WO1995031530A1 - Procede de propagation de virus aviaires - Google Patents

Procede de propagation de virus aviaires Download PDF

Info

Publication number
WO1995031530A1
WO1995031530A1 PCT/US1995/005544 US9505544W WO9531530A1 WO 1995031530 A1 WO1995031530 A1 WO 1995031530A1 US 9505544 W US9505544 W US 9505544W WO 9531530 A1 WO9531530 A1 WO 9531530A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
avian
cell line
hepatocellular carcinoma
ilt
Prior art date
Application number
PCT/US1995/005544
Other languages
English (en)
Inventor
Deoki N. Tripathy
William M. Schnitzlein
Original Assignee
The Board Of Trustees Of The University Of Illinois
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The University Of Illinois filed Critical The Board Of Trustees Of The University Of Illinois
Priority to AU24687/95A priority Critical patent/AU2468795A/en
Publication of WO1995031530A1 publication Critical patent/WO1995031530A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18051—Methods of production or purification of viral material

Definitions

  • ILT infectious laryngotraceitis
  • ILT infectious laryngotraceitis
  • ILT is a respiratory disease of chickens that occurs worldwide and that ranges in severity from mild clinical signs, such as conjunctivitis and reduced egg production, to acute respiratory distress and high mortality. See, Hanson, L.E réelle et al. Lar ngotraceitis. In: Diseases of Poultry, 9th ed pp 485-495. Calnek, H. J. et al, eds.
  • the ILT virus is typically propagated either in chorioallantoic membrane of developing chicken embryos or in primary cells of avian origin.
  • the ILT virus was initially propagated in the chorioallantoic membrane of developing chicken embryos.
  • the ILT virus could be propagated in adult chicken kidney cells as well as in a variety of chicken embryo epithelial cells originating from kidney, liver, and lung. See Hanson, L.E. et al. supra, Tripathy, D.N. et al. supra and Hughes, C.S., et al., Avian Pathol. 27:295-303 (1988).
  • these cell cultures have a number of problems.
  • the present invention fills this need by providing for a method of propagating an avian virus in an avian hepatocellular carcinoma cell line.
  • the method comprises inoculating an avian virus capable of growing in an avian hepatocellular carcinoma cell line into the cell line under conditions in which the hepatocellular carcinoma cells are infected with the avian virus and the avian virus grows within the hepatocellular cells.
  • avian viruses which are able to grow in the hepatocellular cell line are infectious laryngotracheitis (ILT) virus, reovirus/tenosynovitis virus, bursal disease virus, avian pox virus, and Newcastle disease virus.
  • ILT infectious laryngotracheitis
  • reovirus/tenosynovitis virus reovirus/tenosynovitis virus
  • bursal disease virus avian pox virus
  • Newcastle disease virus Newcastle disease virus.
  • the hepatocellular carcinoma cells originate from a chicken and is called an LMH cell line.
  • the LMH cell line has been deposited pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S. A under ATCC Accession No. CRL 11597. Also claimed are hepatocellular carcinoma cells infected with avian viruses.
  • a practical application of the present invention is the production of high avian virus titers for use in vaccines, or diagnostic kits.
  • the present invention is a method to propagate avian viruses in a heptocelluar carcinoma cell line.
  • Any avian virus which grows in a hepatocellular carcinoma cell line can be used.
  • a hepatocellular carcinoma cell line is a particularly valuable cell line in which to propagate viruses because of its viability, lack of variability and ease of maintenance.
  • a hepatocellular carcinoma cell line can be produced from one of the above-mentioned birds by administering diethylnitrosamine to the bird.
  • the birds are generally given drinking water containing 100 parts per million (ppm) diethylnitrosamine for 9 weeks and then weekly or biweekly intramuscular injections of 20 to 100 mg of diethylnitrosamine/kg for 30 weeks.
  • Tumorous tissues are then taken from several nodules of the liver separately, transferred onto plastic plates containing 5 ml of Waymouth's medium supplemented with 10% fetal calf serum and kanamycin (6 ⁇ g/ml), minced, and cultured at 37°C in a CO2 incubator. The medium is changed the next day and twice weekly thereafter.
  • a preferred cell line which was produced in this manner is the LMH cell line produced from the liver of a chicken, Kawaguchi, T. et al. Cancer Research 47:4460-4464 (1987).
  • a hepatocellular carcinoma cell line such as the LMH cell line, is typically grown as cells attached to plastic or glass surfaces in a single monolayer of cells.
  • the cell line can be grown in any suitable medium such as, for example, Waymouth's Medium with 10% fetal calf serum (FCS), CELLGRO® (Mediatech Inc.) with 1-5% FCS, DME-F12 (Sigma Inc.) with 10% FCS, Eagles MEM (Gibco Inc.) with 10% FCS, Medium 199 (Gibco, Inc.) with tryptose phosphate broth and 10% FCS, and Medium 1640 with 10% FCS.
  • FCS fetal calf serum
  • CELLGRO® Mediatech Inc.
  • DME-F12 Sigma Inc.
  • Eagles MEM Gibco Inc.
  • Medium 199 Gibco, Inc.
  • FCS Medium 1640 with 10% FCS.
  • the carcinoma cells are passaged every 5 to 7 days using a 1:5 or 1:10 expansion ratio.
  • the resulting cell monolayers are not allowed to reach a high density before passage since they become difficult to trypsinize which would result in the subsequent formation of poor monolayers.
  • the monolayers are drained of medium and then rinsed briefly with a solution containing trypsin, preferably a mixture of trypsin and ethylene diamine tetraacetic acid (EDTA).
  • the cell monolayer allowed is allowed to disperse and the individual cells are then diluted in growth medium to allow for a 1:5 or 1:10 seeding of new flasks.
  • the new cell cultures are then incubated at 37° C in a CO2 incubator.
  • An avian virus can be grown on the avian hepatocellular carcinoma cells by inoculating the cells with the virus.
  • To inoculate the cell line with an avian virus confluent or nearly confluent monolayers of cells are drained of medium and the virus inoculum added to the monolayer. The inoculum is allowed to adsorb for about an hour at 37°C and then fresh medium is added and the cultures returned to the incubator.
  • Virus-inoculated cultures are incubated until monolayers show maximum cytopathic effect (CPE).
  • CPE cytopathic effect
  • the ILT virus requires 1 to 3 days incubation depending on the titer of virus in the inoculum.
  • the ILT virus can be obtained by known methods as described by
  • ILT virus strains can be also obtained commercially from Schering-Plough Animal Health (Omaha, NE) and from the National Veterinary Services Laboratory, (Ames IA).
  • Additional viruses which have been propagated according to the process of the present invention are the infectious bursal disease virus, Newcastle disease virus, avian pox virus and the reovirus/tenosynovitis virus.
  • the Newcastle virus also known as the PMV-1 virus, is a species of the Paramyxovirus genus which is a member of the Paramyxoviridae virus family. Different isolates and strains of Newcastle disease virus may induce a number of different clinical manifestations including hemorrhagic lesions of the digestive tract, and produce neurologic and respiratory indications.
  • the avian pox virus is a common viral disease of domestic birds characterized by the development of discrete, nodular, proliferative skin lesions on the unfeathered parts of the body (cutaneous form) or fibrino- necrotic and proliferative lesions in the mucous membrane of the upper respiratory tract, mouth, and esophagus (diphtheritic form).
  • the avian reovirus/tenosynovitis is a virus which causes arthritis, and chronic respiratory disease in chickens.
  • the reovirus has also been associated with other disease conditions including ruptured gastrocnemius tendons, pericarditis, myocarditis, hydropericardium, cloacal pasting, early mortality in poults, and malabsorption syndrome.
  • Infectious bursal disease virus causes an acute, highly contagious viral infection of young chickens that has lymphoid tissue as its primary target with a special predilection for the bursa of Fabricius severely affecting the birds' immune system.
  • a virulent vaccine strain of ILT virus, L608, and a modified live virus, LT-IVAX® were provided by Schering-Plough Animal Health Corp. (Omaha, NE).
  • a reference challenge strain (NVSL ILTV) was obtained from the National Veterinary Services Laboratory (Ames, Iowa).
  • LMH cells were grown in Waymouth's MB 752/1 medium containing 10% fetal bovine serum (FBS) and 10 ⁇ g/ml gentamicin (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). The cells were grown in monolayers and kept at 37°C in a humidified atmosphere of 4% C0 2 .
  • FBS fetal bovine serum
  • GEBCO BRL Life Technologies, Inc. Gaithersburg, MD
  • ILT virus-containing inocula from each of the three viral strains were allowed to adsorb to monolayers of three separate cultures of LMH cells for 60-120 min at ambient temperature with constant rocking. After adsorption, the LMH cell monolayers were overlaid with Waymouth's medium supplemented with 10% fetal bovine serum (FBS) and 10 ⁇ g gentamicin/ml.
  • FBS fetal bovine serum
  • the monolayers were frozen and thawed twice.
  • the resulting cell lysates were used as a source of viral inocula for future infections.
  • LTL608 in LMH cells, a cytopathic effect became evident, which was characterized by the formation of giant cells.
  • all virus strains produced plaques, which were approximately 1mm in diameter by 4-5 days postinfection.
  • a culture of the LMH cell line was inoculated with a strain of Reovirus/Tenosynovitis virus provided by Schering-Plough, Omaha, NE according to the procedure of Example 1. This procedure resulted in the successful propagation of Reovirus/Tenosynovitis virus in the LMH cell line as evidenced by a cytopathic effect of the growing virus in the LMH cells.
  • Example 5 A culture of the LMH cell line was inoculated with a strain of avian pox virus provided by Schering-Plough, Omaha, NE according to the procedure of Example 1. This procedure resulted in the successful propagation of avian pox virus in the LMH cell line as evidenced by a cytopathic effect of the growing virus in the LMH cells.
  • Example 5 A culture of the LMH cell line was inoculated with a strain of avian pox virus provided by Schering-Plough, Omaha, NE according to the procedure of Example 1. This procedure resulted in the successful propagation of avian pox virus in the LMH cell line as evidenced by a cytopathic effect of the growing virus in the LMH cells.
  • Example 5 A culture of the LMH cell line was inoculated with a strain of avian pox virus provided by Schering-Plough, Omaha, NE according to the procedure of Example 1. This procedure resulted in the successful propagation of avian pox virus in the LMH cell
  • a culture of the LMH cell line was inoculated with a strain of
  • Newcastle virus provided by Schering-Plough, Omaha, NE according to the procedure of Example 1. This procedure resulted in the successful propagation of Newcastle virus in the LMH cell line as evidenced by a cytopathic effect of the growing virus in the LMH cells.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Un procédé permet de propager des virus aviaires dans une lignée cellulaire de carcinome hépatocellulaire aviaire. On peut citer comme exemples de virus pouvant être ainsi propagés ceux de la laryngotrachéite avairie, un réovirus ou le virus de la ténosynovite, celui de la bursite infectieuse, le poxvirus aviaire, et celui de la maladie de Newcastle. Les lignées cellulaires infectées par ces virus, notamment la lignée cellulaire LMH, sont aussi revendiquées dans le cadre de l'invention.
PCT/US1995/005544 1994-05-12 1995-05-10 Procede de propagation de virus aviaires WO1995031530A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU24687/95A AU2468795A (en) 1994-05-12 1995-05-10 Process for propagating avian viruses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24153194A 1994-05-12 1994-05-12
US08/241,531 1994-05-12

Publications (1)

Publication Number Publication Date
WO1995031530A1 true WO1995031530A1 (fr) 1995-11-23

Family

ID=22911074

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/005544 WO1995031530A1 (fr) 1994-05-12 1995-05-10 Procede de propagation de virus aviaires

Country Status (2)

Country Link
AU (1) AU2468795A (fr)
WO (1) WO1995031530A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804233A1 (fr) * 1993-07-13 1997-11-05 Solvay Animal Health, Inc. Procedes de mise en culture du virus infectieux responsable de la laryngotracheite et du virus du syndrome de chute de ponte
CN107126558A (zh) * 2017-04-26 2017-09-05 广州博恒生物科技有限公司 鸡传染性法氏囊病灭活疫苗及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420759A1 (fr) * 1989-09-29 1991-04-03 Institut National De La Recherche Agronomique Procédé de préparation de composition immunisante
WO1995002417A1 (fr) * 1993-07-13 1995-01-26 Solvay Animal Health, Inc. Procedes de mise en culture du virus infectieux responsable de la laryngotracheite et du virus du syndrome de chute de ponte

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420759A1 (fr) * 1989-09-29 1991-04-03 Institut National De La Recherche Agronomique Procédé de préparation de composition immunisante
WO1995002417A1 (fr) * 1993-07-13 1995-01-26 Solvay Animal Health, Inc. Procedes de mise en culture du virus infectieux responsable de la laryngotracheite et du virus du syndrome de chute de ponte

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CONDREAY ET AL: "EFFICIENT DUCK HEPATITIS B VIRUS PRODUCTION BY AN AVIAN LIVER TUMOR CELL LINE", JOURNAL OF VIROLOGY, vol. 64, no. 7, pages 3249 - 32558 *
DATABASE MEDLINE FILE SERVER STN KARLSRUHE; SCHNITZLEIN ET AL: "PROPAGATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS IN AN AVIAN LIVER CELL LINE" *
KAWAGUCHI ET AL: "ESTABLISHMENT AND CHARACTERIZATION OF A CHICKEN HEPATOCELLULAR CARCINOMA CELL LINE,LMH", CANCER RESEARCH, vol. 47, 15 August 1987 (1987-08-15), pages 4460 - 4464, XP002570486 *
SCHOLZ ET AL: "AN AVIAN HEPATOMA CELL LINE FOR THE CULTIVATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS AND FOR THE EXPRESSION OF FOREIGN GENES WITH A MAMMALIAN PROMOTOR", JOURNAL OF VIROLOGICAL METHODS, vol. 43, no. 3, pages 273 - 286, XP025818335, DOI: doi:10.1016/0166-0934(93)90146-I *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804233A1 (fr) * 1993-07-13 1997-11-05 Solvay Animal Health, Inc. Procedes de mise en culture du virus infectieux responsable de la laryngotracheite et du virus du syndrome de chute de ponte
EP0804233A4 (fr) * 1993-07-13 1998-06-10 Solvay Animal Health Inc Procedes de mise en culture du virus infectieux responsable de la laryngotracheite et du virus du syndrome de chute de ponte
CN107126558A (zh) * 2017-04-26 2017-09-05 广州博恒生物科技有限公司 鸡传染性法氏囊病灭活疫苗及其制备方法
CN107126558B (zh) * 2017-04-26 2020-06-23 广州渔跃生物技术有限公司 鸡传染性法氏囊病灭活疫苗及其制备方法

Also Published As

Publication number Publication date
AU2468795A (en) 1995-12-05

Similar Documents

Publication Publication Date Title
AU628063B2 (en) Ibdv production in continuous lines
US6919084B2 (en) Duck pneumovirus and corresponding vaccines
WO2012033328A2 (fr) Lignées cellulaires dérivées de mdck adaptées à la culture sans sérum et à la culture en suspension et procédé de préparation d'un virus de la vaccine utilisant lesdites cellules
JP4148980B2 (ja) ニワトリ貧血因子ワクチン
TANAKA et al. Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan
DK163133B (da) Biologisk ren praeparation af en smitsom bronchitis-virusstamme
WO1999024068A1 (fr) Utilisation d'une lignee cellulaire aviaire immortelle pour cultiver des virus aviaires ou animaux destines a produire des vaccins
Guneratne et al. Some observations on the isolation and cultivation of avian reoviruses
Abdel-Alim et al. Immunogenicity and antigenicity of very virulent strains of infectious bursal disease viruses
AU759179B2 (en) Novel antigenic class of avian reoviruses
US4195130A (en) Propagation of feline infectious peritonitis virus in tissue cultures
WO2003039593A1 (fr) Vaccin contre l'adenovirus aviaire
US6410222B1 (en) In ovo vaccination of marek's disease type I virus
WO1995031530A1 (fr) Procede de propagation de virus aviaires
NAKAMURA et al. Enhanced replication of orbiviruses in bovine testicle cells infected with bovine viral diarrhoea virus
US5686287A (en) Marek's disease virus vaccine
EP0748867A1 (fr) Vaccin contre la maladie de Marek
Callinan et al. An outbreak of disease in pigeons associated with a herpesvirus
JPH10175881A (ja) 伝染性気管支炎ウイルス培養物及びそれからのワクチン
US7824689B2 (en) Chicken astrovirus type 2
Sutjipto et al. Physicochemical characterization and pathogenicity studies of two turkey adenovirus isolants
Cho An improved method for extracting cell-free herpesviruses of Marek's disease and turkeys from infected cell cultures
WO2014061914A1 (fr) Souche k40/09 du virus de la bronchite infectieuse et vaccin pour la bronchite infectieuse l'utilisant
桐沢力雄 et al. In vivo and in vitro infections of chicken lymphocytes with Marek's disease virus or herpesvirus of turkeys.
EP0727484A1 (fr) Procédé pour la croissance de virus

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AU BB BG BR BY CA CN CZ EE FI GE HU IS JP KG KR KZ LK LR LT LV MD MG MN MX NO NZ PL RO RU SG SI SK TJ TM TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA