LIVER PHYSIOLOGY AND DISEASE

Transcription

1 GASTROENTEROLOGY 1982;82:502-6 LIVER PHYSIOLOGY AND DISEASE Non-A, Non-B (Type 1) Hepatitis Agent Capable of Inducing Tubular Ultrastructures in the Hepatocyte Cytoplasm of Chimpanzees: Inactivation by Formalin and Heat HIROSHI YOSHIZAWA, YUKIO ITOH, SHIGENORI IWAKIRI, KOJI KITAJIMA, AKIRA TANAKA, TOSHIAKI TACHIB ANA, TETSUO NAKAMURA, YUZO MIY AKA WA, and MAKOTO MA YUMI Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science; the Japanese Red Cross Blood Center; Immunology Division, Jichi Medical School; Institute of Immunology; and the Third Department of Internal Medicine, University of Tokyo, Japan We have reported two kinds of viruslike particles derived from human sera that induced morphologically and serologically different types of non-a, non B hepatitis in chimpanzees. A chimp serum containing one such agent capable of inducing a type of hepatitis with cytoplasmic tubular ultrastructures (non-a, non-b, type 1) was titrated for its infectivity in chimps. Two chimps who received 1 ml of a 10-2 dilution of the original serum developed the characteristic morphological changes in the liver together with elevated serum transaminase levels, while the other two who received 1 ml of a 10-4 dilution failed to show such changes. The two who escaped the infection were proven to be susceptible to the agent, because they developed non-a, non-b, type 1 hepatitis when they were challenged by 1 ml of a 10-1 dilution of the same serum on the 23rd week after the first inoculation. One milliliter of a 10-1 dilution containing more than 10 chimp infecting units was inactivated in the presence of 1/2000 formalin or by heating at 100 C for 5 min and then given to four other chimps. None of them developed clinical or histologic evidence of non-a, non-b, type 1 hepatitis, thereby indicating that both formalin and heat could destroy the ability of the agent to induce this type of hepatitis. Received September 23,1981. Accepted October 16, Address requests for reprints to: Makoto Mayumi, M.D., Hepatitis Division, The Tokyo Metropolitan Institute of Medical Science, 3-18 Honkomagome, Bunkyo-ku, Tokyo 113, Japan by the American Gastroenterological Association /82/ $02.50 Non-A, non-b (NANB) hepatitis is attracting increasing attention from hepatologists, blood bankers, and epidemiologists since today it accounts for at least 90% of all hepatitis developing after transfusion with blood units from which hepatitis B surface antigen had been excluded (1,2). Identification of the agents responsible for the transmission of NANB hepatitis and inactivation of such agents may help prevent the transmission of hepatitis and provide the basis for the eventual development of NANB vaccines. We have previously reported finding two kinds of viral agents each associated with different types of NANB hepatitis, provisionally designated NANB-1 and NANB-2 (3,4). The NANB-1 hepatitis agent of documented infectivity in humans, when injected into susceptible chimps, induced the typical liver changes characterized by microtubular structures composed of two unit membranes with electronopaque materials in between (5,6). In contrast, the other type of viral agent, which had been identified in the serum of donors with a high serum glutamic pyruvic transaminase (SGPT) level, induced chemical and morphologic evidence of hepatitis in chimps but without cytoplasmic tubular structures (NANB- 2). The antigenic difference between these two kinds of NANB agents was clearly demonstrated by reinjection and cross-challenge studies in chimps (4). We have titrated the infectivity of the serum containing NANB-1 viral agent in chimps, and then tried to inactivate the NANB-1 agent by treatments known to be effective for hepatitis B virus.

2 March 1982 INACTIVATION OF A NON-B HEPATITIS AGENT 503 Table 1. Chimpanzees imd Inocula Inoculum (1 mil Chimp Weight Group No. Sex (kg) Dililtiono Inactivation 1 38 F M M F F 8.1 io-1 Formalin 45 M it Formalin 4 40 F C, 5 min 44 M C, 5 min Inoculum No.1, the preacute serum of chimp 37 who contracted NANB-l hepatitis with cytoplasmic tubular ultrastructures (3), was dii~ted with physioiogical saline supplemented with 0.1% fetal calf serum and a l-ml amount was irijected intravenously into chimps with or without inactivation. Materials and Methods Chimpanzees Eight chimps were used in the experimental infection of NANB-1 hepatitis. All of them were wild~caught and kept in individual stainiess shiel cages with care and feeding as described by Barker et al. (7). None of them had previously been used for experimental transmission of hepatitis and all were negative for serological markers of hepatitis A virus (HA V) or hepatitis B virus thbv) infection. Preinoculation. serum samples collected weekly failed to exhibit elevated levels of serum glutamic oxaloacetic transaminase (SGOT) or SGPT for 3 rno or longer (in~ house normal values did not exceed 30 Katmim units/ml). Eight chimps were divideci into four groups of 2 ariimals (inale and female) and each group received intravenously 1 ml of a dilution of the inoculum of documented infectivity for NANB-1 hepatitis, or the inoculum containing 10 or more chimp infecting units of NANB-1 viral agent, whkh had been inactivated (see below). The specifka~ion of animais and the design of the experiinents are given in Table 1. After the inoculation, serum and liver biopsy specimens were collected at weekly intervals. Manipulations such as intravenous inoculation, bleeding, and biopsies were pedoril,led while the chimps were sedated with ketamine hydrochloride, a drug without recognized hepatotoxicity. Inoculum Only Ii singie inoculum was used throughout the experiment. It was inoculum No. 1 w~.ich previously had been used for transmission of NANB-1 hepatitis iil chimps and which contained the virus like particles. The inoculum represented acute-phase serum of chinip 37 who had been injected willi serum containing NANB-1 virus like particles obtained from an apparently healthy blood donor (s.6.) with elevated serum transaminase levels. In inoculum No. -1, viruslike particles morphologically indistin- guishable from those found in the serum of the donor were detected by immune electron microscopy; they measured 27 ± 2 nm in diameter with a density of 1.28 glcm 3 in CsCl. When 2 ml of undiluted inoculum was injected into three susceptible chimps, all of them developed NANB-1 hepatitis characterized by cytoplasmic tubular structures after an incubation period of 2-3 wk (3,4). Titration and Inactivation of the Infectivity in Inoculum No. 1 Inoculum No. 1 whi'ch had been aliquoted and stored at -70"C was thawed on the day of injection and diluted to 10-2 and 10-4 with pp-ysiological saline supplemented with 0.1% (vol/vol) fetal calf serum. Chimps 38 and 42 received 1 ml of a 10-2 dilution, and chimps 39 and 43 received 1 ml oj a 10-4 dilution. For the inactivation with formalin, the inoculum was diluted to 10-1 with physiologieal saline supplemented with 0.1 % fetal calf senim, mixed with formalin [370/0 (wtl vol) solution of formaldehyde, Wako Chemical Co., Ltd., Tokyo] to achieve a final concentration of , and then incubated at 37 C for 72 h with occasional agitation. The formalin-treated inoculum containing 1 ml of a 10-1 dilution of inoculum No.1 was given to chimps 41 and 45. For heat inactivation, 3 ml of a 10-1 dilution of inoculum No.1 was transferred into a glass tube (i x 6.5 cm, i mid thick) and swij;led in boiling water for 5 min. The tube was rapidiy cooled in ice water and a 1-ml aliquot was given to chimps 40 and 44. MorpholQgical Examination Coded liver biopsy specimens were examined by light and electron microscopy for the diagnosis of hepatitis. They Were cut into two fragments, one of which was fixed in 10% formalin, embedded in paraffin, cut at 2-#Lm thickness, and stained with hematoxylin and eosin. For electron microscopy, another fragment was immediately fixed in 4% parafor:rp.aldehyde, postfixed with osmium and embedded in Epon 812, and cut at A thickness. They were double-stained with uranium acetate and lead citrate and then observed in a JEM 100C electron microscope operating at a plate magnification of x : The liver section was spread on a 200-mesh carbon-coated grid and at least?o square fields were examined for the presence of cytoplasmic tubular structures. Resuits Figure 1 summarizes the results of the titration of inoculum No.1 and the inactivation of its infectivity by treatment with formalin or heat. Chimps 38 and 42, 'who had received 1 ml of a 10-2 dilution of the inoculum; developed histologic evidence of acute hepatitis along with characteristic tubular structures in their hepatocyte cytoplasm (Figure 2). Cytoplasmic tubular structures were observed during the period from 3 to 13 wk after

3 504 YOSHIZAWA ET AL. GASTROENTEROLOGY Vol. 82, No.3 TUBULAR ULTRASTRUCTURES IN HEPATOCYTES GROUP 1 (NANB-1, 10-2 ) 38 DD DDDDDDDDDDDD 42 DD DDD GROUP 2 (NANB-1, 10-4 ) 39 DDDDDDDDDDDDDDDDDDDDDDDDD 43 DDDDDDDDDDDDDDOOOOOOOOOOO GROUP 3 (NANB-1, to- 1, formolin) 41 OOOOOOOOOOODDDDDDDDDDDDDD 45 ODDDDODDDDDDDDDDDDDDDDDDD GROUP 4 (NANB-1, to- 1,100oC,5min) 40 DODODDDDDDDODDDODDDOODDDD 44 DODDDODDDDDDDDDODDDDOODOD,,,, a WEEKS AFTER INOCULATION Figure 1 Titration of the infectivity and inactivation of inoculum No.1 which contained NANB-1 virus like particles with the infectivity of NANB-1 hepatitis. Chimps 38 and 42 received 1 ml of a 10-2 dilution of the original serum, chimps 39 and 43 1 ml of a 10-4 dilution, chimps 41 and 45 1 ml of a 10-1 dilution inactivated by formalin, and chimps 41 and 44 1 ml of a 10-1 dilution inactivated by heating. Closed squares represent liver biopsy specimens with, the open squares without, cytoplasmic tubular structures characteristic of N ANB-1 hepatitis (5,6). inoculation in chimp 38 and from 3 to 22 wk in chimp 42. A mild elevation of SGPT was observed during the acute phase of illness in both chimps. The clinical course of chimp 38 is depicted in Figure 3. In contrast, chimps 39 and 43 who received 1 ml of a 10-4 dilution of the same inoculum did not develop histologic evidence of hepatits or tubular structures in the hepatocyte cytoplasm, nor did they show an appreciable elevation of serum transaminases. In order to disprove that these chimps were resistant to the NANB-l viral agent, they were challenged with 1 ml of a 10-1 dilution of the same inoculum on the 23rd wk after the initial injection. Both of them developed NANB-l hepatitis with characteristic tubular structures in the hepatocyte after an incubation period of 4 wk, indicating that they were susceptible to this agent and that the initial injection contained insufficient NANB-l viral agents for transmission of hepatitis. Accordingly, inoculum No.1 was estimated to contain between 10 2 and 10 4 chimp infecting units of NANB-l viral agent per milliliter. The other four chimps were each injected with 1 ml of a 10-1 dilution of inoculum No.1 (containing 10 or more chimp infecting units) after it had been inactivated by formalin or heating. Chimps 41 and 45 received the inoculum which had been treated with formalin and chimps 40 and 44 received the same inoculum after it had been heated at 100 C for 5 min. During a 25-wk period after the injection, none of them developed histologic changes indicative of viral hepatitis that were detectable by light microscopy or appreciable elevation of serum transaminase values, nor did they display the characteristic cytoplasmic tubular structures visible by electron microscopy. Chimps 41 and 45 were challenged by 1 ml of a 10-1 dilution of inoculum No.1 on the 23rd wk after the first inoculation. Both of them contracted NANB- 1 hepatitis 3 and 4 wk after the injection, respectively, thereby indicating that they had remained susceptible to the agent. None of the 8 animals revealed serologic evidence of concomitant infection with HAV, HBV, cytomegalovirus, and Ebstein-Barr virus at any time during the observation period. Discussion We have titrated the infectivity of the inoculum which contained NANB-l virus like particles and induced a type of NANB hepatitis exhibiting the cytoplasmic tubular structures originally described by Shimizu et al. (5) and Jackson et al. (6). The inoculum, the preacute serum of chimp 37 who contracted NANB-l hepatitis (3), could transmit hepatitis to two susceptible chimps when 1 ml of a 10-2 dilution was given, but could not infect the other two injected with 1 ml of a 10-4 dilution. This indicates that the, original inoculum contained somewhere between 10 2 and 10 4 chimp infecting units of the NANB-l viral agent per milliliter. The possibility that the two chimps who escaped the infection were resistant to NANB-l agent was excluded by the subsequent successful transmission of NANB-l hepatitis to them by inoculum that contained a sufficient amount of the same agent. Considering that inoculum No.1 was the preacute-phase serum of a chimp with presumed high infectivity, the observed titer was disappointingly low. For example, plasma of asymptomatic carriers of HBV contains more than 10 6 chimp infecting units of hepatitis B per milliliter (8). Our results appear to suggest a low concentration of the virus and related antigens of NANB-l in the circulation of infected persons. The paucity of viral antigens in carrier sera might retard the rapid development of immunologic methods for a practical in vitro detection of the agent. The infectivity of the inoculum containing more than 10 chimp infecting units of NANB-l agent was completely destroyed by the treatment with formalin or heat. Formalin at 1/2000 concentration or heating at 100 C has been successfully applied for the inactivation of materials containing HBV (9,10); NANB-l agent was demonstrated to be equally sensitive to such treatments. Recently, Tabor and Gerety also reported the successful inactivation of a NANB viral

4 March 1982 INACTIVATION OF A NON-B HEPATITIS AGENT 505 Figure 2. A hepatocyte of chimp 38 with NANB-1 hepatitis who underwent biopsy 5 wk after the inoculation. Numerous tubular structures that appear circular in cross section are observed. The other biopsy specimens of chimp 38 and those of chimp 42 taken during the period indicated by closed squares in Figure 1 showed similar changes. The bar indicates 1,",m. agent (NANB-l, NANB-2 or another type) by treatment with formalin (11). The dosis of inactivated NANB-l agent used in the present experiment, however, was insufficient to TUBULAR ULTRASTRUCTURES IN HEPATOCYTES NANB-1 CHIMP 38 OL- L- ~~ ~ ~~ ~ ~ ~ o WEEKS AFTER INOCULATION Figure 3. Clinical course of chimp 38 injected with inoculum No.1 containing live NANB-1 agent. Solid circles and solid line represent SGPT; open circles and dotted line represent SGOT. Closed squares represent liver biopsy specimens with. and open squares without. tubular structures characteristic of NANB-1 hepatitis (5.6). induce an immune state in the host to overcome a further challenge by the same agent. When the two chimps who had been injected with the formalininactivated NANB-l agent subsequently were challenged by an inoculum containing live NANB-l agent 23 wk after the first inoculation, both of them developed chemical and histologic evidence of NANB-l hepatitis. This implies that the inactivated NANB-l viral agent corresponding to 10 or more chimp infecting units was inadequate to raise protective antibodies. Future efforts will have to evaluate whether a higher dosis of the same inoculum, after inactivation by formalin or heat, will be able to induce an immune state to protect animals against the subsequent challenge by liver agent. Certainly, this would be a prerequisite for the future development of NANB-l vaccine. Non-A, non-b hepatitis today represents one of the major public health care problems, taking over the place which formerly had been occupied by hepatitis A and B. The chronic carrier state of NANB agents recognized among asymptomatic persons (12) warns us of the possible transmission of NANB hepatitis by body fluids or close physical contact, just as has been proven for hepatitis B. At present, however, the

5 506 YOSHIZAWA ET AL. GASTROENTEROLOGY Vol. 82, No. 3 mode of horizontal and possibly maternofetal transmission of NANB hepatitis remains obscure owing to the lack of practical methods to detect serum markers of NANB hepatitis infection. In the interim, the fact that the NANB-l agent can be inactivated by formalin or heating may be helpful whenever contamination with this agent is suspected. References 1. Alter HI, Purcell RH, Holland PV, et al. Clinical and serological analysis of transfusion-associated hepatitis. Lancet 1975;2: Knodell RG, Cqnrad ME, Ginsberg AL, et al. Efficacy of prophylactic gamma-globulin in preventing non-a, non-b post-transfusion hepatitis. Lancet lq76;2: Yoshizawa H, Akahane Y, Itoh Y, et al. Virus like particles in a plasma fraction (fibrinogen) and in the circulation of apparently healthy blood donors capable of inducing non-a/non-b hepatitis in humans and chimpanzees. Gastroenterology 1980;79: Yoshizawa H, Itoh Y, Iwakiri S, et al. Demonstration of two different types of non-a, non-b hepatitis by reinjection and cross-challenge studiell in chimpanzees. Gastroenterology 1981;81: Shimizu YK, Feinstone SM, Purcell RH, et al. Non-A, non-b hepatitis: ultrastructural evidence for two antigens in experimentally infected chimpanzees. Science 1979;?05: Jackson D, Tabor E, Gerety RJ, et al. Acute non-a, non-b hepatitis: specific ultrastrugtu~al alteratiqns in endopillsmic reticulum of infected hepatocytes. Lancet 1979;1; Barker LF, Chisari FV, McGrath PP, et al. Transmission of type B viral hepatitis in chimpanzees. J Infect Dis 1973; 127: Shikata T, Karasawa T, Ape K, et al. Hepatitis-B e antigen and infectivity of hepatitis-b viru!>. J Infect Dis 1977;136: Purcell RH, Gerin JL. Hepatitis B subunit vaccine: a preliminary report of safety and efficacy tests in chimpanzees. Am J Med Sci 1975;270: Krugman S, Giles JP. Viral hepatitis, type B (MS-2 strain). Further observations on natural history and prevention. N Eng! J MEld 1973;288: Tabor E, Gerety RJ. Inactivat~on of an agent of human non-a, non-b hepatitis by formalin. J Infect Dis 1980;142:767-:-: Hoofnagle JH, Gerety RJ, Tabor E, et al. Transmission of non A, non-b hepatitis. Ann Intern Med 1977;87:14-20.