VIRUS INOCULATION ON EMBRYONATED CHICKEN EGGS

By:
Name : Pratiwi Kusuma Kurniawati
SID : B1B017007
Entourage : III
Group :8
Assistant : Agung Wiriat Putra Pratama Hadi

VIROLOGY LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGI
PURWOKERTO

2019
 I. INTRODUCTION

A. Background

Newcastle disease (ND) virus is caused by a virulent strain of avian paramyxovirus

type 1 (APMV-1) from the Avulavirus genus subfamily Paramyxovirinae, the
Paramyxoviridae family. NDV is a pathogen capable of producing a devastating
disease in domestic fowl, with vast social and economic consequences. (Dimitrov et
al., 2016). The incidence of NDV that attacks poultry shows manifestations of
gastrointestinal, respiratory, and neurological disorders that cause deaths of up to
100% depending on the viral pathotype. Paramyxovirus isolated from poultry have
been classified serologically and phylogenetic analysis in ten subtypes, namely
APMV-1 to APMV-10. Virulent of NDV strains are endemic to poultry in most of
Asia, Africa, and some northern and southern states of America. This disease can also
be transmitted to humans. ND disease first epidemic in Java Island, Indonesia in 1926
and spread pandemically in chickens and other poultry subsequently found in
Newcastle upon Tyne, England in 1927 (Qosimah et al., 2018).
Newcastle disease viruses into three main pathological groups there are
velogens, mesogens, and lentogens. Velogens induce severe systemic infections with
high mortality rates. Mesogens cause disease and death primarily for chickens younger
than eight weeks and produce mainly respiratory disease. Lentogens are avirulent and
cause mild enteric, respiratory or subclinical disease. According to the World
Organisation for Animal Health (OIE), virulent Newcatle disease virus strains, which
include both mesogenic and velogenic strains, must meet one of the following criteria:
have an intracerebral pathogenicity index ICPI) in day-old chicks (Gallus gallus) of
0.7 or greater or have multiple basic amino acids at the C terminus of the F2 protein
and phenylalanine at residue 117, which is the N terminus of the F1 protein. The term
“multiple basic amino acids” refers to the presence of at least three arginine or lysine
residues from positions 113 through 116. (Dimitrov et al., 2016).
The clinical manifestation of a velogenic strain of Newcastle disease virus,
typically results in a primarily intestinal infection charac‑ terized by hemorrhagic
lesions found in the intestines of dead birds (termed viscerotropic velogenic) or a
predominantly respiratory and neurological infection (referred to as neurotropic
velogenic). Neurological signs are also often observed with velogenic infections,
especially in birds with partial immunity who often go on to develop a chronic
infection, and the signs typically include tremors, ataxia, torticollis, and paresis or
paralysis of the wings or legs which develop several days post-infection. Both wild
and domestic species can develop similar neurotropic signs. Mesogenic viruses can
cause clinical disease which typically include respiratory and neurological signs but
the infection is self-limiting, and mortality is rare in older birds unless there are
secondary bacterial infection. Lesions associated with both velogenic and mesogenic
strains of NDV infection are most often detected in the central nervous system (CNS),
alimentary tract, renal system, or respiratory tract and viruses virulent in wild bird
populations tend to afect the CNS or kidneys or to cause systemic disease that results
in rapid mortality in the absence of recognizable gross or histopathological lesions
(Brown & Bevins, 2017).
Newcastle disease virus is primarily transmitted via inhalation or ingestion of
virus shed in feces and respiratory secretions by infected birds for variable lengths of
time. Some virus isolates have been found to be transmitted through the egg to the
hatching chick. Furthermore, virus has been found to be present in all parts of the
carcass and is able to persist for many months on both chicken skin and bone marrow
if kept at refrigerated temperatures (Brown & Bevins, 2017). Newcastle disease virus
is in respiratory air, feces, chickens that experience pain and carcasses of chickens that
die because Newcastle disease. Besides chicken, the spread of the disease can be
through domestic birds or wild birds that are around or into the cage. The role of the
various factors above in the transmission of the Newcastle disease virus depends on
various management factors and the environment in which a farm operates. The
success of the transmission of the Newcastle diseases virus is closely related to the
ability of the virus to survive in chicken carcasses or excretions from sick chickens. In
infected chicken carcasses, the Newcastle disease virus can last for several weeks at
low temperatures or for several years if stored at freezing temperatures. Stool can
contain a large number of Newcastle disease virus, at a temperature of 37○C the virus
is still alive for more than one month (Akoso, 1998).
Mechanisms of virus entry the animal cell host can trough membrane fusion,
endocytosis, and direct penetration. In membrane fusion pathway, virus can fuse either
directly to the plasma membrane (receptor mediated fusion) or after being swallowed
into an endosome. Which of these routes is followed depends on the type of virus. In
fusion with the plasma membrane, the virus binds to a protein in the cell membrane.
The function of this cellular protein (a receptor for the virus, shown in green) is
perverted to induce a conformational change in the viral fusion protein, leading to
fusion. For virus that is triggered within an endosome, the endosome’s acidic
conditions induce fusion. In either case, the viral genome passes through a fusion pore
into cytosol, and infection is initiated (Cohen, 2016).
In endocytosis pathway, endocytic vesicles help to carry the incoming particles
deep into the cytoplasm unobstructed by cytoplasmic crowding and obstacles such as
the cytoskeleton. In the process of intracellular maturation of endocytic vacuoles, the
host cell exposes the viruses to changing conditions including a drop in pH that many
viruses use as a cue to activate penetration. Endocytic entry of viruses occurs in a
stepwise manner involving attachment to the cell surface, clustering of receptors,
activation of signaling pathways, formation of endocytic vesicles and vacuoles,
delivery of viral cargo to endosomal compartments, sorting, and escape into the cytosol
(Cossart & Helenius, 2014).
The penetration event involves the delivery of the genome and accessory
proteins to the cytosol. It is one of few events during virus entry that requires an active
process initiated by the virus. In the case of enveloped animal viruses, penetration
invariably involves membrane fusion which is mediated by specific viral
glycoproteins. As a result of fusion, viral capsids are released into the cytosol.
Endosomes and macropinosomes are the most common sites for these fusion events.
Here, the viruses fuse their envelope with the limiting membrane of the endocytic
vacuoles from the lumenal side. The mechanisms of penetration by non-enveloped
viruses are less well characterized. For the example, Adenoviruses cause the lysis of
endosomes, allowing escape into the cytosol. Picornaviruses undergo a conformational
change that allows the particles to form a pore through which the viral RNA is released
into the cytosol. Parvoviruses have acid-activated phospholipase activity, which is
thought to help their escape from vacuoles (Yaumachi & Helenius, 2013).

The objectives of this laboratory activity are to know kinds of virus inoculation,
to know virus inoculation method in embryonated chicken’s egg, and to know the
characteristics of the virus infected chicken’s embryo (Newcastle Disease).
 II. MATERIAL AND METHOD

A. Material

The tools used in this laboratory activity are cotton, syringe injection, needle,
and petri dish.
The materials used in this laboratory activity are embryonated chicken’s egg
age of 9 – 12 days, alcohol 70 %, candle, suspension of NDV.

B. Method

The method used in this laboratory activity are:

1. Viral Inoculum Making
Organ of chicken such as brain, trachea and lungs that been infected by
NDV was cut and mashed by mortar and pestle. The extract was scaled 1 gram
by digital scale. The extract was added by PBS 9 mL, then centrifuged at 2500
rpm for 15 minutes. The extract was sterilized using milipore 0,22 µm. Then the
inoculum was gotten.
2. Inoculation of Virus
Egg was punched by sterile needle. At the hole, Inoculum was injected
by syringe 45o at chorioallantioc space. The hole was covered by candle melt.
The egg was ready to incubated for 3x24 hours at 37o.
 III. RESULT AND DISCUSSION

Table 3.1 Observation Table of Virus Inoculation in Chicken’s Embryonated

Egg Entourage III
Group/ Volume Lesion Hemorrage
Entourage
(mL) Head Body Leg

1 0,2 - - - -

2 0,4 Head ++ + +

3 0,2 Head + - -

4 0,4 Head+body ++ - ++

5 0,2 - ++ - ++

6 0,4 Beak + - +

7 0,2 Head+body ++ ++ -

8 0,4 - - - -

Note:
- : No symptom
+ : Few symptom
++ : Moderate symptom
+++ : Many symptom
Based on the result from group 1 and 8 with 0,2 mL and 0,4 mL suspension of
NDV give no effect for embryoated chicken egg, the embryonated chicken egg give
negative interpretation which is the egg is not infected by Newcastle disease virus. In
group 2 with 0,4 mL suspension of NDV affect lesion in head, moderate symptom of
hemorrhage in head and few symptom of hemorrhage in body and legs. In group 3
with 0,2 mL suspension of NDV affect lesion in head, few symptom of hemorrhage in
head and no symptom in body and legs. In group 4 with 0,4 mL suspension of NDV
affect lesion in head and body, with moderate symptom of hemorrhage in head and
legs and no symptom of hemorrhage in body. In group 5 with 0,2 mL suspension of
NDV have no symptom of lesion and hemorrhage in body, but moderate symptom of
hemorrhage in head and legs. In group 6 with 0,4 mL suspension of NDV affect lesion
in beak, few symptom of hemorrhage in head and legs, and no symptom of hemorrhage
in body. In group 7 with 0,2 mL suspension affect lesion in head and body, moderate
symptom of hemorrhage in head and body, and no symptom of hemorrhage in legs.
Almost in all groups shows the symptom that caused by suspension of NDV, only
group 1 and 8 that no affected by the symptom from NDV. According to York & Betts
(1967), the criteria of infections, which will vary according to the virus and the time
and route of inoculation, include the formation of lesions on the chorioallantoic
membrane or lungs, death of the embryo, and the appearance of hemagglutinins or
complement fixing antigens in the allantoic or amniotic fluid. The chorioallantoic
membrane inoculation is used because in this case, the virus which injected can
directly transferred into membrane. A neutralization test for antibodies for Newcastle
disease virus using 9 to 12 days old chick embryos. The chick died in 3 - 4 days, fluids
were collected from all chick embryos and titrated for hemagglutin for chicken
erythrocytes.

Figure 1. The Control of Chicken’s Embryo

Based on the figure 1 of the control chicken’s embryo, the embryo which
doesn’t attacked by Newcastle disease Virus has no lesions and haemorrhage
symptom, and the condition of the embryo is normal.

Figure 2. Chicken’s Embryo That Inoculated by NDV

 Based on the figure 2, the chicken’s embryo that predicted infected by
Newcastle disease virus, but in the truth chicken’s embryo does not infected by
Newcastle disease virus that not show any symptom. Success and failure in inoculating
NDV into embryonic chicken eggs can be caused by viral titers given to chicken
embryos or caused by several factors such as the immune system, besides the age of
the embryo used, it can be possible that the embryonic chicken eggs from our group
are not really 9 -12 days. In addition, it can be caused when we inject the NDV
suspension, not fully suspended into the embryonic eggshell. This is in accordance
with the statement of Alexander and Senne (2011), the success in isolating and
developing the virus depends on several conditions namely inoculation route, embryo
age, incubation temperature, incubation time after inoculation, volume and dilution of
the inoculum used, immune status of the group where chicken eggs are.
 CONCLUSION AND SUGGESTION

A. Conclusion
Based on the result and discussion be conclude that several types of animal
viral inoculation are in ovo, in vivo, and in vitro. Viral inoculation method in
embryonated chicken’s egg are inoculation in chorioallantoic space, in the
chorioallantoic membrane, and yolk sac. The characteristics of the virus infected
chicken’s embryo are lesion on the embryo, the green color of the embryo’s leg, and
death embryo. In our group, after inoculation of Newcastle disease virus there are no
symptom either lesion or haemorrhage that affected on embryonated chicken egg.

B. Suggestion
The laboratory activity will be better if the student is more careful in finding
embryonated egg that exactly have 9-12 days old, to do the method more carefully and
use the lab activity correctly, so the result will not failed. The tools may checked before
used so the tools can be used well during lab activity.
 REFERENCE

Alexander, D. J. & Senne, D. A., 2011. Newcastle Disease, Other Avian

Paramyxovirus and Pneumovirus Infection In: Disease of Poultry. Lowa:
Blackwell Publishing

Akoso, B. T., 1998. Kesehatan Unggas. Yogyakarta: Penerbit Kanisius.

Brown, V. R. & Bevins, S. N., 2017. A review of virulent Newcastle disease viruses
in the United States and the role of wild birds in viral persistence and spread.
Veterinary Research, 48(68), pp. 1-15.

Cohen, F. S., 2016. How Viruses Invade Cells. Biophysical Journal, 110, pp. 1028-
1032.

Cossart, P. & Helenius, A., 2014. Endocytosis of Viruses and Bacteria. Cold Spring
Harbor Perspective in Biology, 6, pp. 1-28

Dimitrov, K. M., Lee, D., Coplin, D. W., Olivier, T. L., Miller, P. J. & Afonso, C. L.,
Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show
Unexpectedly High Genetic Similarity with Historical Virulent Isolates from the
1940’s. Journal of Clinical Microbiology, 1(3), pp. 1-26.

Qosimah, D., Murwani, S., Sudjarwo, E. & Lesmana, M. A., 2018. Effect of
Newcastle disease virus level of infection on embryonic length, embryonic
death, and protein profile changes. Veterinary World, 11, 1316-1320.

Yaumachi, Y. & Helenius, A., 2013. Virus entry at a glance. Journal of Cell Science,
126, pp. 1289-1295.

York & Betts., 1967. Viral and Rickettsial Infections of Animals. New York: Academic
Press.
 ATTACHMENT (PORTOFOLIO)

Group : 8
Entourage : III
QUESTION
Expalin the characteristics of the Newcastle disease virus and the various
modes of transmission !
1. Characteristics
a. Newcastle disease have classification:
- Group : V (-) ssRNA
- Order : Mononegavirales
- Family: Paramyxoviridae
- Genus : Avulavirus
- Species: Newcastle disease
b. Size : 100-500 nm
c. Shape :pleomorphic and have envelope
d. Attack to the central nevous system in avian
e. Other name: Sampar ayam, pseudofowl pest, pseudovogel pest, avian
pest, tetelo, pseudopoultry plague, etc.
2. Mode of transmission
a. Aerosol : through air particle
b. Feces : through avian feces
c. Oral : through feed that eaten by avian infected with NDV
d. Ovarial : virus enters embryonic chicken eggs through the pores. Virus
originates from the parent feces