EMBRYONATED EGG CULTURE FOR

INFLUENZA A VIRUS: ISOLATION, CULTURE, AND

IDENTIFICATION

Prodi S1 Farmasi
Universitas Sriwijaya
Syllabus
01 Overview

02 Material

03 ECE Method

04 Analysis
OVERVIEW
IAVs cause seasonal epidemics that are responsible for 3–5 million severe human cases and
≥250,000 deaths worldwide annually, as well as sporadic pandemics that can induce an even
higherdisease burden and loss of human life

Seasonal epidemics are caused by antigenic drift, a process facilitated by a combination of

immune pressure and an intrinsically high IAV mutation rate

INFLUENZA A VIRUS
SPREAD
 Influenza A Virus

Influenza A Virus Spread

The hemagglutinin (HA) protein of a circulating IAV acquires
mutations that enable immune evasion, such a virus can spread
efficiently in humans who lack preexisting antibodies against the
antigenically drifted variant.

For this reason, seasonal IAV vaccines must be reformulated every

1–3 years.

IAVs possessing genes derived from avian, human and/or swine

viruses—to which most humans lack immunity—are transmitted to
humans from reservoir species (i.e., birds and swine)

IAVs are diverse. Virus subtypes are categorized on the basis of the
antigenic properties of the HA and neuraminidase (NA) virion surface
proteins, and to date 18 HA subtypes (e.g., H1, H2 and so on) and 11
NA subtypes (e.g., N1, N2 and so on) have been identified
 Embryonated Chicken Eggs are used for the cultivation of some viruses. The viruses
grow in the cells of the embryo and membranes

Specimens are inoculated into pathogen-free fertilized eggs of 10-11 days and are
incubated for 2-9 days before harvesting the viruses. Growth and multiplication of the
viruses are indicated by the death of the embryo, or by the formation of typical pocks or
lesions on the egg membranes.

EMBRYONATED
EGG CULTURE
(ECE)
Embryonated Egg Culture (ECE)
Salient features of AIV Culture in ECE and Egg Culture
 Embryonated Egg Culture (ECE)
PROTOCOL

Sample
ASSAY
Collection

Identification Cultivation
Sample Collection
 Sample Collection

Most IAV surveillance samples are collected from humans

exhibiting respiratory disease symptoms, and they are processed
and analyzed in local, national and global laboratories throughout the
world

Swab Samples
from humans (nasal, nasopharyngeal or oropharyngeal); avian species
(oropharyngeal, tracheal, cloacal or fecal); and nonhuman, mammalian
species (nasal, naso- or oropharyngeal, tracheal, rectal or fecal)

Liquid samples
from environmental sources (e.g., water from lakes or troughs in live
poultry markets); experimentally infected animals (e.g., nasal wash,
bronchoalveolar lavage); and humans (nasopharyngeal aspirates, naso- or
oropharyngeal gargles, bronchoalveolar lavage, sputum or tracheal
aspirates)

Tissue samples
from experimentally killed animals, or from slaughtered or dead animals
(tissues may include blood, respiratory organs (nasal turbinate, trachea,
bronchus and lung), intestinal tract, liver, kidney, spleen, pancreas, brain
and/or other tissues)
Identification
 Identification Workflow

Protocol parts and subcomponents are indicated by the dark and light boxes, respectively. Specific text sections describing the steps for
each technique are indicated at the right. The gold standard workflow is shown by solid lines with arrows, and the fast workflow is
shown by dotted lines with arrows.
Cultivation
 VIRUS CULTURE
PROPAGATION

• Take pathogen-free fertilized chicken eggs of 11-12 days.

• Locate a non-veined area of the allantoic cavity which is located

just below the air sac placing the egg in front of a light source.
Mark the area with a pencil

• Make a small nick in the marked area using a jeweler’s scribe.

• Drill a hole at the top of the egg with a Dremel motorized tool. This
is done to decrease the pressure of the air sac to prevent the
leakage of inoculum.

• Inoculate the eggs using a tuberculin syringe (a 1 ml syringe fitted

with a 1/2 inch, 27 gauge needle).

• Pass the needle through the hole in the shell, through the
chorioallantoic membrane, and inject the inoculum in the allantoic
cavity, which is filled with allantoic fluid.

• Seal these two holes of the shell with melted paraffin.

• Incubate the eggs at 37 degrees C for 48 hours.

 VIRUS CULTURE
HARVESTING

STEP 1
Remove the top of the eggshell (the part covering the air sac).

STEP 2
Pierce the shell membrane and chorioallantoic membrane with a
pipette.

STEP 2
Withdraw about 10 mL of allantoic fluid per egg.

