Acute Non-A, Non-B Hepatitis

Transcription

1 GASTROENTEROLOGY 76: , 1979 Acute Non-A, Non-B Hepatitis Prolonged Presence of the nfectious Agent in Blood EDWARD TABOR, M.D., MLTON APRL, D.V.M., LEONARD B. SEEFF, M.D., and ROBERT J. GERETY, M.D., Ph.D. Hepatitis Branch, Division of Blood and Blood Products, and Division of Pathology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland; Department of Medicine, Veterans Administration Hospital, Washington, D.C.; and Georgetown University School of Medicine, Washington, D.C. Non-A, non-b hepatitis, previously transmitted to chimpanzees by inoculation of human serum, was serially transmitted through a second and third passage to additional chimpanzees using serum drawn during acute non-a, non-b hepatitis. Sera obtained at weeks 4 and 5 after inoculation from two different chimpanzees, and from one chimpanzee at week 13 after inoculation, were shown to cause elevation of serum aminotransferase levels and abnormal liver biopsies in recipient chimpanzees, with no serologic evidence of hepatitis A or B, cytomegalovirus, or Epstein-Barr virus infection. Serum obtained 3 wk after inoculation did not cause elevation of aminotransferase levels in the recipient chimpanzee, although a single abnormal biopsy was obtained. Thus, the non-a, non-b hepatitis agent was present in serum during acute disease near the time of the first aminotransferase elevation (week 4; perhaps also week 3), and persisted at least until 1 week after the peak aminotransferase level (week 13). Since the discovery of hepatitis B surface antigen (HBsAg)t and hepatitis A virus {HAV),2 it has become evident that a third type of hepatitis exists, for which no agent has yet been identified. This has been designated "non-a, non-b hepatitis".3.4 t is now apparent that non-a, non-b hepatitis is responsible for greater than 89% of the cases of post- Received September 18, Accepted November 21, Address requests for reprints to: Edward Tabor, M.D., Hepatitis Branch, Bureau of Biologics, 8800 Rockville Pike, Bethesda, Maryland This study was presented in part at the Symposium on Viral Hepatitis, San Francisco, California, March 17, 1978, and at the nternational Transfusion Society, Paris, France, July 28, The authors thank Mr. A. J. Shawver, Mr. D. Gilbert, Mr. F. Mitchell, and Mr. J. Cogan for technical assistance. transfusion hepatitis in the United Stafes, since the requirement that HBsAg-positive donor blood be excluded.' s The existence of a transmissible agent in this disease was suggested by a recent study of stored serum specimens from studies in human volunteers conducted in the 1950s,6 and was confirmed by the transmission of non-a, non-b hepatitis to chimpanzees 7 8 by inoculation of human serum, and by the first successful serial passage of the agent to other chimpanzees. 9 This study was conducted to confirm the transmissibility of non-a, non-b hepatitis to chimpanzees and to identify the period during which this agent is present in the blood during the acute disease. Materials and Methods Chimpanzees Six chimpanzees (Pan troglodytes) were studied. All were born in a U.S. breeding colony (nternational Center for Environmental Safety, Alamagordo, N. Mex.; FDA contract No ); at the start of the study, each was approximately 19 mo old and weighed between 7.5 and 9 kg. From birth to 12 mo of age, they had been housed together in a nursery and fed an infant formula; the care and feeding after age 12 mo have been described elsewhere. to There was little likelihood of prior exposure to hepatitis viruses; the only potential sources were their human caretakers who were tested monthly for serum aspartate aminotransferase (AST) and alanine aminotransferase (AL T), and other infant chimpanzees which were tested for AST and ALT. The parents of these infant chimpanzees, while not in contact with them after birth, were also monitored at regular intervals for AST and ALT. Serologic Studies Beginning 4 wk before inoculation and continuing throughout the course of this study, serum specimens

2 April 1979 NON-A. NON-B HEPATTS 61\1 from each of the six chimpanzees were tested weekly for AST and ALT with a biochromatic analyzer (ABA-100, Abbott Laboratories, North Chicago, ll.)" (normal, 5-40 V/liter), for isocitric dehydrogenase (lcd) by the Sigma method 12 (normal, Sigma Units, S.U.), for HBsAg by radioimmunoassay (RA) (Ausria, Abbott Laboratories),13 for antibody to HBsAg (anti-hbs) by RA (Ausab, Abbott Laboratories),14 for antibody to hepatitis B core antigen (anti-hbc) by complement fixation15 and by a solidphase RA'6 (reagents kindly provided by Dr. L. R. Overby, Abbott Laboratories), and for hepatitis B e antigen (HBeAg) and its antibody (anti-hbe) by agar gel diffusion.17 Selected serum samples were tested for antibody to HA V (anti-hav) by immune adherence hemagglutination, for antibody to cytomegalovirus (anti-cmv) by RA,20 and for antibody to the capsular antigen of the Epstein-Barr virus (anti-ebv) by indirect immunofluorescence. 21 AST and AL T levels remained below 40 V throughout the quarantine period except for chimpanzee No. 959 whose AST and ALT remained below 58 V. Four of the chimpanzees had no detectable anti-hay and had not been previously inoculated with any other serum; chimpanzees Nos. 911 and 920, which had recovered from experimentally induced HA V infection after inoculation with chimpanzee plasma or human plasma, respectively, had anti-ha V titers of 1:1000. Liver Biopsies Liver biopsy specimens were obtained weekly. The chimpanzees were anesthetized with cyclohexylamine, a drug with no known liver toxicity. The specimens, obtained with a 14 gauge Vim-Silverman needle, were stained with hematoxylin and eosin. Biopsies were assessed by the criteria of Barker et al.' and were considered positive if lymphocytic infiltration of the sinusoids or portal areas together with eosinophilic degeneration or acidophilic bodies were observed. Biopsy specimens were evaluated under code by two of the authors, both of whom agreed in their interpretations of each biopsy in this study. Table 1. Passage of Non-A, Non-B Hepatitis in Chimpanzees Enzyme levels of donor blood Donor Recipient chimpanzee Week AST ALT chimpanzee No. obtained (U) (U) No. nfectivity / _ o One abnormal liver biopsy. Enzyme levels normal or near normal throughout the study. Patient ~ Nurse noculum Chimpanzee #930 week 3 week 4 week 5 week 13 Chimpanzee #920 Chimpanzee # 911 Chimpanzee #916 Chimpanzee #946 week 4 week 5 Chimpanzee # 953 Chimpanzee #959 Figure 1. Serial passage of human non-a. non-b hepatitis in chimpanzees. nocula: Second Passage Aliquots of serum samples from donor chimpanzee No. 930, previously infected with acute, self-limited non A, non-b hepatitis,7 had been frozen at -70 C as soon as possible after venipuncture. As previously reported,7 chimpanzee No. 930 had developed serum aminotransferase elevations beginning at week 3 after inoculation, and these had remained elevated until week 24 after inoculation. One milliliter of serum from week 3 after inoculation was inected intravenously (Lv.) into chimpanzee No. 920, from week 4 into chimpanzee No. 911, from week 5 into chimpanzee No. 916, and from week 13 into chimpanzee No. 946 (Figure 1). The AST and ALT of donor chimpanzee No. 930 at the time the inocula were obtained are shown in Table 1. nocula: Third Passage Serum samples from donor chimpanzee No. 916 were obtained during the second passage and were frozen at -70 C. One milliliter of serum from chimpanzee No. 916 obtained at week 4 after inoculation was inected Lv. into chimpanzee No. 953, and 1 ml of serum from week 5 was inected Lv. into chimpanzee No. 959 (Figure 1). The AST and AL T of donor chimpanzee No. 916 at the time the inocula were obtained are shown in Table 1. Results Five of six chimpanzees which received sera from chimpanzees with acute non-a, non-b hepatitis subsequently developed hepatitis (Figures 1-3). AST or ALT first became elevated between 3 and 5 wk after inoculation in three chimpanzees during the second passage (Nos. 911, 916, and 946), and at 1 and 3 wk after inoculation in the two chimpanzees infected during the third passage (Nos. 953 and 959). Peak aminotransferase values ranged from AST 93 to 324 V and AL T 142 to 386 V in the second passage, and AST from 49 to 140 V and ALT from 86 to 397 V in the third passage. n two of the second passage chimpanzees (Nos. 916 and 946) and in the two third passage chimpanzees (Nos. 953 and 959), elevation of AST, ALT, and led occurred in a biphasic pattern similar to that described in the 'original non- l

3 682 TABOR ET A. GASTROENTEROLOGY Vol. 76, No.4 No. 920 No No. 911 No Figure 2. Second passage of human non-a, non-b hepatitis in chimpanzees. Normal values for aspartate aminotransferase (AST) and alanine aminotransferase (AL T) are ~ 4 0 V/liter, and have been represented in the figures as 40 V/liter for clarity WEEKS AFTER NOCULATON A, non-b hepatitis in chimpanzees infected by inoculation of human sera,' characterized by an initial mild sustained aminotransferase elevation followed by a greater peak occurring between weeks 9 and 12. n chimpanzee No. 911, a monophasic pattern of enzyme elevations was seen, with peak enzyme levels at week 8. n each chimpanzee, CD levels were elevated at approximately the same time as AST and ALT elevations, reaching a peak of 500-1,350 S.U. in the second passage and S.U. in the third passage (data not shown). Chimpanzee No. 920 had normal or near-normal liver enzyme levels throughout this study (Figure 2). Histologic evidence of hepatitis was detected at the time of elevated liver enzymes (Figures 2 and 3). n chimpanzees Nos. 916, 946, 953, and 959, abnormal histology was found during each of the biphasic peaks in liver enzymes; in chimpanzee No. 911, liver histology was abnormal at the time of the single peak in liver enzymes. Abnormal liver histology was characterized by lobular disarray, eosinophilic degeneration and acidophilic bodies, extensive sinusoidal lymphocytic infiltration, and in some cases centrilobular inflammation (No. 916) or periportal inflammation (Nos. 946, 953, and 959). Marked nuclear changes were observed during enzyme elevations, occurring before and during the peak of liver enzymes, characterized by marginal displacement of the chromatin and central placement of the nucleolus, with a "bull's eye" appearance, and multinucleate cells with as many as five nuclei per cell. Biopsies from chimpanzee No. 920 remained normal, except for a single biopsy at week 11 which showed eosinophilic degeneration, focal necrosis, and sinusoidallymphocytic infiltration. All six chimpanzees remained negative for HBsAg, anti-hbc, anti-hbs, HBeAg, and anti-hbe. Anti-HAV remained negative in all chimpanzees except Nos. 911 and 920, in whom the preexisting titer of 1:1000 remained unchanged. All chimpanzees remained negative for anti-cmv, and no change occurred in the preexisting titers of anti-ebv in any of the chimpanzees (data not shown). Discussion This study documents a prolonged period during which the agent of non-a, non-b hepatitis circulates in the blood during experimentally induced acute non-a, non-b hepatitis in chimpanzees. nfection was transmitted by serum obtained from each of two chimpanzees at weeks 4 and 5 (second and third passages) and at week 13 from one chimpanzee (second passage) after inoculation. Serum from week 3 after inoculation may have transmitted this disease also, but the evidence is less convincing. A single abnormal liver biopsy in chimpanzee No. 920, at an appropriate interval after inoculation, may

4 April 1979 NON-A, NON-B HEPATTS 683 have represented non-a, non-b hepatitis, or may have been unrelated to the non-a, non-b hepatitis inoculation. Such clear abnormalities in liver histology are very unusual in chimpanzees, even when biopsied serially for many months; in fact, liver histology may remain completely normal in chimpanzees even during mild HBV infection. to Whether a subclinical non-a, non-b hepatitis infection occurred in chimpanzee No. 920 will have to be determined when a serologic test for non-a, non-b hepatitis becomes available. Nonetheless, these data show that the agent of non-a, non-b hepatitis appears in the blood near the time of first elevation of serum aminotransferases. Although other investigators have shown that the agent of non-a, non-b hepatitis may be present in the blood of infected humans as early as 12 days before clinical hepatitis!2 the possible presence of the agent before aminotransferase elevation has not been evaluated, nor can this possibility be determined from the data presented here. nfectivity remained present until at least 1 week after the peak aminotransferase level, the 13th wk after inoculation, even in an apparently acute, self-limited case. Thus, non-a, non-b hepatitis resembles HBV infection, and is unlike HAV infection!3 in that infectivity of the serum persists for a prolonged period after the onset of serum enzyme elevations. The conclusion that the non-a, non-b hepatitis in these chimpanzees was transmitted by the inocula used is based on several observations. n each chimpanzee, the incubation period from the time of inoculation to the time of aminotransferase elevations and the pattern of aminotransferase elevations were similar to that observed in our original studies of the transmission of human non-a, non-b hepatitis to chimpanzees. 7 While the present study was in progress, no hepatitis occurred in two colony-born chimpanzees of the same age housed in the same room, which had been inoculated with other serum samples, or in four uninoculated colony-born chimpanzees of the same age housed in the adacent room. Non-A, non-b hepatitis has been documented to occur as a chronic asymptomatic infection in some humans, by the transmission of this disease to other humans and to chimpanzees by their blood or serum.7 The specific period during which the agent persists in chronic non-a, non-b hepatitis, as opposed to acute, self-limited non-a, non-b hepatitis, was not addressed here. The two donor chimpanzees (Nos. 930 and 916) which provided the inocula for these studies appeared to recover fully, as did all but one of the chimpanzees (No. 953) in this and previous studies.' This conclusion is based on the return to normal or near-normal levels of AST, 80 ~ ~ 40 No. 953 f- No. 959 (f) «~ ::L L - ~ ~ Liver Histologv Liver HistologV ~ ~ ~ WEEKS AFTER NOCULATON ~ Figure 3. Third passage of human non-a, non-b hepatitis in chimpanzees. Normal values for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are 540 U/liter, and have been represented in the figures as 40 U /liter for clarity. ALT, and lcd, and the complete resolution of histopathologic changes on liver biopsy. However, in each chimpanzee which developed non-a, non-b hepatitis, AST and AL T levels did not return to values as low as those before infection, but did decrease to normal or near normal values. n some cases, occasional AST or AL T values after recovery were between V on a single bleeding, returning to less than 40 V the following week. The assumption that this represented residual changes in liver physiology or structure unrelated to active infection cannot be made with certainty in the absence of a serologic test for non-a, non-b hepatitis. This study confirms the transmissibility of non-a, non-b hepatitis by serial transmission in chimpanzees, and establishes a period of time during acute disease in which the agent is present in the blood. Although this period may vary in different individuals, the agent was shown here to be present in the blood at weeks 4 (in two donor chimpanzees) and 5 (in two donor chimpanzees) after inoculation near the time of onset of aminotransferase eleva-

5 684 TABOR ET A. GASTROENTEROLOGY Vol. 76, No. 4 tions. and remained present at week 13 in one chimpanzee. This study suggests that the incubation period from inoculation to time of enzyme elevations was shorter in the third passage. but the small number of chimpanzees in this passage prevents any definite conclusions. There was no apparent difference in the severity of the disease in chimpanzees after serial passage; the degree of enzyme elevations and histologic evidence of liver damage appeared to remain constant. References 1. Blumberg BS: Polymorphism of serum proteins and the development of isoprecipitins in transfused patients. Bull NY Acad Med 40: , Feinstone SM. Kapikian AZ. Purcell RH: Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness. Science 182: Feinstone SM. Kapikian AZ, Purcell RH, et al: Transfusionassociated hepatitis not due to viral hepatitis type A or B. N Engl J M ed 292: , Alter HJ. Purcell RH. Holland PV, et al: Clinical and serological analysis of transfusion-associated hepatitis. Lancet 2: , Seeff LB. Zimmerman HJ. Wright EC. et al: A randomized, double-blind controlled trial of the efficacy of immune serum globulin for the prevention of post-transfusion hepatitis. Gastroenterology 72: Hoofnagle JH, Gerety RJ, Tabor E, et al: Transmission of non A. non-b hepatitis. Ann ntern Med 87: Tabor E, Gerety RJ. Drucker JA. et al: Transmission of non-a, non-b hepatitis from man to chimpanzee. Lancet 1: Alter HJ, Purcell RH. Holland PV. et al: Transmissible agent in non-a, non-b hepatitis. Lancet 1: Tabor E, Gerety RJ, Drucker JA, et al: Experimental transmission and passage of human non-a. non-b hepatitis in chimpanzees. n: Viral Hepatitis. Edited by GN Vyas, SN Cohen. R Schmid. Philadelphia. The Franklin nstitute Press, p Barker LF, Chisari FV, McGrath PP. et al: Transmission of type B viral hepatitis to chimpanzees. J nfect Dis 127: , Humoller FL, Holthaus JM, Walsh JR: mproved method for the colorimetric determination of glutamic-oxalacetic transaminase activity. Clin Chern 3: Sterkel RL. Spencer JA, Wolfson SK, et al: Serum isocitric dehydrogenase activity with particular reference to liver disease. J Lab Clin Med 52: Ling CM. Overby LR: Prevalence of hepatitis B virus antigen as revealed by direct radioimmunoassay with 125-antibody. J mmunol 109: , Hollinger F. Vorndam V, Dreesman G: Assay of Australia antigen and antibody employing double antibody and soidphase radioimmunoassay technique and comparison with the passive hemagglutination methods. J mmunol 107: , Hoofnagle JH, Gerety RJ, Barker LF: Antibody to hepatitis-bvirus core in man. Lancet 2: , Overby. LR. Ling, CM: Radioimmune assay for anti-core as evidence for exposure to hepatitis B virus. Rush-Presbyterian-St. Luke's Medical Bulletin 15(2):1-10, Magnius LO: Characterization of a new antigen-antibody system associated with hepatitis B. Clin Exp mmunol 20: Moritsugu Y. Dienstag JL. Valdesuso J, et al: Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination. nfect mmun 13: Miller WJ. Provost PJ, McAleer WJ. et al: Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologic investigations. Proc Soc Exp Bio Med 149: Smith KO. Gehle WD, McCracken AW: Radioimmunoassay techniques for detecting naturally occurring viral antibody in human sera. J mmunol Methods 5: Henle G, Henle W: mmunofluorescence in cells derived from Burkitt's lymphoma. J Bacteriol 91: Hollinger FB. Gitnick GL, Aach RD, et al: Non-A. non-b hepatitis transmission in chimpanzees: a proect of the Transfusion-Transmitted Viruses Study Group. ntervirology 10: Havens WP: Period of infectivity of patients with experimentally induced infectious hepatitis. J Exp Med 83: , 1946