The document outlines the cultivation of viruses, particularly focusing on methods using embryonated hens' eggs and cell culture. It details the advantages of using embryonated eggs for virus isolation and vaccine production, as well as the procedures for inoculation and incubation. Additionally, it discusses alternative cell culture methods for vaccine production and highlights challenges and considerations associated with both egg-based and cell-based systems.

2. Viruses are Different From Other Microbes
• Viruses are obligate intracellular parasites. They depend
totally on their host cells for their existence. Their total host
dependence makes it, extremely difficult to get good insight
of them natural conditions, because the internal
characteristics of the host cells are likely to interfere with the
observations. Due to these reasons, it has been found
desirable that viruses are cultivated or grown in the
laboratory itself.

3. Isolation of Virus
• Laboratory animals
• Fertilized Hen’s Egg
– Chorioallantoic membrane
– Allantoic cavity
– Amniotic cavity
– Yolk sac
• Organ/Tissue/Cell Culture
• Growth identified by serological method like neutralization.

4. Virus Culture
Embryonated Egg Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/ Tissue cultures Primary
Diploid/ Secondary
Continuous
Animal inoculation Suckling

5. Embryonated Hen’s Egg
Cultivation of Viruses and Bacteria
• Chorioallantoic membrane (CAM) – visible lesions called pocks. Each
infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
• Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
• Amniotic cavity – primary isolation of Influenza virus
• Yolk sac – Chlamydia, Rickettsia & some viruses

6. Embryonated eggs:
• The Embryonated hen’s egg was first used for cultivation of viruses by
Good Pasteur and Burnet (1931). Cultivation of viruses in organized
tissues like chick embryo necessitates a different type of approach.. For all
practical purposes they all themselves behave as tissue cultures. The
process of cultivation of viruses in embryonated eggs depends on the type
of egg which is used. The egg used for cultivation must be sterile and the
shell should be intact and healthy.

7. Burnet as Director of the Hall Institute, 1944-1965
F.M. Burnet in the laboratory
in the early 1950's,
was experimenting
on influenza virus genetics,
using the developing hen's egg

8. Only Embryonated Eggs Are Suitable
for Growing Virus
Inoculated eggs are candled
daily to see the chicken
embryos inside.

9. Eggs are Used for Mass Vaccine Production
in Influenza
 Animals and chick embryo
were the first method that was used
to cultivate virus. This method is rarely
used as it is not convenient.
However, when preparing for bulk virus,
(e.g. antigen or vaccine production)
the usage of chick embryo is useful.

10. Advantages of Fertile Eggs
 Fertile chicken eggs provide
a convenient, space-saving incubator
for many kinds of animal viruses.
Different viruses can be injected
into an egg at different sites and
the egg can be easily observed
for viral replication throughout
the development of the chicken embryo.

11. Advantages of Using Embryonated Eggs
• Isolation and cultivation of many avian and few mammalian
viruses
• Ideal receptacle for virus to grow
• Sterile & wide range of tissues and fluids
• Cost- much less
• Maintenance-easier
• Less labor
• Readily available

12. Advantages of Fertilized Eggs are
 Free from bacteria and many latent viruses.
 Free from specific and
non specific factors of defense.

13. Structure and Utility of Fertilized Egg

14. Routes of Injecting the Fertilized Eggs

16. Cultivation of Virus in Eggs
• To cultivate viruses in
eggs, the procedure
adopted should be very
simple. The eggs are kept
in incubator and embryos
of 7-12 days old are
used. The egg containing
embryo usually has an air
apace at the larger end.
The position of this sac is
first determined.

17. Begin you Exercise with
Candling Eggs
• Candling is the process of
holding a strong light above
or below the egg to observe
the embryo. A candling lamp
consists of a strong electric
bulb covered by a plastic or
aluminum container that has
a handle and an aperture.
The egg is placed against
this aperture and illuminated
by the light. If you do not
have a candling lamp,
improvise. Try using a torch.

18. Marking the inoculation site:
1. Hold the blunt end of the egg
against the aperture of the
candling lamp and note the
position of the head of the
embryo.
2. Turn the egg a quarter turn away
from the head.
3. Draw a line on the shell marking
the edge of the air sac.
4. Draw an X approximately 2 mm
above this line.
5. The X marks the inoculation
site.