Depending on the virus strain, one or two eggs will

produce sufficient virus to produce one 15 microgram dose
of vaccine.
 VIRUS CULTURE
INOCULATION SITES

Approximate structure of the avian embryo and inoculation sites (a) relative to the inoculation sites for (b) CAS, (c)
CAM, and (d) YS inoculation methods. Approximate placement of the syringe is shown. Extra holes in the air cell for
CAS and CAM inoculation are shown with an open circle
 VIRUS CULTURE
PROPAGATION AND HARVESTING

Embryonated egg inoculation and allantoic fluid collection.

(a) A graphical longitudinal section of an embryonated egg’s

interior anatomy

(b) Photograph of a candled, 10-d-old embryonated chicken egg

with an inoculation puncture. Anatomic regions, as described in a,
are indicated.

(c) Photographs of an ‘in-house’ egg-piercing tool consisting of a 20-

gauge, 1.5-inch catheter punched through a rubber stopper (i); the
generation of the inoculation puncture by using the egg-piercing tool
(ii); and the egg inoculation procedure (iii).

(d) Photographic representation of the egg allantoic fluid collection

procedure. After the egg was chilled at 4 °C overnight, its shell
above the air sac was cracked and removed (i); the allantoic
membrane was pulled back (ii); and the allantoic fluids were
collected (iii).
 VIRUS CULTURE
PROPAGATION AND HARVESTING

Approximate structure of the avian embryo and inoculation sites (a) relative to the inoculation sites for (b) CAS, (c) CAM, and (d) YS inoculation
methods. Approximate placement of the syringe is shown. Extra holes in the air cell for CAS and CEmbryonated egg inoculation and allantoic
fluid collection. (a) A graphical longitudinal section of an embryonated egg’s anatomy showing the different parts. Inoculation sites are
highlighted in bold. (b) Standard setup used for chicken egg inoculation, showing the main materials needed for the procedure and a detailed
picture of the virus injection. (c) Standard setup used for virus harvesting from infected chicken eggs, showing the main materials needed for the
procedure and a detailed picture of the virus collection procedureAM inoculation are shown with an open circle
ASSAY
 HEMAGGLUTINATION ASSAY
HEMAGGLUTINATION ASSAY

WHY
Widely used, inexpensive, rapid an standard screening
method for the detection of IAVs is the hemagglutination assay

PRINCIPLE
based on the ability of the viral HA protein to bind to and
agglutinate red blood cells (RBCs) mediated by α2,3- and/or
α2,6-linked sialic acid moieties at the RBC surface

PERFORM
performed by mixing dilutions of virus-containing samples with a
standard amount of RBCs in microtiter plates and observing
agglutination patterns after a period of incubation
 HEMAGGLUTINATION ASSAY
HEMAGGLUTINATION ASSAY

WHY
Widely used, inexpensive, rapid an standard screening
method for the detection of IAVs is the hemagglutination assay

PRINCIPLE
based on the ability of the viral HA protein to bind to and
agglutinate red blood cells (RBCs) mediated by α2,3- and/or
α2,6-linked sialic acid moieties at the RBC surface

PERFORM
performed by mixing dilutions of virus-containing samples with a
standard amount of RBCs in microtiter plates and observing
agglutination patterns after a period of incubation
 HEMAGGLUTINATION ASSAY
SEVERAL FACTORS NEED TO BE CONSIDERED

• a positive hemagglutination assay indicates the presence of

viral HA protein, but it does not require infectious virus;

• some IAVs do not agglutinate RBCs efficiently, even if their

infectivity is high;

• the origin of the RBCs (e.g., chicken, turkey, guinea pig or

horse) can markedly affect the hemagglutination activity of an
IAV, owing to species-specific expression patterns of α2,3- and
α2,6-linked sialic acid moieties on RBC surfaces42; and

• hemagglutinin proteins from other viruses(e.g.

paramyxoviruses) can induce RBC agglutination, so a positive
hemagglutination assay result should always be followed with a
secondary assay that can specifically identify the IAV
 RT-PCR ASSAY

WHY
It is preferable to use a method that will detect viruses
irrespective of antigenic differences in the HA protein, especially
when the HA subtype of the prospective virus is unknown

PREFERABLE
Real-time RT-PCR is favored because results can be obtained
more rapidly; however, M gene sequences amplified by using
standard RT-PCR can be evaluated by using gel
electrophoresis

PREFERABLE
Although IAV genomic sequences vary widely across subtypes
and species-specific virus groups, the M gene segment is
relatively highly conserved.
IMMUNOFLOURESCENCE ASSAY
MATERIAL AND EQUIPMENT
M&E
Thank You