WO1990010060A1 - Culture de cellules d'hepatocytes de l'hepatite non-a, non-b - Google Patents

Culture de cellules d'hepatocytes de l'hepatite non-a, non-b Download PDF

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WO1990010060A1
WO1990010060A1 PCT/US1990/000915 US9000915W WO9010060A1 WO 1990010060 A1 WO1990010060 A1 WO 1990010060A1 US 9000915 W US9000915 W US 9000915W WO 9010060 A1 WO9010060 A1 WO 9010060A1
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nanbh
vitro culture
nanbh virus
virus
hepatocytes
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PCT/US1990/000915
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Robert E. Lanford
Kenneth H. Burk
James R. Jacob
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Southwest Foundation For Biomedical Research
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Priority to AU51666/90A priority Critical patent/AU647384B2/en
Publication of WO1990010060A1 publication Critical patent/WO1990010060A1/fr

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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/39—Steroid hormones
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24251—Methods of production or purification of viral material

Definitions

  • the present invention relates to an in vitro cell culture medium capable of maintaining in culture Non-A, Non-B hepatitis (NANBH) virus. More particularly, this invention relates to a serum-free primate hepatocyte cell culture medium which can maintain NANBH virus in culture.
  • NANBH Non-A, Non-B hepatitis
  • Non-A, Non-B hepatitis has long been recognized as a vj rus-induced disease, distinct from other forms of viral-associated liver diseases, including hepatitis A virus (HAV) and B virus (HBV), and the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV) .
  • HAV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis A virus
  • HBV hepatitis
  • the present invention provides an in vitro cell culture of NANBH virus which includes NANBH-infected primate hepatocytes sustained in a serum-free medium comprising a basal cell culture medium, a hepatocyte proliferogen, serum albumin, a corticosteroid such as hydrocortisone, one or both of somatotropin or prolactin, a growth/releasing factor, cholera toxin and ethanolamine.
  • NANBH virus which includes NANBH-infected primate hepatocytes sustained in a serum-free medium comprising a basal cell culture medium, a hepatocyte proliferogen, serum albumin, a corticosteroid such as hydrocortisone, one or both of somatotropin or prolactin, a growth/releasing factor, cholera toxin and ethanolamine.
  • the present invention also provides a serum-free, cell-free isolate of NANBH virus.
  • the isolate of NANBH virus is obtained as either a culture medium supernatant or a lysate of the in vitro cell cultured NANBH virus- infected hepatocytes.
  • the present invention includes methods of producing NANBH virus infection in chimpanzees.
  • Such controlled NANBH virus infection of chimpanzees should provide an experimental model for the study of NANBH virus infection and serve as reservoir for production of antibodies to NANBH virus.
  • the NANBH virus infection is induced by inoculating chimpanzees with an infectious amount of an inoculum comprising an in vitro culture of NANBH virus-infected hepatocytes, a cell-free supernatant of the in vitro culture, a lysate of the in vitro culture, or cultured hepatocytes separated from their in vitro culture medium.
  • the present invention provides a method of confirming NANBH viral infection in a host.
  • the method involves excising hepatocytes from the host, culturing the hepatocytes, and observing cytopathic effects of the cultured hepatocytes after about two to four weeks.
  • the cytopathic effects of the hepatocytes is indicative of NANBH virus infection.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following described serum-free media exemplifies the formulations of the present invention which are useful in sustaining NANBH virus in primate hepatocyte cell culture.
  • Williams Medium E served as a basal medium.
  • WME Williams Medium E
  • WME is presently preferred as the basal medium of the serum-free medium of the present invention, it will be understood by those skilled in the art who have the benefit of this disclosure that other commercial media formulations can be expected to give satisfactory results.
  • hepatocyte proliferogen refers to one or more of the several growth factors or hormones, such as epidermal growth factor (EGF) , insulin, liver growth factor (LGF), and glucagon, all of which have been implicated in controlling liver growth in vivo (Salas-Prato, supra, at G15) .
  • EGF epidermal growth factor
  • LGF liver growth factor
  • glucagon glucagon
  • transport protein refers to those proteins found in the serum which include as one of their functions the transport of certain substances in the blood.
  • proteins include serum albumin, which may be advantageously used in the commonly available form of bovine serum albumin (BSA) , and transferrin.
  • BSA bovine serum albumin
  • transferrin transferrin
  • the trace metal specifically contemplated for use in the medium of the present invention is selenium; however, WME contains copper, zinc, cobalt and iron and either WME, or other basal media, can be additionally supple ⁇ mented with either or both of zinc and/or copper depending upon the original condition of the hepatocytes and whether the basal medium includes either or both of those trace metal(s).
  • TRF thyrotropin-releasing factor
  • fibroblast growth factor fibroblast growth factor
  • platelet-derived growth factor platelet-derived growth factor
  • multiplaction-stimulating activator multiplaction-stimulating activator
  • ECGS endothelial cell growth supplement
  • the supplements are set out in specific proportions in the following table, it will be understood by those skilled in the art who have the benefit of this disclosure that those proportions can be, and in some circumstances, must be, varied. For instance, ECGS has been found not to be required for maintenance of the hepatocytes. The concentration of glucagon in the media can be reduced. Also, there is some interchangeability between certain of the supplements. For instance, the addition of soybean lipids may be substituted for linoleic acid. In addition, the quality of the hepatocytes obtained from different isolations may require the use of different hepatocyte proliferogens .
  • the media of the present invention therefore, includes a range of proportions of each of the supplements as shown in Table 2. TABLE 2
  • Insulin >_ 2 ⁇ g/ml 5- ⁇ 10 ⁇ g/ml
  • Glucagon > 0.5 ⁇ g/ml 0. 5-10 ⁇ g/ml
  • a media formulation includes, for instance, l ⁇ ⁇ 6 M TRF, the media includes about 10 ⁇ 6 M TRF.
  • Example 1 of the present invention The several studies that were performed to evaluate the ability of the media formulation of Example 1 of the present invention to support NANBH viral replication will now be described.
  • the isolation of NANBH virus-infected hepatocytes from chimpanzees is described in Examples 2 and 4.
  • the serum-free media formulation utilized WME as a basal medium supplemented with 10 mM HEPES, pH 7.4,
  • Thyrotropin Releasing Hormone 50 ⁇ l 200 ⁇ g/ml LGF
  • WME was purchased with L-glutamine and without NaHCO- ⁇ from Hazelton Research Products, Inc. (Denver, Penn.).
  • NANBH virus-infected hepatocytes In order to obtain NANBH virus-infected hepatocytes for in vitro experimentation, a parenteral NANBH virus infection was induced in chimpanzee PTTx7, a 14-year-old female, by inoculation with 5 ml of a 20-fold concentrate of acute phase plasma of unknown titer derived from a second passage of the Hutchinson strain of NANBH virus. Progression of the NANBH virus infection was monitored by ALT/AST enzyme fluctuations from weekly blood samples and by histopathologic examination of periodic liver needle punch biopsies. All biopsies were processed identically using conventional techniques.
  • liver biopsies were fixed for 1-3 hours in neutral buffered 3.7% formalin, processed manually according to standard procedures, embedded in paraffin, sectioned at 4 microns 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 significantly delayed, a second inoculation of 1.5 ml
  • NANBH vi.rus Hutchm. son i.noculum was administered on week 10 to assure infection.
  • the appearance of elevated ALT on week 12 indicated that the second inoculum either exacerbated the primary infection or was not required.
  • the ALT profile of the animal exhibited a rise above normal values from 12-19 weeks post-inoculation, and a second ALT elevation occurred on week 39.
  • Liver wedge survery was performed on week 14 at the onset of definitive ALT elevation.
  • Microscopic exami ⁇ nation of liver tissue taken at this time revealed occasional collections of lymphocytes and macrophages in hepatic triads and in focal parenchymal areas. There were no other changes indicating a significant inflam ⁇ matory response. Although minimal inflammation was present, this finding could be representative of normal liver tissue.
  • Hepatocytes were isolated on week 14 under the pretense that maximal virus replication would occur prior to or during this stage of the disease manifestation.
  • Ketamine hydrochloride was used as the immobilizing and pre-anesthetic agent. Surgery was performed under general anesthesia with non-hepatotoxic sodium pentobarbital. A liver wedge of approximately 10 g was perfused using a modification of established protocols (Maslansky, C. J. and G. M. Williams, In Vitro Models for Cancer Research, Vol. II: Carcinomas of the Liver and Pancreas, M. M. Weber and L. I. Sekely (Eds.), CRC Press: Boca Raton, Fla., pp. 43-60 (1985)). A two-step perfusion procedure was employed with all solutions maintained at 37°C throughout the perfusion procedure.
  • the initial perfusion lasted 10 minutes using 1 liter of Ca , Mg -free Hanks Balanced salt solution supplemented with 10 mM HEPES (pH 7.4), 0.5 mM EGTA, and 100 ⁇ g/ml gentamycin sulfate.
  • the next perfusion was for 20 minutes at approximately 60 nil/min. of Williams Medium E (WME) supplemented with 10 mM HEPES (pH 7.4), 100 ⁇ g/ml gentamycin sulfate, and 200 units/ml collagenase Type I (300 units/mg, Sigma).
  • WME Williams Medium E
  • the liver capsule was then removed with fine forceps and hepatocytes were dislodged by gentle agitation in 100 ml of collagenase solution.
  • the hepatocyte suspension was filtered through several layers of gauze pads into an equal volume of cold WME containing 5% fetal bovine serum (FBS), 10 mM HEPES (pH 7.4), and 100 ⁇ g/ml gentamycin sulfate. Hepatocytes were sedimented at 50 x g for 5 minutes and cell pellets were resuspended in WME 5% FBS. Sedimentation was repeated twice, pellets were resuspended in 10 ml WME 5% FBS, and viability and cell density were determined by trypan blue exclusion.
  • FBS fetal bovine serum
  • HEPES pH 7.4
  • gentamycin sulfate 100 ⁇ g/ml gentamycin sulfate. Hepatocytes were sedimented at 50 x g for 5 minutes and cell pellets were resuspended in WME 5% FBS. Sedimentation was repeated twice, pellets were resuspended in 10 m
  • PRIMARIA plates were coated with rat tail collagen (Michalopoulos, G. and H. C. Pitot, "Primary culture of parenchymal liver collagen membranes,” Exptl. Cell. Res. 94: 70 (1975)) for 6 minutes at room temperature, the excess collagen was removed, and plates were dried overnight under U.V. light. Viable cells were plated at a density of 3-4 x 10 cells/60 mm dish. Cell attachment occurred during a 3-hour incubation at 37°C, 10% C0 2 in WME 5% FBS, at which time cell monolayers were gently washed one time with WME and re-fed with serum-free medium formulation prepared as described in Example 1 above. The medium was changed 24 hours after isolation and at 48-hour intervals thereafter.
  • the cultured hepatocytes displayed a typical hepatocyte morphology as observed by phase-contract microscopy on day 5 of culture. This morphology was maintained until days 21-28 when the cultures exhibited a degenerative process.
  • NANBH Hepatocyte Cell Culture Characteristics The synthesis and secretion of albumin, apolipoprotein A-I, and apolipoprotein E were monitored by immunoblotting of aliquots of tissue culture medium. Briefly, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE,) and were electrophoretically transferred to Nylon-X nitrocellulose filters (Fisher) at 100 mA for 16 hours at 4°C. Unoccupied binding sites were blocked in 10% nonfat dry milk in phospate buffered saline (PBS) for 2 hours at 37°C.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Membranes were incubated for 2 hours at 37°C in PBS-milk-Tween (PBS containing 5% nonfat dry milk, 0.3% Tween-20), using primary antibodies directed against human apolipoproteins A-I and E. Membranes were washed three times with PBS-Tween and incubated 1 hour at 37°C in PBS-milk-Tween with antibodies directed against each of the primary antibodies. Membranes were washed three times with PBS-Tween and incubated 1 hour at 37°C in PBS-milk-Tween with [ I] protein A (8.5 ⁇ Ci/ ⁇ g, NEN) . Membranes were washed three times with PBS-Tween and air dried. Immu ⁇ oblots were autoradiographed at -85°C on XAR-5 film (Kodak) with intensifying screens.
  • Albumin detected by this immunoblot procedure remained at constant levels throughout the culture period. Although albumin is a marker for differentiated hepatocytes, it is not as stringent of a marker for the differentiated state as is lipoprotein synthesis. The decline in lipoprotein synthesis after 28 days in culture paralleled a degeneration in the hepatocyte cultures. The degeneration of primary hepatocytes after 3-4 weeks of culture was evident in cultures derived from two different N ⁇ NBH-infected chimpanzees, but was not observed in cultures from a normal chimpanzee or chim ⁇ panzees with HBV infections. Normal hepatocyte cultures generally survive more than 100 days in the serum-free media. Further experimentation will be required to determine whether the degenerative process is due to viral-induced cytopathic effect.
  • liver specific plasma proteins were analyzed. On day 17, cul- Lures were labeled for 24 hours with [ 35S]methionine.
  • Plasma proteins were immune precipitated from the labeled medium and analyzed by SDS-PAGE.
  • Hepatocyte cultures grown on coverslips were analyzed at various times during the culture period for the presence of a novel NANBH virus-associated antigen that can be detected by irnmunocytochemical staining (Burk et al., "Detection of non-A, non-B hepatitis antigen by irnmunocytochemical staining," Proc. Natl. Acad. Sci. U.S.A. 81:3195-3199 (1984)). Typical cytoplasmic staining was observed in all samples examined with a tendency for the percentage of cells expressing this marker to increase with time in culture. However, the number of cells with definitive staining never increased above 10%.
  • the active replication of the virus in tissue culture was suggested by the presence of a NANBH virus-associated cytoplasmic antigen.
  • the degeneration of the primary chimpanzee hepatocytes after 4 weeks of culture may have been due to the replication of the virus.
  • the production of infectious NANBH virus in the hepatocyte cultures was assayed by inoculation of a chimpanzee with tissue culture medium and monitoring the animal for disease manifestation.
  • Previous experimentation with HBV suggested that the lirrited number of cells infected in cultures resulted in lower viral titers than those observed in vivo.
  • Tissue culture medium as described in Example 2 was collected at two-day intervals and passed through 0.45 ⁇ m filters and stored at -100°C. Equal amounts of each sample, days 3 through 31, were collected (190 ml total) and concentrated by pressure dialysis under N penal gas at 4°C with an exclusion membrane of 30,000 MW (YM30, A icon) . The eight-fold concentrate (22 ml) was stored at -100°C until use as exemplified by Example 4.
  • PTTxl96 a 12-year-old male chimpanzee, received 10 ml of an eight-fold concentrate of tissue culture medium (Example 3) derived from hepatocyte cultures isolated during the acute phase of the experimental NANBH virus infection of PTTx7. 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. A slight increase in ALT occurred on week 4 and microscopic examination of a liver punch biopsy at this time revealed minimal foci of hepatocellular necrosis with two or three neutrophils associated with the necrosis. This represents a minimal change that can be occasionally observed in normal tissue but was of interest under these conditions.
  • ALT/AST inversion occurred on week 8 and a second liver needle punch biopsy taken at this time exhibited essentially normal tissue with no microscopic lesions recognized. Similar findings of normal tissue were observed in biopsy material taken on week 12. Due to the delay in onset of clinical hepatitis, a second injection of the same inoculum (7 ml) was administered on week 12. This was followed by an elevation in ALT values beginning 3 weeks later. A persistent ALT elevation was observed 16-24 weeks after the first inoculation. The long incubation period may reflect the low titer of our initial inoculum. However, microscopic examination of a liver punch biopsy taken on week 14 exhibited signs of hepatitis.
  • the liver biopsy was fixed x-jith cold 3% glutaraldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.4) and postfixed for 1 hour at 4°C in 1% osmium tetroxide. Dehydration in ethanol and propylene oxide was followed by embedding in Epon 812. Sections were cut with a diamond knife on an LKB UM I ultra- microtome, stained with saturated aqueous uranyl acetate and lead citrate, and examined with an AEI EM6B electron microscope. Magnification scales were calibrated using a carbon grating replica (54,800 lines/inch); E. F. Fullam Inc., Schenectady, N.Y.).
  • Plasma samples taken from PTTxl96 on weeks 0, 16 and 22 of this experimental NANBH infection were analyzed for seroconversion in response to CMV, EBV, HBV, HSV and spumavirus. These agents may cause hepatitis or be transmitted by this methodology. No increase in antibody titer was observed by specific assay for CMV, EBV, HBV, spumavirus, and HSV. These results confirm that the disease transmitted to PTTxl96 was caused by an NANBH agent. Without a definitive probe to monitor viral expression in the medium of these hepatocyte cultures, it was necessary to obtain conclusive evidence for the active replication of NANBH virus by the induction of hepatitis in a chimpanzee with medium derived from the infected cultures.

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Abstract

L'invention concerne la culture in vitro de cellules virales de l'hépatite non-A, non-B (NANBH). On a isolé et cultivé des hépatocytes primaires provenant d'un chimpanzé pendant la phase aiguë d'une infection expérimentale par le virus NANBH. La culture de cellules d'hépatocytes différentiées a été maintenue dans un milieu sans sérum comprenant un milieu de Williams E, un proliférogène d'hépatocyte, de la transferrine, de l'albumine de sérum, un corticostéroïde, de la prolactine, un facteur de libération de thyrotropine, une toxine du choléra et l'éthanolamine. Les hépatocytes cultivés se sont révélés positifs pour l'expression d'un antigène cytoplasmique associé à NANBH. La présence de ce marqueur cytoplasmique a suggéré la persistance de l'infection in vitro. La production de virus infectieux in vitro a été confirmée en inoculant une culture de tissus infectés par le virus NANBH et en documentant par la suite le développement de NANBH chez le chimpanzé.
PCT/US1990/000915 1989-02-24 1990-02-22 Culture de cellules d'hepatocytes de l'hepatite non-a, non-b WO1990010060A1 (fr)

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AU51666/90A AU647384B2 (en) 1989-02-24 1990-02-22 Non-A, non-B hepatitis hepatocyte cell culture

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US31528889A 1989-02-24 1989-02-24
US315,288 1989-02-24

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WO1991015574A1 (fr) * 1990-04-03 1991-10-17 Southwest Foundation For Biomedical Research Virus purifies de l'hepatite c et proteines et peptides de ces virus
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
WO1994025064A1 (fr) * 1993-05-04 1994-11-10 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMANSERVIC ES Propagation de cellules virales de l'hepatite c et procedes associes
US5436318A (en) * 1990-04-06 1995-07-25 Genelabs Technologies, Inc. Hepatitis C virus epitopes
WO1996024662A1 (fr) * 1995-02-10 1996-08-15 Consiglio Nazionale Delle Ricerche Procede de propagation 'in vitro' du virus de l'hepatite c (vhc) dans les cultures de cellules animales non lymphoblastoides, et produits obtenus
US5851758A (en) * 1995-10-25 1998-12-22 Childrens Research Institute Cytopathic replication of hepatitis C virus in a new cell line
US5998130A (en) * 1990-06-25 1999-12-07 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
WO1999067362A1 (fr) * 1998-06-24 1999-12-29 Institut National De La Sante Et De La Recherche Medicale I.N.S.E.R.M. Procede de replication in vitro du virus de l'hepatite c
CN101463345B (zh) * 2008-12-26 2012-03-21 青岛易邦生物工程有限公司 一种病毒繁殖促进剂及其应用
CN108998569A (zh) * 2018-08-13 2018-12-14 郑州安图生物工程股份有限公司 一种乙型肝炎病毒血清样本保存稀释液及其制备方法

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US4464474A (en) * 1980-07-09 1984-08-07 Connaught Laboratories Limited Non-A, non-B hepatitis assay and vaccine

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EP0261233A4 (fr) * 1986-04-01 1988-08-29 Genelabs Inc Cellules tissulaires immortalisees a specificite virale.

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US4464474A (en) * 1980-07-09 1984-08-07 Connaught Laboratories Limited Non-A, non-B hepatitis assay and vaccine

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Biochemical Journal, Volume 243, issued 1987, DIAZ-GIL et al", Identification of a liver growth factor as an albuminbilirubin complex", pages 443-448. See antire Article. *
Biological Abstracts, Volume 733, Issued 1981, BURK et al, "Ultrastructural changes and Virus like particles localized in Liver Hepatocytes of CHIMPANZEES infected with Non-A Non-B Hepatitus", Abstract # 18396. See entire Abstract . *
In Vitro Cellular and Developmental Biology Volume 24 No. 3, March 1988, SALAS-PRATO et al, "Attachment and multiplication, Morphology and protein production of human fetal primary liver cells cultured in hormonally defined media", pages 230-238. See entire Article. *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350671A (en) * 1987-11-18 1994-09-27 Chiron Corporation HCV immunoassays employing C domain antigens
WO1991015574A1 (fr) * 1990-04-03 1991-10-17 Southwest Foundation For Biomedical Research Virus purifies de l'hepatite c et proteines et peptides de ces virus
US5843639A (en) * 1990-04-06 1998-12-01 Genelabs Technologies, Inc. Hepatitis C virus epitopes
US5436318A (en) * 1990-04-06 1995-07-25 Genelabs Technologies, Inc. Hepatitis C virus epitopes
US5443965A (en) * 1990-04-06 1995-08-22 Genelabs Incorporated Hepatitis C virus epitopes
US5998130A (en) * 1990-06-25 1999-12-07 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
US6217872B1 (en) 1990-06-25 2001-04-17 The Research Foundation For Microbial Diseases Of Osaka University Non-A, non-B hepatitis virus genomic cDNA and antigen polypeptide
WO1994025064A1 (fr) * 1993-05-04 1994-11-10 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMANSERVIC ES Propagation de cellules virales de l'hepatite c et procedes associes
WO1996024662A1 (fr) * 1995-02-10 1996-08-15 Consiglio Nazionale Delle Ricerche Procede de propagation 'in vitro' du virus de l'hepatite c (vhc) dans les cultures de cellules animales non lymphoblastoides, et produits obtenus
US5851758A (en) * 1995-10-25 1998-12-22 Childrens Research Institute Cytopathic replication of hepatitis C virus in a new cell line
WO1999067362A1 (fr) * 1998-06-24 1999-12-29 Institut National De La Sante Et De La Recherche Medicale I.N.S.E.R.M. Procede de replication in vitro du virus de l'hepatite c
EP0972828A1 (fr) * 1998-06-24 2000-01-19 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procés pour la réplication in vitro du HCV
CN101463345B (zh) * 2008-12-26 2012-03-21 青岛易邦生物工程有限公司 一种病毒繁殖促进剂及其应用
CN108998569A (zh) * 2018-08-13 2018-12-14 郑州安图生物工程股份有限公司 一种乙型肝炎病毒血清样本保存稀释液及其制备方法

Also Published As

Publication number Publication date
EP0460056A4 (en) 1992-06-24
AU647384B2 (en) 1994-03-24
AU5166690A (en) 1990-09-26
EP0460056A1 (fr) 1991-12-11
JPH04505700A (ja) 1992-10-08
NZ232640A (en) 1992-09-25

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