19. • Eggs: 9-day old or 10-day old embryonated eggs. Candle the eggs
and mark the inoculation sites as described in Section 5. Eggs
should be placed in an egg rack with the inoculation site uppermost.
• Egg shell punch.
• Cotton wool.
• A 70 percent alcohol solution in water.
• Syringe 1 mL.
• Needles preferably 25 gauge, 16 mm.
• Stationery tape (also called cello or sticky tape) or melted wax to
seal the inoculation site.
• Inoculum. This must be free of microbial contamination.
• Discard tray.
Materials Needed for Egg
Inoculation

20. 1. Use cotton wool and 70 percent alcohol to swab the end of the
eggs to be inoculated. Allow the alcohol to evaporate.
2. Swab the eggshell punch with 70 percent alcohol solution.
Place used cotton wool in discard tray.
3. Pierce a hole in the end of the egg at the marked inoculation
site.
4. Attach needle to 1 mL syringe.
5. Draw inoculum into 1 mL syringe.
Inoculation of the Allantoic
cavity

21. 6 Keeping the needle and syringe vertical, place the needle
through the hole in the eggshell. The needle will need to
penetrate approximately 16 mm into the egg to reach the
allantoic cavity.
7. Inject 0.1 mL of inoculum into the egg.
8. Withdraw the needle from the egg.
9. Seal the hole in the shell with stationery tape or melted wax.
10. Discard the used needles and syringes.
11. Place the inoculated eggs into a second incubator. Check the
temperature and humidity of incubate
Inoculation of the Allantoic cavity

22. Piercing a hole in the egg shell
• A dental drill can be
used if it is available.
In most laboratories a
tool called an eggshell
punch can be
improvised using
materials that are
cheap and easy to
procure.

23. Routes of Egg Inoculation

24. Inoculating the Specimens
• The rest of the embryo then
gets exposed and ready for use.
Virus suspension to be
cultivated is taken in dropper
and gently spread over the
exposed embryo. After
inoculation is thus completed,
the open area of the shell is
sealed eggs are incubated for
one week as in hatching. The
virus particles infect the
membrane at random and create
pock marked appearance
against the transparent
background. This indicate viral
basis.

25. Chorioallantoic membrane (CAM):
• CAM is inoculated mainly for
growing poxvirus. Herpes
simplex virus is also grown. Virus
replication produces visible
lesions, grey white area in
transparent Cam. Each pock is
derived from a single virion.
Pocks produced by different virus
have different morphology. Under
optimal conditions, each
infectious virus particle can form
one pock. Pock counting,
therefore can be used for the
assay of pock forming virus such
as vaccinia.

26. Piercing the Chorioallantoic Membrane
• Little holes are drilled
through the egg shell for
infection of the chorio-
allantoic membrane

27. Can be used in few Fungal Infection
• They provide a complex
environment, including
phagocytic cells, to study
fungal host-pathogen
interaction, but are of a lower
developmental stage than
adult mice.

29. Piercing the Shell with Needle

30. Injecting Infective Material with
Needle

31. Overview of Inoculating Sites

32. • Inoculation into the allantoic cavity provides a rich
yield of influenza and some paramyxoviruses.
Allantoic inoculation is employed for growing the
influenza virus for vaccine production. Other
allantoic vaccines include Yellow fever (17D strain),
and rabies vaccines. Duck eggs are bigger and have a
longer incubation period then hen’s egg. They
therefore provide a better yield of rabies virus and
were used for the preparation of the inactivated non-
neural rabies vaccines.
Allantoic cavity:

33. ALLANTOIC ROUTE –
INOCULATION SITE DETERMINATION

34. Amniotic cavity:
• The amniotic sac is
mainly inoculated
for primary
isolation of
influenza a virus
and the mumps
virus.

35. Amniotic Route of Inoculation

36. Yolk sac:
• It is inoculated for
the cultivation of
some viruses as
well as for some
bacteria like
Chlamydia and
Rickettsia.

37. YOLK SAC ROUTE

38. Influenza Vaccine Development in
Fertilized Eggs

39. Influenza Vaccine Traditional Methods- Influenza
Examining the infected eggs Vaccine

40. How Vaccines are Produced in Eggs
• In egg culture, flu viruses are
injected into chicken egg
embryos, where they multiply.
After several days of incubation
a machine opens the egg and
harvests the virus, which is then
purified and chemically killed.
On average it takes one or two
eggs to produce a single dose of
annual flu vaccine.
In cell culture, the virus is
grown in animal or human
cells, which are available in
unlimited supply.

41. How the Reassortant Vaccines for
Influenza Produced in Eggs
• The egg is inoculated
with a mixture of the
epidemic influenza virus
strain (red) and a
standard strain (green)
that can replicate in
chicken eggs. Both
strains replicate
themselves, but as they
do so their genetic
material becomes mixed,
producing hybrid viruses
known as reassortants

42. Eggs as Tools for Developing
Influenza Vaccines
• Influenza vaccine manufacture
in eggs, computer artwork.
Fertilized chicken eggs can be
used to produce vaccines
against influenza viruses. The
reassortants are analyzed, and
those which have the epidemic
strain surface proteins but
other genes of the standard
strain will be selected. These
are injected into different eggs
to replicate before harvesting.

43. Eggs are Used in Mass Scale
Development of Vaccines

44. Egg Allergies and Vaccines
• No suitable cell culture
system exists and egg
inoculation is the method
of choice. Influenza virus
vaccines are still
cultivated in eggs, and
hence people with egg
allergies cannot tolerate
the influenza vaccines.

45. Follow all the Biosafety
Considerations
• All procedures involving
the manipulation of
infectious materials are
conducted within
biological safety
cabinets, specially
designed hoods, or other
physical containment
devices, or by personnel
wearing appropriate
personal protective
clothing and equipment.

46. EGG-BASED PRODUCTION
Advantages:
 Well established and cost-effective
 Lower cost
•Disadvantages:
• Extensive planning: long timeline for million eggs procurement
• Limited flexibility in case of exponentially increasing demand
(pandemic not contained & defeated):
•production takes too long
•eggs don’t grow on demand
• Potential impurities in eggs (antibiotics, other viruses) may
cause sterility problems
• Risk of allergies against egg albumin
• Growth of epidemic viruses in eggs result in variants that are
antigenically distinct from the original viruses
• Emerging endemic viruses sometimes do not grow at all in eggs

47. Cell Culture Vaccine Production

48. Potential Advantages
In response to the urgent and growing need for alternative means for influenza vaccine
production, a number of vaccine manufacturers have considered new approaches.
Of particularly interest has been the potential use of tissue culture cell lines to be
used either in additional to, or in lieu of, egg-based production. The chief
advantages of such a system are summarized below:
 Enables utilization of the same basic and clinically proven approach used in egg-
based production systems – i.e., production of whole-viruses, multiple disrupted
(“split”), more highly purified influenza virus antigens, or live attenuated vaccines
– while at the same time eliminating the long lead times and supply-chain
vulnerabilities required for egg-based production systems .
 Enables a more robust, consistent and reproducible means of vaccine production,
utilizing a scalable and a closed (or largely closed) bioreactor process, which may
be initiated at any time and extended for a prolonged period if needed.
 Enabling the capability of producing influenza vaccines with avian strains, which
generally cannot grow in eggs without genetic modification

49. Ideal characteristics of cell lines that could be utilized for production
 Replication of influenza strains to sufficient titer. Aside from embryonated eggs,
chicken embryo fibroblasts, and other avian cells ,replication of influenza
viruses has otherwise been documented in hamster cells (BHK21-F and
HKCC), MDBK cells, PER.C6 cells, Vero cells, and MDCK cells. A
prerequisite for a successful infection is the addition of proteases to the
medium, preferably trypsin or similar serine proteases, as these proteases
extracellularly cleave the precursor protein of hemagglutinin (HA0) into active
hemagglutinin (HA1 and HA2). Only cleaved hemagglutinin leads to the
adsorption of the influenza viruses on cells with subsequent virus assimilation
into the cell, which leads to further replication. Of the various continuous
mammalian cell lines that have been adequately tested – including diploid cell
lines (MRC-5, WI-38, and FRhl-2) and continuous cell lines (PER.C6, NIH-
3T3, BHK, CHO, Vero and MDCK) – only Vero, PER.C6 and MDCK have
consistently yielded influenza viruses titers that are sufficiently high enough to
be considered commercially viable.

50.  Established Regulatory Precedent: Immortalized (continuous) cell
lines are the only mammalian cell lines that have been documented to
support sufficient replication of influenza viruses. Of the three leading
candidates (Vero, PER.C6 and MDCK) Vero cells have been the only ones
to have been used in a widely used, commercially available vaccine. The
main regulatory concern related to these cell lines is that each of them has
been documented to be tumorigenic in animals at some point during their
passage history. Of the three, MDCK cells are the most tumorigenic, the
mechanism for which remains unknown. This and other regulatory aspects
related to cell line characterization have posed significant challenges for the
approval of mammalian cell-derived influenza vaccines

51.  Ability to Propagate Cells in a Chemically Defined Medium Under Serum-
free Conditions. In order to develop a process suitable for large-scale
commercial production, cells can be propagated and maintained as either
anchorage-dependent (adherent) or non-anchorage dependent. In the case
of adherent cells, after the initial proliferation phase, the nutrient medium is
removed and fresh medium is added to the cells, with infection of the cells
with influenza viruses taking place simultaneously or shortly thereafter.
After a specified time post infection, a protease (most often trypsin) is
added in order to obtain an optimum virus replication. The initial and
subsequent additions of trypsin later on in the typical process are generally
labor-intensive, with an increased potential for contamination of the cell
culture by adventitious agents. A more cost-effective alternative is cell
proliferation in fermenter systems utilizing cells growing adherently on
microcarriers under serum-free conditions, but such a process also requires
opening of the culture vessels several times and thus brings with it an
increased risk of contamination. The most desirable system would be the
propagation of cells in suspension. This not only eliminates the need for
trypsin, but also reduces cost, labor and the risk of contamination and also
allows for the formation of microvilli on the entire cell surface, thus
improving process yield and efficiency.

52. Types of Cell Substrates Used in Viral
Vaccines
 Primary Cells or Tissues: used without passage in
tissue culture
 Diploid Cells: cells with a finite lifespan and passage in
tissue culture
 Continuous Cell Lines: immortal, neoplastic cells with
unrestricted passage in tissue culture
 Non-tumorigenic

53. Cell substrates used for vaccine production
• Primary cells & Tissues (1954)
– Calf lymph for smallpox vaccines
– AGMK cells for polio vaccines
– Embryonated hens’ eggs for influenza, yellow fever vaccines
– Chicken embryo cell culture for measles, mumps vaccines
– Mouse brain for inactivated JEV vaccine
• Human diploid cells (introduced in 1960s)
– MRC-5, WI-38 for rubella, varicella vaccines
• CHO cells for highly purified, subunit investigational vaccines (1980s)
• Vero cells at non-tumorigenic passages for highly purified, inactivated
vaccine (IPV) (1980s)
• Vero cells at non-tumorigenic passages for live-attenuated vaccines (late
1990s)
• In vitro transformed human cells (e.g., 293, PER.C6) for defective vaccines
(early 2000s)

54. Viral Vaccines: Primary Cells or Tissues
Cell Substrate Live Vaccines Inactivated
Vaccines
Mouse brain Japanese
Encephalitis
Calf lymph Smallpox
Embryonated hens’ eggs Yellow Fever Influenza
Influenza
Monkey kidney cells Poliovirus
Chicken embryo Measles Rabies
fibroblasts (CEFs) Mumps

55. Viral Vaccines: Diploid Cell Strains
Cell Substrate Live Vaccines Inactivated Vaccines
Rhesus fetal lung:
FRhL-2 Rotavirus Rabies
Human fetal lung:
WI-38 Rubella
Adenovirus
MRC-5 Varicella Poliovirus
Hepatitis A
Rabies

56. Viral Vaccines: Continuous Cell Lines
Cell Substrate Live Vaccines Inactivated
Vaccines
African green monkey kidney:
Vero Poliovirus Poliovirus
(Europe) (U.S.)

57. Cell-based manufacturing steps

58. Cell propagation occurs in large
fermenters
Propagation of cells
Seed
cells Pre-culture
Production
fermenter
Virus infection
Virus propagation

59. Flu vaccine production: a complex
biological process
Optimized strains are
inoculated into millions of
specially prepared eggs or
cell culture bioreactors to
amplify the virus
Traditional egg based
OR
Flu cell culture
• Long lead times
• Open handling
steps
• Secure egg-
supply required
• Highly scalable
and responsive,
closed process
• Readily available
raw materials
• Closed system
bioreactors
WHO/CDC/EA
recommend strains
Seed viruses distributed to
manufacturers to begin the
production process
Seed optimization for
production
a) Concentration and
purification of whole virus
b) Disruption of virus for
split or subunit vaccines
c) Separation of virus from
other components for
subunit vaccines
d) Sterilization
Blending of virus in final
dose and presentation
(with or without adjuvant)
•Three strains for
seasonal vaccine at 15
micrograms each
•One strain for
pandemic vaccine as
low as 3.75 micrograms
Quality controls and batch
release
Multi
Dose Vial
Pre- filled
syringe
Packaging into final
boxes with Patient
Information Leaflets
and cartons for
shipping
Strain selection Bulk production
Virus antigen
isolation
Formulation, filling
and release
Packaging
Whole
virus
Subunits
purified
X month X month X month X month X month
Overall process is a 4- 6 month cycle
Or

60. The vaccine landscape is changing and is
moving to new manufacturing methods
Egg-based
production
technology
Cell Culture-based
production
technology
Cell Free
production
technology
System Eggs Mammalian Cells Insect and Bacterial Cells Recomb Cells
Lead Times 6-9 months ~3-6 months ~3 months Days?
Maturity On the market New on the market In 2-4 years time -

61. Facility design options
Disposable Bioreactors
Disposable DSP
Traditional Fixed Manufacturing
• Reduced capital investment
• Faster set up/ project throughput
• Easily moved
• Opportunity to ‘stockpile’ capacity
• Capital intensive manufacturing
• Long set up time
• Significant validation costs
• Highly localized
500 liter Wave Bioreactor
(GE Healthcare)

62. Influenza vaccine production capacity build
up with disposables – to save time
Traditional Flu Vaccine Production Facility Commissioning & Validation:
Build Facility
Validate Equipment
Commission Facility
Validate Process
Build Facility
Validate Equipment
Commission Facility
Validate Process
Time Saved
Saving
~60% in
time
Single-use Insect Cell Culture-Based Flu Vaccine Production: