Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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MOLECULAR AND SEROLOGICAL IDENTIFICATION OF
NEWCASTLE DISEASE VIRUS PROPAGATED IN EMBRYONATED
CHICKEN EGGS
Firas T. Mansour Al-Mubarak*, Ammal F. Ghanim**, Hazim T. Thwiny*** ,
Ibraheem K. Younus***
*Department of Microbiology, College of Medicine, University of Basrah, Basrah, Iraq
**Kut Technical Institute, Middle Technical University, Wasit , Iraq
***Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine,
University of Basrah, Basrah , Iraq.
Corresponding Author: [email protected]
Key words: Egg inoculation, hemagglutination, RT-PCR.
ABSTRACT
The aim of this study is to propagate the non-virulent Newcastle disease virus in the
laboratory, determination the cytopathic effects in the inoculated chicken embryos, and
confirmation of virus growth by serological and molecular techniques by performing
haemagglutination and reverse transcriptase polymerase chain reaction (RT-PCR) tests,
respectively. LaSota virus strain which is a live vaccine was used for this purpose. Nine-dayold
embryonated chicken eggs were inoculated with the virus and further incubated for 48
hours; and the allantoic fluid was collected for further processing. Petechial haemorrhages and
congestions were observed in the inoculated embryos while in the un-inoculated eggs; the
embryos were normal and did not show any lesion. Virus growth in the allantoic fluid was
confirmed by performing haemagglutination and RT-PCR tests. These results support the
isolation of other viruses in our laboratories, which will contribute to perform other
experiments such as studying virus characteristics and observation of its pathological effects on
the embryos, preparation of viral antigens, sequencing of viral genome, and possibly
discovering new viruses.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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INTRODUCTION
Newcastle disease has been one of the most important poultry diseases worldwide. The
causative agent of this disease is Avian Paramyxoviruses type-1 which is classified under the
genus Avularius, family Paramyxoviridae (1). Newcastle disease viruses (NDV) are divided
into three main groups: virulent (Velogenic), moderately virulent (Mesogenic) and non-virulent
(Lentogenic). These strains differ in the number of amino acids at the site of cleavage of the
fusion protein (2,3). Clinical signs of the infected birds are vary greatly according on the
species of bird, strain of virus and preexisting immunity (4,5). The non-virulent strain mainly
LaSota has been approved to be used as a live vaccine throughout the world, which gives good
protection against the virulent and moderately virulent strains if they administered correctly to
health birds (6).
Diagnosis of the disease is usually done in the laboratory using some techniques such as
reverse-transcriptase polymerase chain reaction (RT-PCR), haemagglutination (HA),
haemagglutination inhibition (HI), and Enzyme Linked Immunosorbent assay (ELISA). In
addition, virus inoculating the allantoid cavity of the embryonated eggs is widely used (7). The
HA assay is the most common indirect method for estimating the quantities of virus particles,
generated from allantoic fluid or cell culture supernatants. This assay is based on the fact that
several viruses contain a protein that can bind and clump red blood cells (8).
The aim of this study is to propagate Newcastle disease virus (non-virulent strain) in
embryonated chicken eggs and to detect virus growth by serological and molecular techniques.
MATERIALS AND METHODS
VIRUS CULTIVATION AND HARVESTING
Fertile hen’s eggs were used to propagate the viruses used in this study. LaSota virus
strain. Working virus concentration of LaSota strain was prepared by diluting the virus stock
1:3000 in PBS containing 1% antibiotics (100 U/ mL penicillin and 100 ug/ mL streptomycin).
The fertilized eggs were first incubated at 37.5˚C for 9 days. During the period of
incubation, the embryos were candled in a dark room using an egg-candling box. To determine
the site of injection which is the opposite site of the embryo head, the air sacs were highlighted
with a pencil. All the eggs that did not have developing embryos were discarded. The egg
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
243
surface was sterilized by wiping with ethanol. A small hole was made at the injection site using
a special drill without any damage in the shell membrane. A small syringe (1 ml) was used to
inoculate the eggs with the virus. The needle was passed through the chorionic membrane. The
virus (0.1 ml) was injected into the allantoic cavity filled with allantois fluid. The hole was
carefully closed with tape, and eggs were incubated at 37.5 ° C for 48 hours, and the allanoic
fluid was then collected. Embryos were collected and moved in plastic Petri dishes to observe
the cytopathic effects. Photos were then taken for documentation.
DETECTION OF VIRUS BY HEMAGGLUTINATION
Virus growth was confirmed by processing allantoic fluid to hemagglutination assay.
Six well ceramic agglutination plate was prepared for the experiment. One drop purified RBC
was mixed with another drop of allantoic fluid in the well. The negative control was
represented by mixing one drop of RBC with another drop of PBS. After a few minutes of
incubation at room temperature, the reaction was visualized and photos were taken for
documentation.
VIRAL RNA EXTRACTION AND QUANTIFICATION
Extraction of viral RNA from allantoic fluid was performed using a QIAamp viral RNA
purification kit (Qiagen, Germany) according to the manufacturer’s protocol. The concentration
of the extracted RNA was determined using NanoDrop spectrophotometer by UV absorption.
Eluted viral RNA samples were kept at –20˚C for further use.
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION
One Step RT–PCR kit (Bioneer) was used this purpose. A forward primer 5’-TAC AAC
AGG ACA TTG ACC ACT TTG CTC AC-3’ and reverse primer 5’-TGC ATC TTC CCA
ACT GCC ACT GC-3’ were used to amplify 299 base pare (bp) fragment. Gene specific
primers were used for cDNA synthesis and PCR amplification in a single tube. Starting
material of viral RNA used in cDNA synthesis was 100 ng/μl. The PCR conditions were:
cDNA synthesis at 45◦C for 30 min, initial denaturation at 95◦C for 2 min followed by 30 cycles
of: denaturation at 95◦C for 10 s, annealing at 58◦C for 20 s, and extension at 72◦C for 30 s. The
reaction was then held at 72◦C for 5 min, and then cooled down at 4◦C for 5 min. The amplified
PCR product was detected using 1.5% agarose gel prepared with agarose (Promega) in TAE
buffer. The amplicon size was estimated by comparison with a standard DNA ladder.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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RESULTS
DETECTION OF CYTOPATIC EFFECT IN CHICKEN EMBRYOS
Cytopathic effects represented by represented by petechial haemorrhages and embryo
distortion were observed on the embryos following inoculation with the virus. In comparison,
the uninfected embryos (control group) showed no sign of pathological lesions (Figure1).
Figure 1: Chicken embryos inoculated with the virus.
DETECTION OF VIRUSES BY HEMAGGLUTINATION
Positive haemagglutination results were visualized after mixing allantoic fluid with
RBCs, which confirm the growth of the virus. A negative result (no evidence of
haemagglutination) was shown in the control group (Figure 2).
Figure 2: Confirmation of virus growth by haemagglutination test.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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DETECTION OF VIRUSES BY RT-PCR
PCR product of partial polymerase (F) gene of Newcastle disease virus (LaSota) were
separated on 1.5% agarose gel pre-stained with ethidium bromide. The results showed the
amplification of 299 bp from allantoic fluid collected from inoculated eggs with the virus
(Figure 3).
300
200
100
bp
299 299 400
Figure 3: Detection of virus growth by RT-PCR.
DISCUSSION
Newcastle disease virus is a good example of studying determinants of viral
pathogenicity. There are certain strains of this virus that play a role in causing important
agricultural diseases in birds in general and poultry in particular with a high mortality rate. On
the other hand, there are non-virulent strains (such as the LaSota strain) and are often used as
vaccines. The use of this type of virus in the laboratory, and because of the safety of dealing
with it, is of great importance in studying the pathogenicity of the virus on chicken egg
embryos as well as cell cultures (9).
In this study, a non-virulent virus was successfully grown in embryonated chicken eggs
to obtain a high titer of virus stock, which is necessary for performing further in vitro
experiments. Among these experiments are studying the structure of viruses by determining the
nucleotide sequence in the viral genome, as well as preparing viral antigens, which are
important in performing various laboratory techniques such as ELISA, neutralization tests,
haemagglutination and haemagglutination inhibition. In addition, amplification of viral genes
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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would be useful for the preparation of DNA probes to detect the full length of viral RNA by
northern blotting technique, and for sequencing the viral genome to detect any nucleotide
variations between virus strains (10).
The LaSota virus strain has been cultivated in chicken eggs for the production of
vaccines (7). In this study, virus was not grown for virus production, but was for obtaining
stock of viruses important to perform further studies. In this regard, it is highly recommended
to prepare antigens from the new generation of viruses using various laboratory methods.
Moreover, it is important to determine which viral antigen should be isolated (for example, the
type of antigen used in the ELISA differs from that used in the haemagglutination test).
Furthermore, it is also recommended to sequence the PCR product and align the sequences with
GenBank to highlight differences between other strains and to study the effect of these
differences.
The types of methods described above may lead to the potential new results, which will
help in preparing the different types of antigens needed for a variety of in vitro techniques, and
thus require further studies.
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