Method of making a virus using duck embryonic derived stem cell lines

1. A method of producing a virus, the method comprising:

a) isolating a duck embryo around oviposition;

b) dissociating the embryo of step a) into cells;

c) seeding the dissociated cells of step b) in basal medium comprising insulin like growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), animal serum, and a layer of feeder cells;

d) culturing the cells of step c) for at least one passage;

e) withdrawing the IGF-1 and CNTF from the culture of step d);

f) culturing the cells of step e) for at least one passage;

g) progressively withdrawing the feeder cells from the culture of step f);

h) culturing the cells of step g) for at least one passage;

i) progressively withdrawing the animal serum from the culture medium of step h) over several passages;

j) adapting the cells of step i) to suspension culture conditions such that a duck cell line capable of proliferating in basal medium in the absence of exogenous growth factors, a feeder layer, and animal serum is obtained, wherein the duck cell line does not produce replication-competent endogenous retrovirus particles;

k) proliferating the duck cell line obtained in step j) in a serum-free medium;

l) infecting the duck cell line of step k) with said virus;

m) culturing said infected cells of step l) to allow virus replication; and

n) harvesting said virus.

2. The method of claim 1 , further comprising adding glutamine and D-glucose to the medium of steps k), l), and m).

3. The method of claim 2 , wherein the adding glutamine and D-glucose to the medium of steps k), l), and m) occurs on a daily basis.

4. The method according to claim 2 , wherein the glutamine concentration is maintained between 1 mM to 3 mM, preferably around 2 mM.

5. The method according to claim 2 , wherein the D-glucose concentration in the culture medium of steps k), l), and m) is maintained between 1 g/l to 10 g/l, preferably around 2 to 3 g/l.

6. The method of claim 1 , wherein the cell density in step l) is of at least 1.5 million cells/ml.

7. The method of claim 1 , wherein steps k), l), and m) are performed in a cultivation vessel that is a continuous stirred tank bioreactor.

8. The method of claim 1 , further comprising adding glutamine and D-glucose to the medium of steps l) and m).

9. The method of claim 1 , further comprising adding glutamine and D-glucose to the medium of step l).

10. The method of claim 1 , further comprising adding a proteolytic enzyme to the culture medium of step m).

11. The method according to claim 1 , wherein steps k), l), and m) are performed at a temperature of 33° C.

12. The method according to claim 1 , wherein the withdrawal of IGF-1 and CNTF from the culture medium in step e) is performed simultaneously.

13. The method according to claim 1 , wherein said duck is a Pekin duck.

14. The method according to claim 1 , wherein said duck is a Muscovy duck.

15. The method according to claim 1 , wherein said duck cell line is diploid.

16. The method according to claim 1 , wherein the virus is an endogenous avian leucosis virus (ALV-E) or an endogenous avian retrovirus (EAV).

17. The method according to claim 1 , wherein said virus is selected from the group comprising poxviruses, orthomyxoviruses, paramyxoviruses, herpes viruses, hepadnaviruses, adenoviruses, parvoviruses, reoviruses, circoviruses, coronaviruses, flaviviruses, togaviruses, birnaviruses, and retroviruses.

18. The method according to claim 17 , wherein

the poxvirus is selected from the group consisting of Modified Vaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16m8 vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus, ALVAC, NYVAC, juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, and Turkey pox virus;

the paramyxovirus is selected from the group consisting of measles virus, mumps virus, rubella virus, Sendai virus, Respiratory Syncythial virus (RSV), human para-influenza types I and III, Rinderpest virus, canine distemper virus, Newcastle disease virus, and duck para-influenza virus;

the orthomyxovirus is selected from the group consisting of human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, and feline influenza virus;

the togavirus is selected from the group consisting of Sinbis virus, Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, and a recombinant togavirus thereof;

the retrovirus is selected from the group consisting of reticulo-endotheliosis virus, duck infectious anemia virus, duck spleen necrosis virus, and a recombinant retrovirus thereof;

the parvovirus is a duck parvovirus;

the adenovirus is selected from the group consisting of fowl adenovirus, goose adenovirus, duck adenovirus, pigeon adenovirus, and a recombinant adenovirus thereof;

the birnavirus is Infectious Bursal Disease virus; or

the flavivirus is selected from the group consisting of Dengue virus, Japanese encephalitis virus, and West Nile virus.