is required to fully characterize the occurrence and course of disease. For example, numerous investigators have shown that the presence of HBcAg in liver is correlated with HBV infection (Arnold et al., 1975; Bianchi and Gudat, 1979; Huang and Neurath, 1979; Yamada et al., 1978; Barker et al., 1973).

311 In addition, serum anti-HBc is a reliable marker for past or ongoing HBV infection (Hoofnagle et al., 1979a). In vaccine efficacy studies by Szmuness et al. (Szmuness, 1979; Szmuness et al., 1980), anti-HBc was the only serological event to demonstrate that some of the subjects were later infected by HBV. The anti-HBc marker has also been used to reassess the efficacy of hyperimmune gammaglobulin in the prevention of HBV infection (Krugman et al., 1979; Hoofnagle et al., 1979b; Grady et al., 1982). Similar methods to detect WHcAg and anti-WHc in woodchucks would therefore permit further development of this model of human disease. In the present study, we describe specific and sensitive RIAs for the detection of WHcAg and the homologous antibody (anti-WHc). The RIA for WHcAg was able to detect core antigen in liver homogenates from marmots undergoing acute or chronic WHV infections. Core particles from liver homogenates were found by electron microscopy at the peak of WHcAg activity in CsCl gradients. Both heavy (1.35 g/cm3) and light (1.31 g/cm3) core particles were found in the WHV system in association with the acute phase of infection. The existence of heavy and light cores in the WHV system parallels observations in the HBV system (Gerin et al., 1975; Moritsugu et al., 1975; Shih et al., 1980; Hess et al., 1981) in which the heavy cores contain virus DNA. In a previous study (Ponzetto et al., 1984a), we found that detection of WHcAg in liver by immunofluorescence was correlated with WHV infection; the latter is also consistent with observations in HBV-infected humans and chimpanzees (Arnold et al., 1975; Bianchi and Gudat, 1979; Huang and Neurath, 1979; Yamada et al., 1978). A serological survey of NIAID woodchucks demonstrated positivity for anti-WHc in all but one animal having at least one marker of past or ongoing WHV infection. This particular animal had low titers of anti-WHs, possibly indicating a long elapsed period since infection. Animals recovered from acute WHV infection (i.e., anti-WHs positive) have been shown to exhibit low titers of anti-WHc (lo*) (Ponzetto et al., 1984a; Fig. 5). Thus, anti-WHc in this single woodchuck may have been present, but at very low titers (i.e., beyond the limit of sensitivity of the present assay). In contrast, anti-WHc titers in sera of chronically infected animals and in animals during late phase of acute experimental infection were in the range of lo5 to 104, respectively (Ponzetto et al., 1984a; Fig. 5). We have shown that the commercial assay for anti-HBc (CORAB; Abbott Laboratories) does not detect anti-WHc in most of our woodchucks; the latter observation is also consistent with that made by Millman et al. (1982), who found only a small percentage of carrier woodchucks with ‘anti-HBc activity’ at 1 : 2 dilutions of serum. Of 64 anti-WHc positive sera tested in the CORAB assay, only one was positive (Table 2). The anti-WHc titer in the latter serum sample was greater than lo5 by the WHV-specific assay, but was negative in the CORAB assay beyond a 1 : 10 dilution. This indicated at least a thousand-fold difference in sensitivity between HBV and WHV specific RIAs. Previous observations reporting cross-reactivity between human and woodchuck core antigen and antibody could be accounted for by the presence of cross-reacting antibodies in high titered anti-WHc sera from chronic WHV carrier woodchucks (Werner et al., 1979; Stannard et al., 1983). These reports were based on electron microscopic examination of immunoprecipitates, which detects antigen-antibody

312 interaction. On the other hand, detection of cross-reacting antibodies in serum with competitive inhibition methods is limited by the nature of the detecting probe; for example, cross-reactive antibodies in serum may ordinarily bind to a heterologous antigen, but not displace a radiolabeled homologous probe. The latter could arise due to lower relative antibody affinity or the absence of a counterpart antibody in the probe. Interestingly, serum from a ground squirrel chronically infected with GSHV demonstrated far less anti-WHc activity in our competition RIA than would be expected considering the close phylogenetic relationships between GSHV and WHV and their respective hosts. To further investigate the issue of cross-reactivity between HBV and WHV core antigens, we tested HBcAg and WHcAg in the heterologous assays. The standard RIA for HBcAg did not recognize highly positive WHcAg preparations, and vice versa (Table 3). When anti-WHc and anti-HBc were used alternatively as immunoadsorbent and probe, partial cross-reactivities were detected (Table 4). The anti-HBc appeared to be more efficient in recognizing WHcAg when compared to anti-WHc recognition of the HBcAg. A similar phenomenon was observed upon immunofluorescent examination of liver tissues; the human fluoresceinated anti-HBc could stain woodchuck substrates (Gerin, 1979), while anti-WHc did not stain HBcAg positive substrates (Ponzetto, unpublished observations). The overall observations suggest differences in the affinities of cross-reactive antibodies present in these reagents. With the present development of RIAs for WHcAg and anti-WHc, a full series of tests are now available to characterize the natural history of WHV infection in woodchucks. Methods to detect other markers of WHV infection have been described and include those for WHsAg and anti-WHs (Cote et al., 1984; Wong et al., 1982) DNA polymerase (Kaplan et al., 1973) and WHV DNA in serum (Ponzetto et al., 1984b) and tissues (Rogler and Summers, 1982; Ogston et al., 1982; Mitamura et al., 1982). Since both IgM and IgG anti-WHc have been found in the woodchuck model, it will be possible to develop specific assays for anti-WHc IgM; as in the case for anti-HBc IgM in man (Cohen, 1978; Overby et al., 1983), the latter should provide an important correlate for recently acquired WHV infection (Kryger et al., 1982) and for the identification of potential WHV carriers that are seronegative for WHsAg and anti-WHs (Hoofnagle et al., 1978). All of the currently available assays noted above are analogous to those used in the characterization of HBV infection in man and are critical to the full development of the woodchuck/WHV model of virus-induced liver disease in man. Further studies of the natural history of WHV infection will lead to an improved understanding of the mechanisms responsible for viral pathogenesis and persistence during chronic infection, and their relationship to the development of hepatocellular carcinoma.

Acknowledgements This research was supported by contract NOl-AI-22665 between the National Institute of Allergy and Infectious Diseases (NIAID) and Georgetown University.

313 The NIAID animal colony is supported by contract NOl-AI-02640 between NIAID and Meloy Laboratories, Inc. A.P. is the recipient of a grant from Regione Piemonte. We thank Charlotte L. Langer for assistance in screening woodchuck serum samples, and Cynthia French for her secretarial assistance in the preparation of this manuscript.

References Arnold, W., Meyer zum Buschenfelde, K.H., Hess, G. and Knolle, J. (1975) The diagnostic significance of intrahepatocellular hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and IgG for the classification of inflammatory liver diseases. Klin. Wochenschr. 53, 1069-1074. Barker, L.F., Chisari, F.C., McGrath, P.P., Dalgard, D.W., Kirschstein, R.L., Almeida, J.D., Edgington, T.S., Sharp, D.G. and Peterson, M.R. (1973) Transmission of type B viral hepatitis to chimpanzees. J. Infect. Dis. 127, 648-662. Beasley, R.P. (1982) Hepatitis B virus as the etiologic agent in hepatocellular carcinoma: epidemiologic considerations. Hepatology 2 (Suppl.), 21-26. Berninger, M., Hammer, M., Hoyer, B. and Germ, J.L. (1982) An assay for the detection of the DNA genome of hepatitis B virus in serum. J. Med. Virol. 9, 57-68. Bianchi, L. and Gudat, F. (1979) Immunopathology of hepatitis B. Prog. Liver Dis. 6, 371-392. Budkowska, A., Shih, J.W.-K. and Germ, J.L. (1977) Immunochemistry and polypeptide composition of hepatitis B core antigen (HBcAg). J. Immunol 118, 1300-1305. Cohen, B.J. (1978) The IgM antibody response to the core antigen of hepatitis B virus. J. Med. Virol. 3, 141-149. Cote, P.J., Engle, R.E., Langer, C.A., Ponzetto, A. and Germ, J.L. (1984) Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays. J. Virol. 49, 701-708. Ey, P.L., Prowse, S.J. and Jenkin, CR. (1978) Isolation of pure IgGt, IgGz,, and IgG,, immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15, 429-436. Frommel, D., Crevat, D., Vitvitsky, L., Pichoud, C., Hantz, O., Chevalier, M., Grimaud, J.A., Lindberg, J. and Trepo, C.G. (1984) Immunopathologic Aspects of Woodchuck Hepatitis. Am. J. Pathol. 115, 125-134. Fudenberg. H.H. and Kunkel, H.G. (1957) Physical properties of the red cell agglutinins in acquired hemolytic anemia. J. Exp. Med. 106, 689-702. Germ, J.L. (1979) The eastern woodchuck (Marmota monux): A potential animal model for virus-induced disease in man. In: Systemic Effects of HBsAg Immune Complexes (Bartoli, E., Chiandussi, L. and Sherlock, S., eds.), pp. 171-175. Piccin Medical Books, Padua. Germ, J.L. (1983) Hepatitis B virus and primary hepatocellular carcinoma. Gastroenterology 84, 869-870. Germ, J.L. (1984) The woodchuck (Marmofa monax): An animal model of hepatitis B virus-like infection and disease. In: Advances in Hepatitis (Chiasari, F., ed.), pp. 40-48. Masson Publishing, Inc., New York. Germ, J.L., Ford, E.C. and Purcell, R.H. (1975) Biochemical characterization of Australia antigen: evidence for defective particles of hepatitis B virus. Am. J. Pathol. 81, 651-662. Grady, G.F., Cyr, K.M. and Werner, B.G. (1982) Serologic reanalysis of the NHLBI needle-stick HBIG/ISG study. In: Viral Hepatitis. 1981 International Symposium (Szmuness, W., Alter, H.J. and Maynard, J.E., eds.), pp. 753-754. The Franklin Institute Press, Philadelphia. Hess, G., Arnold, W. and Meyer zum Buschenfelde, K.-H. (1981) Demonstration and partial characterization of an intermediate HBcAg (Dane particle) population. J. Virol. 7, 241-250. Hoofnagle, J.H., Seeff, L.B., Bales, Z.B., Zimmerman, H.J. and the Veteran Administration Hepatitis Cooperative Study. (1978) Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. N. Engl. J. Med. 298, 1379-1383. Hoofnagle, J.H., Seeff, L.B., Bales, Z.B., Gerety, R.J. and Tabor, E. (1979a) Serologic responses in HB. In: Viral Hepatitis (Vyas, G.N., Cohen, S.H. and S&mid, R., eds.), pp. 219-242, The Franklin Institute Press, Philadelphia.

314 Hoofnagle, J.H., Seeff, L.B., Bales, Z. and Zimmerman, H.J. (1979b) Passive active immunity from hepatitis B immunoglobulins. Ann. Int. Med. 91, 813-818. Huang, S.N. and Neurath, A.R. (1979) Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to its significance in liver injury. Lab. Invest. 40, l-9. Hunter, W.M. and Greenwood, F.C. (1962) Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature (London) 194, 495-497. Kaplan, P.M., Greenman, R.L., Germ, J.L., Purcell, R.H. and Robinson, W.S. (1973) DNA polymerase associated with human hepatitis B antigen. J. Virol. 12, 995-1005. Krugman, S., Overby, L.R., Mushahwar, I.K., Ling, C.-M., Frosner, G.G. and Deinhardt, F. (1979) Viral Hepatitis, Type B. N. Engl. J. Med. 300, 101-106. Kryger, P., Aldershville, J., Mathiesen, L.R. and Nielsen, J.O. (1982) Acute type B hepatitis among HBsAg negative patients detected by anti-HBc IgM. Hepatology 2, 50-53. Marion, P.L., Oshiro, L.S., Regnery, D.C., Scullard, G.H. and Robinson, W.S. (1980) A virus of Beechey ground squirrels that is related to hepatitis B virus of humans. Proc. Natl. Acad. Sci. U.S.A. 77, 2941-2945. Marion, P.L., Knight, S.S., Salazar, H., Popper, H. and Robinson, W.S. (1983) Ground squirrel hepatitis virus infection. Hepatology 3, 519-527. Mason, W.S., Seal, G. and Summers, J. (1980) Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J. Virol. 36, 829-836. Millman, I., Halbherr, T. and Simons, H. (1982) Immunological cross-reactivities of woodchuck and hepatitis B viral antigens. Infect. Immun. 35, 752-757. Mitamura, K., Hoyer, B., Ponzetto, A., Nelson, J., Purcell, R.H. and Germ, J.L. (1982) Woodchuck hepatitis virus DNA in woodchuck liver tissues. Hepatology 2 (Suppl.), 47-50. Moritsugu, Y., Gold, J.W.M., Wagner, J., Dodd, R.Y. and Purcell, R.H. (1975) Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation. J. Immunol. 114, 1792-1798. Ogston, W.C., Jonak, G.J., Rogler, C.E., Astrin, S.M. and Summers, J. (1982) Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinoma of woodchucks. Cell 29, 385-394. Overby, L.R., Mushahwar, I.K., Chau, K. and Decker, R.H. (1983) Serological markers of viral hepatitis. In: Viral Hepatitis (Overby, L.R., Deinhardt, F. and Deinhardt, J., eds.), pp. 115-117. Marcel Dekker, New York. Ponzetto, A., Cote, P.J., Ford, E.C., Purcell, R.H. and Germ, J.L. (1984a) Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus. J. Virol. 52, 70-76. Ponzetto, A., Cote, P.J., Popper, H., Hoyer, B.H., London, W.T., Ford, E.C., Bonino, F., Purcell, R.H. and Germ, J.L. (1984b) Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck. Proc. Natl. Acad. Sci. U.S.A. 81, 2208-2212. Popper, H., Shih, J.W.-K., Germ, J.L., Wong, D.C., Hoyer, B., London, W.T., Sly, D.L. and Purcell, R.H. (1981) Woodchuck hepatitis and hepatocellular carcinoma: correlation of histologic with virologic observations. Hepatology 1, 91-98. Popper, H., Gerber, M.A. and Thung, S.N. (1982) The relation of hepatocellular carcinoma to infection with hepatitis B and related viruses in man and animals. Hepatology 2 (Suppl.), l-9. Purcell, R.H., Germ, J.L., Almeida, J.D. and Holland, P.V. (1974) Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Inter-virology 2, 231-243. Robinson, W.S. (1980) Genetic variation among hepatitis B and related viruses. Ann. N.Y. Acad. Sci. 354, 371-378. Rogler, C.E. and Summers, J. (1982) Novel forms of woodchuck hepatitis virus DNA isolated from chronically infected woodchuck liver nuclei. J. Virol. 44, 852-863. Shih, J.W.-K., Hess, G., Kaplan, P.M. and Germ, J.L. (1980) Characterization of the hepatitis B virus (Dane particles). J. Virol. Methods 1, 47-59. Snyder, R.L. and Summers, J. (1980) Woodchuck hepatitis virus and hepatocellular carcinoma. In: Viruses in Naturally Occurring Tumors. Cold Spring Harbor Conference on Cell Proliferation (Essex, M., Todaro, G. and zur Hausen, H., eds.), pp. 447-457. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Stannard, L.M., Hantz, 0. and Trepo, C. (1983) Antigenic cross-reactions between woodchuck hepatitis virus and human hepatitis B virus shown by immune electron microscopy. J. Gen. Virol. 64, 975-980.

315 Summers, J. (1981) Three recently described animal virus models for human hepatitis B virus. Hepatology 1, 179-183. Summers, J., Smolec, J.M. and Snyder, R.L. (1978) A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc. Natl. Acad. Sci. U.S.A. 75, 4533-4537. Szmuness, W. (1978) Hepatocellular carcinoma and Hepatitis B virus: evidence for a causal association. Progr. Med. Virol. 24, 40-69. Szmuness, W. (1979) Large scale efficacy trials of hepatitis B vaccines in the USA: baseline data and protocols. J. Med. Virol. 4, 327-340. Szmuness, W., Stevens, C.E., Harley, E.J., Zang, E.A., Oleszko, W.R., William, DC., Sadovsky, R., Morrison, J.M. and Kellner, A. (1980) Hepatitis B vaccine. Demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N. Engl. J. Med. 303, 833-841. Werner, B.G., Smolec, J.M., Snyder, R.L. and Summers, J. (1979) Serological relationship of woodchuck hepatitis virus to human hepatitis B virus. J. Virol. 32, 314-322. Wong, D.C., Shih, J.W.-K., Purcell, R.H., Germ, J.L. and London, W.T. (1982) Natural and experimental infection of woodchucks with woodchuck hepatitis virus, as measured by new, specific assays for woodchuck surface antigen and antibody. J. Clin. Microbial. 15, 484-490. Yamada, G., Feinberg, L.E. and Nakane, P.K. (1978) Hepatitis B cytologic localization of virus antigens and the role of the immune response. Hum. Pathol. 9, 93-109. (Manuscript

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