JPH04505700A - Cell culture of non-A, non-B hepatitis hepatocytes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cell culture of non-A, non-B hepatitis hepatocytes 1. Kouri Natsubu! The present invention provides a test that can maintain non-A, non-B hepatitis (NANBH) virus in culture. Concerning in vitro (!n vitro) cell culture media. More particularly, this invention A serum-free medium for cell culture of primate hepatocytes that can maintain NBH virus in culture. related.

2. Lord 1 Summer! ■ Non-A, non-B hepatitis has long been associated with the hepatitis A virus (HAV) and hepatitis B virus. Other forms of viral-associated liver disease, including HBV (HBV), and cytomegalosis virus (CMV) or EB virus (EBV) It has been recognized as a virus-induced disease. However, despite many years of extensive research, However, the NANBH virus has eluded isolation, characterization and in vitro culture. came. A significant amount of data indicates that two or more types of NANBH viruses suggests the existence of Two general types are identified depending on the mode of transmission: parenteral and and intestinal transmission. Of these two, the type that is transmitted parenterally is associated with chronic hepatitis. 5horey, James, Aeaer, Mu Med, Sci 289:251-261 (1985).

Human versus chimpanzee, the only reliable animal model for this disease Ten years have passed since the first experimental transmission of an infectious NANBH agent. Ta. However, to date, tissue culture systems that maintain NANBH virus in culture have not been developed; as a result, the limited availability of animal models and The lack of in vitro tissue culture models limits the isolation and It has seriously hindered characterization. Isolate and characterize the NANBH virus If this is not possible, researchers will develop diagnostic reagents and treatments for this disease. compounds above, and are thwarted in their attempts to develop a vaccine. . Post-transfusion-related hepatitis (post-transfusion-related hepatitis) approximately 90% of ansfusion associated hepatitis) can be attributed to this putative viral agent. Some researchers believe that this Suggests that the infectious agent has the property of being able to defeat the Togaviridae family. There are some too. We found that it has the morphology of Togaviridae by observation using an electron microscope. Particles (pa rticles) is intriguing.

To date, the inability to maintain cultures of differentiated primate hepatocytes has probably et al. (also for the isolation, characterization and in vitro culture of human infectious NANBH virus) That was the single biggest obstacle. duck Replicates hepatitis B ( Cell culture of avian hepatocytes that can be replicated (replicatfng) has been reported (T uttle+wan et al., J. Virol, 58:17 (1985)) , these cell cultures are not conducive to the growth of human hepadna virus. Hepadna virus exhibits a narrow host range: chimpanzees have human hepatitis B It is the only species other than humans that can be infected with the virus. Recently, dimethyl sulfoxy Primary human hepatocytes maintained in medium containing HBV are susceptible to exogenous infection with HBV. It has been proven that. These cultured cells are short-lived and poorly differentiated; And it has not been proven that people are easily infected with the NANBH virus. L et al., J. Virol, 62:4136-41443 (1988).

Recently, a serum-free medium for cell culture of hormonally restricted differentiated primate hepatocytes has been developed. It has been developed. U.S. Patent No. 222.569.

Filed on July 20, 1988, published by Koichi Mu; Shi, Cultured differentiated primate liver cells. The cells offer many benefits for biochemical, viral culture and carcinogenesis research.

Adult primate hepatocytes can be successfully cultured while maintaining cellular functional and morphological differentiation. The system can be used to study acute and chronic viral hepatitis as well as hepatotropin (hepatotropin). tremendous potential for facilitating the isolation of viruses (patotrophic) provide sex.

Therefore, there is a need for a medium for in vitro NANBH viral cell culture. NANB Such a medium in which the H virus can continue to replicate and grow is finally available led to the isolation and characterization of the ANBH virus and eventually the NANBH virus. Diagnostic agents and treatments specific to combating viral infections can lead to the above agents.

3. Hikari sitting weight! The present invention provides a basic medium for cell culture, hepatocyte proliferogen (proliferogen), gen), serum albumin, corticosteroids such as hydrocortisone, Matotropin or prolactin or both, growth/release factors, cholera toxin Maintained in a serum-free medium (Sus NANBH virus test tube containing N-5 stained primate hepatocytes Provide internal cell culture.

The present invention also provides serum-free, cell-free isolates of NANBH virus. NANBH Virus isolates were obtained from the culture medium supernatant or from NANBH cells cultured in vitro. It is obtained as a lysate of virus-infected hepatocytes.

Furthermore, the present invention provides methods for causing NANBH virus infection in chimpanzees. encompasses the law. This controlled outbreak of NANBH virus in chimpanzees is , provides an experimental model for the study of NANBH virus infection and NANBH Acts as a reservoir for producing antibodies against viruses It should stand. NANBH virus infection is caused by NANBH virus-infected hepatocytes. In vitro culture, cell-free supernatant of in vitro culture, in vitro culture cultured hepatocytes isolated for lysate, or their in vitro culture medium. is induced by inoculating chimpanzees with an infectious dose of the inoculum containing the virus.

Furthermore, the present invention provides a method for confirming NANBH virus infection in a host. Ru. This method involves excising hepatocytes from a host, culturing the hepatocytes, and Cytopathic changes in hepatocytes, including observing cytopathic effects in hepatocytes cultured after 4 weeks. Sexual effects indicate NANBH virus infection.

4. t's---゛ The serum-free medium described below is suitable for injecting NANBH virus into cell cultures of primate hepatocytes. Examples of formulations according to the invention that are useful for maintaining Examples are NAN Although in vitro culture of BH virus-infected chimpanzee hepatocytes has been demonstrated, the culture medium The culture and culture techniques include in vitro culture of NANBH virus in human hepatocytes. It should be understood that the same applies.

Among the media listed in Table 1, Williams medium E (Williams M edium E) (WM E) serves as the basal medium. WME is the present invention currently preferred as a basal medium in serum-free media of Therefore, it is reasonable that alternative commercially available media formulations can be expected to give satisfactory results. It will be understood. For example, Dulbecco modified Eagle medium (Dulbecco 's+wodified Eagle's medium) and ham F12 Mixture of medium (Ham's F12 medium) (Salas-Prato , Growth of Ce1ls in Hormonally Deft ned Media, Book A student, G, H, 5ato et al., Eds, , Co1d Spring Harbor Laboratory, No. 615 -624 pages (1982) lit) or RPMI 1640 (Gibco) (Enat et al. + Proc, Natl, AcadSci, USA 81: 1411 (1984) and 5ell, M.A., et al., Long-ter m culture and passage of human fatal 1iver cells thatsynthesize albua+in ,' in vitro Ce1l Dev, Biol, 21=216-2 20 (1985) 11) contains the supplements listed in Table 1. s) should give satisfactory results when supplemented with s).

Table 1 The precise function of the many different additives that supplement basal media is not well defined. However, to facilitate the description of the media formulations of the present invention, some auxiliary agents have been included in the preliminary classification. It can be classified into minutes.

For example, the term "hepatocyte proliferogens" (prol. iferogen) J is epidermal growth factor (EFG), insulin, liver growth factor growth factors (LGF) and glucagon (some growth factors or hormones). All of them involve controlling liver growth in vivo. (Salas-Pr) ato, J Seiko L-at 613).

As used herein, the term "transport protein" means, as one of its functions, refers to a protein found in serum that involves the transport of certain substances in the blood . Such a protein is advantageously a commonly available derivative of bovine serum albumin (BSA). Includes serum albumin, which can be used in accessible form, and transferrin. . However, hepatocytes maintain satisfactory hepatocyte maintenance without the addition of transport proteins ( Transferrin is synthesized so that @aintenance) can be achieved.

A trace metal specifically contemplated for use in the media of the invention is selenium; et al., WME contains copper, zinc, cobalt and iron and WME, or another basic Either of the media may contain zinc and/or copper, or both, to Depending on the initial conditions and whether the basal medium contains either or both of these trace metals. Further supplements may be provided depending on whether or not the

The literature includes thyrotropin-releasing factor (TRF), fibroblast ) Growth factors, platelet-derived growth factors, multiplication stimulating activity Contains sexualizing factors and endothelial cell growth supplements (ECGS). The use of some growth and/or release factors used in hepatocyte culture (For re-examination, Lettert, K., and S. Koch, “Hepatocyte growth regulation” by hormones in cheIlically definedme dia: A two-signal hypothesis”, Growth of Ce1ls in Hormonally Defined Medi a, Book A student, G, H, 5ato et al. (Eds.

) + Co1d Spring Laboratory, pp. 597-613 ( (1982)). One or more of these growth/release factors are most important for hepatocytes. Depending on factors such as the initial conditions and the particular protocol to be utilized , may be added to the culture medium of the present invention.

Although the adjuvants are described in specific proportions in the table below, those skilled in the art having the benefit of this disclosure Depending on the person, these proportions can be changed, and in some situations they cannot be changed. It will be understood that this must be done. For example, ECG5 is for the maintenance of liver cells. It is acknowledged that this is not required. The concentration of glucagon in the medium is reduced obtain. There is likewise interchangeability between some of the auxiliaries. For example, Rirun Addition of soybean fat can be used instead of acid. In addition, the results obtained from various separations The quality of the hepatocytes is determined by various hepatocyte proliferogens (pr.

1iferogen). The culture medium of the present invention is therefore shown in Table 2 containing each auxiliary agent in the proportion ranges indicated.

Table 2 Furthermore, for the proportions of each of these auxiliaries, the media formulation may e.g. ? I When the medium is described as containing TRF, the medium is 5. &10-' M T It will be understood to include RF.

Evaluating the ability of the media formulation of Example 1 of the present invention to support NANBH virus replication. Several studies conducted to evaluate the results are now disclosed. NANBH virus infected liver Isolating cells from chimpanzees is described in Examples 2 and 4.

Example 1 ! 11 prefectures and gold! The serum-free medium formulation was prepared at 10 μM 1 (EP[!S, pi (7 , 4, 2, 75 IIg/ml NaHCOz, and 50 tt g/ml Ge WME supplemented with tammycin was utilized. For the preparation of the culture medium, add the following amount of auxiliary agent. 5 so 1 5 added to WM E 500 ael in a sterile plastic bottle at 0 g/sl BSA, 500 μg/ml lylunic acid 0.5 @11 5 mg/ @l Insulin 0.5 ml 5 B/ml Insulin, 5 mg/ml ) Lanceferrin.

and 5 μg/ml selenium (ITS) 50μ + 10-”M Hydrocortisone 5μm 200μg/ml cholera toxin 0.5-1 100μs/ml EGP50μl 10-”M Ethanolamide 0.5 ml 1 mg/ml Somatotropin 50u11 mg/ml Prora Kuching 0.5 sl 10-”M Thyrotropin releasing hormone 50ul 200u g/vxl LGFlml 2.0 layer g/lar glucagon WME is L-glue Hazelton Re5earc with Tamine and without NaHCOs h Products, Inc. (Denver, Pennsylvania) Purchased.

Example 2 A. Chimpanzee's NANBH, To obtain NANBH virus-infected hepatocytes for in vitro experiments, parenteral NAN BH virus infection was induced in chimpanzee PTTx7, 14-year-old female by NANBH. Acute phase of unknown titer obtained from second passage of Hutchinson strain of virus. It was induced by inoculation with 5 ml of a 20-fold concentrate of plasma. NANBH virus The progression of the infection is monitored by AL T/AST enzyme fluctuations from weekly blood samples. Periodic liver needle punch biopsies s) was monitored by histological examination. All biopsies were performed similarly using conventional techniques. It has been processed. Immediately after collection, the biopsy was placed in neutral buffered 3.7χ formalin for 1 to 3 hours. fixed in a tube, processed by hand according to standard procedures, embedded in paraffin, and exposed to 4 microorganisms. Sections were made in a tube and stained with hematoxylin and eosin. all sections were examined histologically by the same board-certified veterinary pathologist.

Since the onset of clinical hepatitis was considerably delayed, 1.5 + * I was administered to ensure infection. (10” CID5J NANBH virus Hutchinson inoculum 2 The next vaccination was administered at 10 weeks. However, the appearance of elevated ALT at 12 weeks , the secondary inoculum will worsen the primary infection or is not needed. Indicated. The animal's ALT profile was approximately normal from 12 to 19 weeks after inoculation. showed an increase in values, and a second ALT increase occurred at week 30.

Liver wedge surgery was performed at 14 weeks at the beginning of constant ALT elevation. It was. Microscopic examination of the liver tissue performed at this time revealed that the hepatic triad and the parenchymal part of the lesion were showed occasional populations of lymphocytes and macrophages. exhibits a significant inflammatory response There were no other changes. Although minimal inflammation was present, this finding is consistent with normal liver disease. It could correspond to an organization. Maximum viral replication is associated with disease in liver cells. occur before or during this stage of manifestation) Despite what might have happened, they were separated at 14 weeks.

Liver punch biopsy performed after ALT elevation (19 weeks) revealed the portal area and liver parenchyma. showed an increased number of lymphocytes inside.

There were necrotic hepatocytes associated with parenchymal lesions. Central venous area Hepatocytes are often lightly stained and have minimal swelling of the cytoplasm. B) was granular. All these changes described are associated with minimal lymphoid multifocal showed viral hepatitis.

B. NANBH virus-feeling Yutaka L ■ Plate owner Ketamine hydrochloride was used as immobilization agent and pre-anesthetic agent.

The surgical procedure was performed under general anesthesia using non-hepatotoxic bentoparbital sodium. I went down. Liver wedges of approximately Log Perfused (Maslansky.

C. J. and G. M. Williams, in vitro Model s for Cancer Research, Volume 1I: Carcinoma s of the River and Pancreas, M, M, We ber and bird, 1.5ekely ([sound collection), CRC Press: Bo Perfuse all solutions. A two-stage perfusion procedure was used, maintaining the temperature at 37°C throughout the procedure. The first perfusion was 10 mM HEPES (pH 7,4), 0.5 mM EGTA, and 100 1 liter of Ca", Mg" supplemented with μg/ml gentamicin sulfate Flanks Balanced salt solution without 9 was used for 10 minutes. Next Perfusion with 10 mM HEPES (pH 7,4) for 20 minutes.

100μg/a+I gentamicin sulfate and 200 units/1 collagener Williams medium E supplemented with enzyme (300 units/ng, Sigma) (WME) It was about 60 m1/min. The liver capsule is Remove the hepatocytes using sharp tweezers and gently stir the hepatocytes. Moved in Lagenase Solution 1001. 5% fetal bovine serum (FBS), to mM HEPES (pH 7,4), and 100 μg/l gentami The hepatocyte suspension was placed in several layers into an equal volume of cold WME containing synsulfate. It was filtered through a gauze batt. The hepatocytes were spun down at 50 x g for 5 min and the cell pellets were The pellets were resuspended in WME 5% FBS. Repeat the sedimentation twice and remove the pellet. Resuspend in O1WM 25% FBS and determine viability and cell density with trypan. Measured by excluding blue.

Rat tail collagen (Michalo) on PRIMARIA plates (Falcon) poulos, G., and H.C. Pitot, “Pr1w+ary cu lture.

f parenchymal 1iver collagen membrane es”, Exptl, Ce1l.

Res, 94 Near 0 (1975)) for 6 minutes at room temperature to remove excess collagen. was removed and the plates were dried under UV light overnight. Viable cells at a density of 3-4X Blating was performed at 10-' cells/60-dish. Cell adhesion is 37°cSWME5 This occurs during a 3 hour incubation in 10% CO□ in %FBS, when the cell monolayer Serum-free media prepared as described in Example 1 above and gently washed twice with WME. The ground mixture was fed again. 24 hours after isolation and The medium was changed at 48 hour intervals.

Cultured hepatocytes showed typical Hepatocyte morphology was expressed. This morphology occurs when the culture undergoes a denaturing process. It was maintained from 21 to 28 days when showing signs of illness.

C6NANBH's standing The synthesis of albumin, apolipoprotein <A-1, and apolipoprotein E is Briefly, the temperature was monitored by immunoplot of an aliquot of tissue culture medium. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SD Separation by S-PA(1,E) and Nylo at 100 mA for 16 h at 4°C. Electrophoretically transferred to n-X nitrocellulose filters (Fisher). Ta. Unoccupied binding sites were isolated in phosphate-buffered saline (PBS) for 10 min. % nonfat dry milk for 2 hours at 37°C. The membrane was soaked in PBS-milk at 37°C for 2 hours. Tween (PSB containing 5% skim milk powder, 0.3X Tween-2 0) using primary antibodies directed against human apolibotanpo<A-I and E. and incubate it. The membrane was washed three times with PBS-Twean and At 37℃ for 1 hour [1! SIl protein A (8.5μCj/μg, NEN) and Both were incubated in PBS-Milk-Twean. P the membrane 3 times - Washed with Tween and air dried. Immunoplots were XA at -85°C. Autoradiography using an intensifying plate on R-5 film (Kodak) was analyzed.

Levels of Apolipotanpo<A-r and E increased up to 13 days in culture; It remained constant from days 12 to 28 and declined from days 28 to 45.

Albumin detected by this immunoplot method remains constant throughout the culture period. remained at the level. Albumin is a label for differentiated hepatocytes, but Polyprotein synthesis is triggered by the differentiation state marker (stringer + t). not present.

The decline in Rinobotan Bakusho synthesis after 28 days in culture was observed in cultured hepatocytes. Corresponds to degeneration. Degeneration of primary hepatocytes after 3 to 4 weeks of culture was observed in two different NANs. This was evident in cultured cells obtained from BH-infected chimpanzees, but normal observed in cultured cells from chimpanzees or HBV-infected chimpanzees. I couldn't. Cultured normal hepatocytes are generally grown in serum-free medium for more than 100 days. survive. Further experiments showed that the degenerative process was due to virus-induced cytopathic effects. will be needed to determine whether or not.

To further characterize the differentiation state of hepatocytes in vitro, de novo synthesis of plasma proteins was analyzed. On the 17th, culture for 24 hours pss ) Methionine-labeled plasma proteins were immunoprecipitated from the labeled medium. and analyzed by 5DS-PAGE.

Culture was carried out using 250 tt Ci [”S] methionine (> 800 C4/mm 24 hours in 2.5 ml of the serum-free medium of Example 1 supplemented with Incubated for a while. Filter the medium and add 1/10 volume of 10x CHAPS Extraction buffer [final concentration 1.0χ CHAPS (CalBiochem).

0.25mM phenylmethylsulfonyl fluoride, 10a+M EDTA .

0.05 M Tris (pH 8,0), 0.05 M Tris (pH 8,0), 0. IM NaCl, 100 Mix with uM leupeptin J and incubate for 1 hour at 4°C with agitation. I did it. Commercially obtained antibodies directed against human plasma proteins (Cal Biochem, Boehringer Mannhei+*) (20 μl ) to protein A-agarose beads (50g L Repligen) for 1 hour. Bind in CHAPS extraction buffer on ice. Beads are washed twice (deter (C) IAPS extraction buffer + 1% deoxycholic acid and and 0.1x5DS) and labeled medium overnight at 4°C with agitation. Incubate it for a while. Pellet the beads and wash three times with detergent wash buffer. Washed. Binding proteins were prepared in 2% SDS and 2% 2-mercaptoethanol. Elute with 50 μlt pneumophoresis sample buffer containing Heat and analyze by 5O3-PAGE. Gel for fluorography using Autofluor (National Diagnostics) for processed, dried and autoradiographed on XAR-5 film at -85°C. Analyzed by fees.

This analysis shows that the amount of plasma protein synthesized in vitro is determined by the concentration found in plasma. It was suggested that it reflects the degree of The intensities of polypeptide bands in descending order are min, α, -antitrypsin, fibrinogen, transferrin, apoA- I and E, β2-microglobulin, prealbumin, apoA-11 and A -IIt, complements C3, C4 and C5, C-reactive protein, and apoC-2 and It was C-3. α-, a marker of poorly differentiated fetal or malignant liver tissue All the labels tested were detected except for fetoprotein. numerous plasma tans Protein expression indicates that differentiated hepatocytes of parenchymal origin are maintained in culture. did.

Cultured hepatocytes grown on cover glass (cover 5lips) were cultured. New NANs can be detected by immunocytochemical staining at various times during the cultivation period. analyzed for the presence of BH virus-associated antigens (Burk et al., 'Detect ion of non-A, non-8 hapatitis antigen by im+*unocytoche+wical staining', P roc.

Natl Acad, Sci, USA 81:3195-3199 (19 84)), typical cytoplasmic staining, but the proportion of cells expressing this label varies in culture. observed in all samples examined, with a tendency to increase with time. . However, the number of cells with clear staining did not increase by more than 10% .

Active replication of viruses in tissue culture n) was suggested by the presence of NANBH virus-associated cytoplasmic antigens. moreover , degeneration of primary chimpanzee hepatocytes after 4 weeks of culture was the result of viral replication. Based on these findings, it may be possible that infectious NA in cultured hepatocytes Production of NBH virus was tested by inoculating chimpanzees with tissue culture medium and testing the development of disease. analysis by monitoring the animal for sanifestaton. Ta. Preliminary experiments with HBV showed that a limited number of infected cells in culture remained viable. This suggested a lower viral titer than that observed in the body. moreover, It remains to be seen whether cultured cells result in lower virus titers than those observed in vivo. It was knowledge. Furthermore, whether the expression of NANBH agents is temporary during the culture period or not. It was unknown. Therefore, media samples from each time point (3-31 days) should be stored and Concentrated 8 times by filtration and HBV-immune NANBH virus non-immune It was used to inoculate chimpanzees (P, TTx196).

Example 3 NANBH virus・ The tissue culture medium described in Example 2 was collected at 2-day intervals and divided into 0.45 μl fifties. filter and stored at 100°C. Equal amounts of each sample, 3 to 31 days; were collected (total 190 ml) and purified by pressure dialysis at 4 °C under N2 gas for 30 min. It was concentrated using a 000 MW exclusion membrane (YM 30, Amlcon). 8 FIIl condensate (22 ml) was added for 110 min until use as exemplified by Example 4. Stored at 0°C.

Example 4 KaishikNB ratio's soil - NANBH Irus's peak A definitive study to monitor NANBH virus expression in the medium of these hepatocyte cultures. In the absence of probes, chimpanzees can be grown using medium obtained from virus-infected cultures. Confirmation of active replication of NANBH virus by inducing hepatitis in It was necessary to obtain

PTTxlS6.12-year-old male chimpanzees undergo experimental NANBH of PTTx7. of tissue culture media derived from cultures of hepatocytes isolated during the acute phase of viral infection. Received 10 ml of 8x concentrate (Example 3). A second inoculum of the same material (7 ml ) was administered 12 weeks later.

Weekly blood samples and periodic liver needle punch biopsies were taken for analysis. ALT A slight increase in blood pressure occurred at 4 weeks and microscopic examination of a liver punch biopsy at this time showed Shows minimal foci of hepatocellular necrosis with 2 or 3 types of neutrophils associated with death was. This represents the smallest change that can sometimes be observed in normal tissues. But under the circumstances, it was interesting. A L T/A S Reversal of T occurred at 8 weeks and a second hepatic needle punch biopsy taken at this time showed no recognition. It showed essentially normal tissue with no microscopic lesions. normal tissue types Similar findings were observed in a biopsy taken at 12 weeks.

Due to delayed onset of clinical hepatitis, a second injection of the same inoculum (7 ml) was administered at 12 weeks. was administered. After this, ALT values began to increase 3 weeks later. Sustained ALT increase was observed 16-24 weeks after the final inoculation. The long incubation period is However, this may reflect the low titer of our initial inoculum. Microscopic examination of a liver punch biopsy taken at 14 weeks showed signs of hepatitis. inflammation Foci of accumulation of sex cells were present in the liver parenchyma.Occasionally, necrotic liver cells associated with inflammation were present. Cysts (Calancilman corpuscles) were present. Kzupah cell hyperplasia throughout the liver It was obvious. There was also edematous degeneration of hepatocytes in the central venous region. these changes was minimal but similar to that seen in the biopsy taken at 4 weeks. High Examination of a liver punch biopsy taken at week 17 during the period of serum ALT revealed that a small Presents acute hepatitis characterized by hydropic degeneration with loss of hepatocytes in the center of the lobe. Was.

Liver biopsies were diluted with 0.1M Sorens for observation by electron microscopy. harden with cold 3% glutaraldehyde in phosphate buffer (pH 7.4). and post-fixed in 1% osmic acid for 1 hour at 4"C. Ethanol and After dehydration in propylene oxide, embedding in Epon812. die section LKB IIM with Yamond knife [cut with ultramicrotome, saturated aqueous acetate stained with ranyl and lead citrate and using an AEI EM6B electron microscope. A carbon grating replica (carbon grating) was placed on the 0x magnification scale examined. The scale was attached using a replica (54,800 lines/inch); ( E, F, Fullaw Inc, 5chenectady, N, Y,) Electron microscopy observations made on the liver biopsy taken at 17 weeks revealed fine details of the liver cells. It showed the presence of convoluted tubular organs (tubules) in the cytoplasm. these tubes Presence of a morphological organ used as a signature marker for NANBH in chimpanzees has been done. These results further demonstrate that clinical disease is associated with N This has been proven to be the result of ANBH virus inoculation.

Collected from PTTx196 at 0.16 and 22 weeks of this experimental NANBH infection. The collected plasma samples were tested for CMV, EBV, HBV, H3V and spumavirus. The resulting seroconversion was analyzed. These agents can cause or cause hepatitis. can be transmitted by the following methods. CMV, EBV, HBV, subumavirus, No increase in antibody titers was observed by specific assays against and H3V. . These results indicate that the disease transmitted to PTTx196 is caused by NANBH agonists. It is confirmed that this was caused.

A definitive probe for monitoring virus expression in the medium of these hepatocyte cultures. media obtained from infected cultures can induce hepatitis in chimpanzees. It is necessary to obtain confirmation of active replication of the NANBH virus by Ta. Infectious virus detected in tissue culture medium is transferred to hepatocytes at the time of isolation. There is a very small chance that there is any residual virus present. Extensive cleaning, but perfusion /collagenase procedure (2 liters) and pellet of hepatocytes before blating occurs during clarification and resuspension (4 times). Furthermore, within 3 days, the culture The land had been replaced four times. Therefore, this experiment We demonstrate the feasibility of culturing isolated hepatocytes during the acute stage of R. rus infection.

This system has proven to be advantageous for the identification and characterization of NANBH agents. , which should lead to an explanation of its replication mechanism and long-term chronic persistence. 'be.

Those skilled in the art with the benefit of this disclosure will appreciate that variations in the formulation of the serum-free media of the present invention weakens the ability of the medium to maintain long-term culture of primary hepatocytes infected with BH virus. All such changes that one would understand could be made without patenting It is believed to be within the spirit and scope of the invention as set forth in the claims below.

international search report lms+’+Hatake+□611@111116”+81411ml+1rT/Tl9 ()r)znnql<Fd/US90100915

Claims (26)

[Claims] 1. NANBH virus-infected primate hepatocytes, cell culture basic medium, hepatocyte proliferogens, serum albumin, corticosteroids, Somatotropin and/or prolactin, growth/release factors cholera toxin and ethanolamine In vitro culture of NANBH virus containing. 2. The basic cell culture medium is Williams medium E, Dulbecco's modified Eagle medium. The in vitro culture according to claim 1, which is Ham's F12 medium. 3. Hepatocyte proliferogens are linked to insulin, glucagon, liver growth factors, or The in vitro culture according to claim 1, which is one or more epidermal growth factors. 4. The in vitro culture according to claim 1, further comprising trace metals. 5. The in vitro culture according to claim 4, wherein the trace metal is selenium, zinc or copper. 6. The in vitro culture according to claim 4, wherein the trace metal is selenium. 7. The in vitro culture according to claim 1, further comprising transferrin. 8. The in vitro culture according to claim 1, further comprising linolenic acid. 9. Growth/release factors include thyrotropin-releasing factor, fibroblast growth factor, platelet-derived Growth factors, multiplication stimulating activators or endothelium The in vitro culture according to claim 1, which is one or more cell growth aids. 10. The test tube of claim 1, wherein the growth/release factor is thyrotropin releasing factor. Internal culture. 11. The in vitro culture according to claim 1, wherein the primate hepatocytes are chimpanzee hepatocytes. Nutrition. 12. The in vitro culture according to claim 1, wherein the primate hepatocytes are human hepatocytes. 13. Claim wherein the NANBH virus is a parenterally transmitted NANBH virus. In vitro culture according to 1. 14. NANBH virus infection 106-107 cells/60mm tissue chimpanzee -Hepatocyte culture dish, Epidermal growth factor 25-100mg/ml, insulin 2-10μg/ml, Glucagon 0.5-10μg/ml, bovine serum albumin 0.2-2mg/ ml, linoleic acid 0-5μg/ml, Hydrocortisone 10-9-10-6M, selenium 10-9-10-7M, Cholera toxin 1-5ng/ml, Liver growth factor 0-50ng/ml Transferrin 0-10μg/ml, ethanolamine 10-6-10- 6M, prolactin 0-200ng/ml, somatotropin 0-5μg/m l, Testing of NANBH virus cultures containing thyrotropin-releasing factor 0-10-6M In-tube culture. 15. Claim 1, wherein the NANBH virus is a parenterally transmitted NANBH virus. In vitro culture according to 4. 16. A viral composition comprising a serum-free cell-free NANBH virus. 17. The serum-free cell-free NANBH virus is the NANBH virus according to claim 1. The virus composition according to claim 16, which is a supernatant of an in vitro culture of. 18. The serum-free cell-free NANBH virus is the NANBH virus according to claim 1. 17. The viral composition of claim 16, which is a lysate of an in vitro culture of. 19. Claim wherein the NANBH virus is a parenterally transmitted NANBH virus. 16. The virus composition according to 16. 20. In vitro culture of the NANBH virus according to claim 1 to non-immune chimpanzees. to non-immune chimpanzees, characterized by inoculation with an infectious dose of an inoculum containing nutrients. How to cause NANBH virus infection. 21. The inoculum is a lysate of an in vitro culture of the NANBH virus according to claim 1. 21. The method of claim 20, comprising: 22. The inoculum is a cell-free in vitro culture of the NANBH virus according to claim 1. 21. The method of claim 20, comprising a supernatant. 23. 21. The method of claim 20, wherein the inoculum comprises a serum-free cell-free virus. 24. The inoculum is the in vitro cultured NANBH virus-infected virus according to claim 1. 21. The method of claim 20, comprising long hepatocytes. 25. Claim wherein the NANBH virus is a parenterally transmitted NANBH virus. 20. The method described in 20. 26. liver cells are excised from the host, The hepatocytes are cultured in the in vitro culture medium according to claim 1, and After about 2 to 4 weeks, the cytopathic effect of the cultured hepatocytes was observed. indicates NANBH virus infection, A method for confirming NANBH virus infection in a host, characterized by: