VIRUS INOCULATION OF EMBRYONIC CHICKEN EGGS

By:

Name : Nahdlini Salma Sabila

Student ID : B1B017002
Entourage : III
Group :6
Assistant : Okti Yuga Hararenika Tifani

VIROLOGY LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
 I. INTRODUCTION

A. Background

The Newcastle Disease Virus (NDV) is a very contagious and fatal agent that
causes poultry and other poultry species throughout the world. Virulent NDV
strains can cause high mortality (up to 100%) in unvaccinated chickens. NDV is
a member of the genus Avulavirus of the Paramyxoviridae family in the
Mononegavirals order. This virus is usually spherical, though not always
(pleomorphic) with a diameter of 100 - 300 nm (Akin, 2006). It has a single,
non-segmented RNA genome that contains a 3-leader sequence and a 5-trailer,
important for virus transcription and replication, and follows rule six. NDV has
six structural genes: nucleoprotein (N), phosphoprotein (P), matrix (M), fusion
(F), hemagglutinin-neuraminidase (HN) and large polymerase (L). Of these
proteins, N, P and L proteins form the Ribonucleoprotein (RNP) complex, which
is responsible for virus transcription and replication. HN and F are anchored
inside the viral envelope as surface glycoprotein: HN is responsible for the
attachment of the virus to the host cell receptor, and F mediates the fusion of
viral envelopes with the host cell membrane. Protein F is split into F1 and F2 for
fusion activity and the presence of polybasic motives at cleavage sites is the
main determinant of virulence. Both HN and F proteins are capable of
generating neutralizing antibodies (Chumbe et al., 2017).
Based on its virulence, NDV is grouped into three pathotypes namely
lentogenic, velogenic, and mesogenic. Lentogenic is a less virulent viral strain,
mesogenic is a medium virulence and velogenic virus strain is a virulent strain of
virulent virus. Velogenic strains are distinguished into neurotrophic forms with
symptoms of neurological disorders accompanied by abnormalities in the
respiratory system or pneumotropic, and visotrophic forms characterized by
abnormalities in the digestive system. Viserotropic velogenic NDV infection
caused by multifocal necrosis of the spleen, fabricious and thymus bursa
atrophy. The pathogenicity of VND is influenced by viral strains, route of
infection, age of chicken, and environment (Etriwati et al., 2017).
Velogenic viscerotropic is a highly pathogenic form of Newcastle disease
where bleeding lesions in the digestive system are often seen in this form.
Velogenic neurotropic is a form of Newcastle disease that causes high mortality
and is usually followed by disorders of the respiratory and nerve systems.
Velogenic pneumotropic attacks the respiratory system. The mesogenic
newcastle disease shows clinical symptoms of respiratory system disorders but
nervous system disorders are not always seen and mortality is low, whereas
asymptomatic enteric is a form of subclinical infection in the digestive system.
NDV avirulent (lentogenic and mesogenic) strains are used as live vaccines to
improve Newcastle disease control in chickens, but the choice of vaccine type
depends on the condition of the disease. The inactive vaccine is also used in
controlling Newcastle disease. The pathogenicity of NDVs can be determined by
several factors including the level of virulence of NDV and host (Hewajuli &
Dharmayanti, 2011).
Clinical symptoms experienced by chickens in the form of decreased appetite,
comb and cyanosis warts, swelling in the head, sneezing, coughing, snoring, and
greenish-white diarrhea. Velogenic strain virus infections are fatal, often
followed by high mortality. These symptoms are very variable, beginning with
conjunctivitis, diarrhea and accompanied by nerve symptoms such as tremor,
torticollis (paralysis of the neck and wings). Pathognomonic pathological
anatomic changes in Newcastle disease are characterized by ptechie in the
proventiculus, ventriculus, intestine, tonsillar cavity, trachea and lungs. In
addition, respiratory distress, green diarrhea, weakness, loss of appetite, loss of
appetite, decreased egg production can cause high mortality (Kencana et al.,
2012).
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).
NDV is very easily transmitted, transmission of NDV can occur directly
between chickens in a group of infected animals. The source of the virus usually
comes from infected chicken excreta, air contaminated with viruses, equipment,
and cage workers. The pathogenicity of the NDV virus is affected by viral
strains, infection routes, chicken age, environment, and chicken immune status
when infected with the virus. During illness, the chicken secretes large amounts
of the virus through feces (Tabbu, 2000).
The objectives of this laboratory activity are to understanding kinds of virus
inoculation, how to inoculate virus on embryo of chicken egg, and to know the
embryo characteristics which infected by Newcastle Disease Virus (ND).
 II. MATERIALS AND METHODS

A. Materials

The tools that used in this laboratory work are injection syringe 1 cc, needle
and flashlight.
The materials that used in this laboratory work are 11 – 13 days old
embryonic chicken eggs, cotton, Alcohol 70% and Newcastle Disease virus
suspension.
B. Methods

The method used in this laboratory work is inoculation in the chorioalantoic

space. Chicken embryos aged 10-12 days were used and binoculars with
flashlights. Air bag limits and the location of the head of the embryo are
determined then marked. 70% alcohol is swabbed, then 0.4 cc of the virus
suspension is inoculated into the chorioalantoic space over the boundary of the
air bag by means of a needle inserted 1⁄4 inch at an angle of 45o. The hole is
closed again with a candle. Inoculated eggs, incubated for 3 days at 37o.
 III. RESULT AND DISCUSSION

Table 3.1 Observation of Virus Inoculation on Embryonic Chicken Eggs

Hemorrhage
Group Volume Lesion
Head Body Feet

1 0.2 cc - - - -

2 0.4 cc Head ++ + +

3 0.2 cc Head + - -

4 0.4 cc Head, body ++ - ++

5 0.2 cc - ++ - ++

6 0.4 cc Beak + - +

7 0.2 cc Head, body ++ ++ -

8 0.4 cc - - - -

Information: (+) if there is hemorrhage, (++) if there is a lot of hemorrhage, (-) if

there is no hemorrhage
This practice uses eggs treated differently. There are control eggs and test
eggs. Test eggs were inoculated with serum NDV of 0.2 cc and 0.4 cc, while control
eggs were not treated. Group 6 received 0.4 cc serum NDV. Based on the results of
the practicum obtained by group 6, there were lesions on the part of the beak and
hemorrhage in the head and feet, but none of the chicken embryos experienced
greenish discoloration in the feet. This is consistent with the statement of Beard &
Hanson (1984), the characteristics of NDV infected chicken embryos in the form of
embryonic death, lesions in embryos in the form of dwarfism, cutaneous
hemorrhage, enlarged liver and spleen, abnormal muscle and book development,
lesion formation on CAM, greenish discoloration of the feet. Microscopic changes
that occur in the form of hyperemia, edema, hemorrhage, thrombosis, and vascular
necrosis. Reticulohistiositic cells hyperplasia and multifocal necrosis of the liver.
Inoculated NDV embryos will experience reduction in certain organs such as the
liver, trachea and blood vessels. According to Smietanka et al. (2006), NDV injected
into chicken embryos will migrate into various newly formed organs and damage
these organs, such as damage to the liver, lungs, kidneys and intestines in chicken
embryos. This depends on the virulence of each strain of this virus. According to
Putra et al. (2012), organs from chicken embryos that are thought to be used as a
place for viral replication include skin, lungs, intestine, liver, kidney and heart.
Microscopic lesions caused by the ND virus of Salatiga isolates in the form of
congestion and hemorrhage in the lungs, congestion in the intestine, kidneys, liver,
heart and skin congestion accompanied by inflammation. Chicken embryos that were
not infected by the ND virus looked abnormal, due to decay in the control eggs. The
organs of the embryo look microscopically unchanged. This is suspected to be viral
contamination through porous eggshells, which allows the virus to infect chicken
embryos (Qosimah et al., 2017).

Figure 3.1. Infected chicken embryo Figure 3.2. Control egg

 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion above, can be concluded that there are
lesion on the beak. Hemorrhage on head and feet on the chicken embryo that
infected by Newcastle Disease Virus.

B. Suggestion

Suggestion for this laboratory activity are the measurement of organ extract
mass must be accurately measured with scale and mask and gloves must be used
to prevent any microorganism transmission to our body.
 REFERENCES

Beard, C. W. & Hanson, 1984. Newcastle Disease in Disease of Poultry, 8th ed.
USA: Iowa State University Press.
Chumbe, A., Izquierdo-Lara, R., Calderón, K., Fernández-Díaz, M. & Vakharia, V.
N., 2017. Development of a Novel Newcastle Disease Virus (NDV)
Neutralization Test Based on Recombinant NDV Expressing Enhanced Green
Fluorescent Protein. Virology Journal, 14(1), pp.232.
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.
Etriwati, E., Handharyani, E. & Setiyaningsih, S., 2017. Studi Histopatologi Limpa
dan Bursa Fabricious Ayam Berpenyakit Tetelo (Newcastle Disease) pada
Kasus Lapang (Histopathology Studies on Spleen and Bursa of Fabricius of
Newcastle Disease Chickhens From Field Case). Jurnal Veteriner, 18(4),
pp.510-515.
Hewajuli, D. A., & Dharmayanti, N. L. P. I., 2011. Patogenitas Virus Newcastle
Disease pada Ayam. Wartazoa, 21(2), pp.453-464.
Kencana, G. A. Y., Kardena, I. M. & Mahardika, I. G. N. K., 2012. Peneguhan
Diagnosis Penyakit Newcastle Disease Lapang Pada Ayam Buras di Bali
Menggunakan Teknik RT-PCR. Jurnal Kedokteran Hewan, 6(1), pp.28-31.
Putra, H. H., Wibowo, M. H., Untari, T. & Kurniasih., 2012. Studi Lesi Makroskopi
dan Mikroskopis Embrio Ayam yang Diinfeksi Virus Newcastle Disease Isolat
Lapang yang Virulen. Jurnal Sain Veteriner, (30)1, pp.57-67.
Qosimah, D., Murwani, S. & Amalia, I., 2017. Penyakit Viral pada Unggas. Malang:
Universitas Brawijaya Press.
Smietanka, K., Minta, Z. & Blicharz, K. D., 2006. Detection of Newcastle Disease
Virus in Infected Chicken Embryos and Chicken Tissues by RT-PCR. Bull Vet
Inst Pulawy, 50, pp.3-7.
Tabbu, C. R., 2000. Penyakit Ayam dan Penanggulangannya. Yogyakarta: Kanisius.

Yaumachi, Y. & Helenius, A., 2013. Virus Entry at a Glance. Journal of Cell
Science, 126, pp.1289-1295.
 ATTACHMENT (PORTOFOLIO)

Group : 6
Entourage : III

QUESTION
Explain the division of ND virus strains based on virulence level and time of
death!
 Velogenic causes death in less than 60 hours. Velogenic has the
characteristics of causing coughing - sores, sneezing, head coughing, and
paralysis of the wings. Velogenic attacks the nerve (neurotropic), digestive
system (visceraltropic), respiratory system (pneumotropic).
 Mesogenic causes death within 60 to 90 hours. Mesogenic has
characteristics that cause coughing, sneezing, and reduced frequency of
laying eggs.
 Lentogenic causes death in more than 90 hours (subclinical). Lentogenic
has the characteristics of causing a cough and runny nose.