SCVMJ, IVX (2) 2009
55
MOLECULAR CHARACTERIZATION OF NEWCASTLE
DISEASE VIRUS, QUAIL STRAIN, USING ONE STEP RT-PCR
El-Shahidi M. S.*, El-Tarabili M. M.*, Metwally S. M.**, Abdelwahab
Shahira A.*, Abdel-Dayem M.*, Fetaih H. A.*** and Ramzy Neven****
* Dept of Virology, Fac. of Vet. Med., Suez Canal University.
** Anim. Health Inst., Dokki, Giza.
*** Dept of Pathol., Fac. of Vet. Med., Suez Canal University.
**** Lab. Anim. Health Inst., Ismailia.
ABSTRACT
Quails have been a tremendous potential for village and backyard
production as well as an important laboratory animal. Newcastle disease (ND)
has become a threat to poultry industry and transmitted to different species of
birds including quails. Newcastle disease is caused by avian paramyxovirus
serotype – I, an enveloped, single stranded RNA virus with a 15 Kb genome of
negative polarity which codes for six proteins. In this study a reversetranscriptase-PCR assay (RT-PCR) had been used for detection and genetic
characterization of the ND virus from quails in Egypt. Nucleotide sequencing
analysis of selected cases from quails showed that the similarity of the detected
virus was 100% to different published poultry strains. The new isolate was
genetically characterized as mesogenic/ velogenic strain.
INTRODUCTION
Newcastle disease virus (NDV)
is designated as avian paramyxovirus serotype 1 (APMV-1) (Murphy
et al., 1995). It is an enveloped, single stranded RNA virus with a 15
kb genome of negative polarity which codes for six proteins. NDV strains are grouped into five pathotypes
based on the clinical signs induced
in infected chickens (Beard et al.,
1970). Molecularly, the different pathotypes are characterized by differences in the amino acid sequence
surrounding the post-translational cl-
eavage site of the fusion protein (F0)
into the disulphide-linked F1 and F2
proteins, which enables the virus to
fuse with the host cell membrane
and to spread in the infected organism (Farkas et al., 2009b). The F0
of lentogenic strains possesses two
single basic amino acids at the cleavage site that can only be cleaved by
trypsin-like enzymes, found in a limited number of cell types, whereas
the F0 of virulent NDV with two
pairs of basic amino acids at the
cleavage site can be cleaved by an
56
omnipotent protease and therefore
fuse with a wide range of cells, resulting in a fatal systemic infection
(Alexander et al., 1992).
For confirmation of ND, the OIE Standards Commission prescribes NDV
isolation in embryonated chicken eggs,
and identification using haemagglutination (HA) and haemagglutination
inhibition (HI) test with a NDV-monospecific antiserum (OIE, 1996).
Reverse transcription-polymerase chain reaction (RT-PCR) has
already been established to identify
NDV in allantoic fluid and in poultry vaccines (Stauber et al., 1995),
Oberdorfer & Werner, 1998). (Seal
et al., 1995) used RT-PCR to obtain
cDNA for direct nucleotide sequencing of NDV for molecular epidemiological analysis. (Kant et al., 1997)
performed RT-PCR to detect NDV
in tissue and fecal samples. The RTPCR doesn’t show any cross-reactivity with other avian paramyxovirus
serotypes, and additionally, offers the
possibility of subsequent sequencing
of the amplified DNA allowing pathotyping of the isolate (Creelan et
al., 2002). In the current study a
NDV isolated and identified in quails using one step RT-PCR assay
and sequencing for pathotyping of
the current isolate.
El - Shahidi, et al.,
MATERIAL & METHODS:
Quails: Twenty quails, 3 weeks old,
showing mild respiratory and nervous signs were obtained from quail's farm, Faculty of Agriculture,
Suez Canal University, Ismailia, Egypt, and euthanized for serological
examination and isolation of Newcastle disease virus. Another thirty
six healthy quails obtained from another farm for experimental infection.
Experimental infection: The experimental birds were kept under hygienic conditions in batteries, fed drug free ration and watered ad libitum. They were classified into three
groups. The first two groups were
inoculated with the isolated NDV
quail strain, intraperitoneal and intraocular, respectively with a dose of
0.2 X107 /ml. The third group was
kept as a non inoculated group.
Serum and tissue samples: Serum
and tissue samples from all organs
were collected from all field cases
and experimental quails for the purpose of serological tests (HI), virus
isolation and identification (HA and
SPF-ECE inoculation) and for histopathological examination according
to (Drury and Wallington, 1980).
Hemagglutination inhibition test
(HI): ND virus HI antibody quantification was done for all samples
using procedures of Beard (1989).
Egg inoculation and Allantoic fluid
collection: This was carried out in
SCVMJ, IVX (2) 2009
57
Abbasia Institute for Vaccine and
Serum production using specific pathogens free (SPF) eggs. The mean
death time with the minimal lethal
dose was calculated, and allantoic
egg fluid for molecular characterization of the suspected isolate using
the RT-PCR technique.
The superscript reverse transcriptase/Platinum Tag mix was used according to the manufacturer's instructions. cDNA synthesis was carried
out for one cycle at 45°C for 30 min,
with inactivation of Super Script™
and activation of Platinum Taq
polymerase following with one cycle
DNA extraction: For DNA extra- at 94°C for 2 min. Amplification
ction, according to Creelan et al., immediately followed with 40 cycles
(2002) both blood and tissue sam- of 94°C for 15 sec, 48°C for 30 sec
ples were digested with proteinase K and 72°C for 30 sec, followed by
(QIAGEN Inc., Valencia, CA, USA) one final extension cycle of 72°C for
in buffer ATL (QIAGEN Inc.) for a 7 min.
minimum of 12 h at 55 ºC for tissue Agarose gel electrophoresis: The
samples. DNA extractions were per- PCR products were electrophoresed
formed using the QIAGEN DNeasy in 1.7% agarose gel (Sigma-Aldrich
tissue kit (QIAGEN Inc., Valencia, Co. Saint Louis, MO, USA) prepared
CA, USA) according to the manu- by melting 1.7 gram of agarose in
facturer instructions. For each of the 100 ml 1X TBE buffer (89 mM Tris,
DNA extraction steps, strict proto- 89 mM Boric acid and 2mM EDTA,
cols were followed to avoid cross- pH 8.3) obtained from Fisher Sciecontamination of samples. Samples ntific International Inc. (Fair lawn,
were stored at -80 ºC until used as New Jersey, USA). Ethidium brotemplates for amplification.
mide was added to the gel at a
concentration of 0.5 ug/ml. ElectroReverse transcriptase-polymerase chain reaction (RT- PCR): The one phoresis was conducted at 100 volt
step RT-PCR was performed using (5V/cm) for 20 minutes. The PCR
products were visualized by the UV
the published primers and protocol
designed by (Creelan et al., 2002) illumination using the UVP's Bioimforward primer NDV F, 4829 5'- aging system.
GGTGAGTCTATCCGGARGATA
CAAG-3' 4893; NDV R, 5031 5'TCATTGGTTGCRGCAATGCTCT
-3' 5008. Sequence numbering is according to previously published sequence (accession number AF 077761).
These primers are targeting a 202 bp
size of F protein gene.
DNA sequencing: the DNA product
was excised from the gel and purified using a commercially available
gel extraction kit. The forward
(NDV F) primer was used as described previously. Sequencing of the
purified DNA was done at the Laboratory Research Department at
58
VACSER. Company using 310 genetic analyzer using the big dye terminator Kit.
RESULTS:
Clinical symptoms and pathological
findings: The field and the experimentally infected quails showed mild respiratory and nervous manifestations. Natural infection caused morbidity in 10% of birds and mortality
of 1.6%, while the experimental
infection caused 25% morbidity and
mortality in the first and second week Post infection (PI). Pathological
examination of died and moribund
quails showed focal hemorrhages
allover the body, congestion, pneumonic spots in the lung tissue, focal
necrotic changes and degeneration
of parenchymatous organs, lymphoid depletion in spleen and mild inflammatory changes in the digestive
tract. Brain was also affected with
edema and neuronal degeneration.
Serological study: Serological screening of the field cases using HI test
proved a seroconversion in the diseased birds. The mean HI titre increased from 1/32 to 1/256 at log4.
The agar gel precipitating antibodies
El - Shahidi, et al.,
(AGP) had also increased. The NDV
could be isolated from 3 out of 20
diseased quails, as confirmed by HA
and SPF-ECE inoculation. The isolated quail strain was found to be of
intermediate virulence (mesognenic)
and thereafter was used for experimental infection and for molecular
characterization.
Amplification of DNA sequences
from the field isolates of NDV in
quails by RT-PCR: The amplified
DNA product from quail isolate
propagated in allantoic fluid showed
202 bp DNA product while sample
from different organs tissue showed
negative result (Figure 1).
Sequence alignments and BLAST
search indicated that the RT-PCR
products amplified from the quail
sample was identical and had 100 %
similarity to the published sequence
of several strains of NDV (Figure 2).
In this study the amino acid sequence analysis of the targeted area showed that the current quail isolate
belongs to the mesogenic/ velogenic
strains, as it contains more basic
amino acids.
SCVMJ, IVX (2) 2009
59
1000
500
100
L
1
2
3
4
Fig. 1: L- ladder 100 bp. 1- Egg fluid sample. 2- Negative control sample for egg fluid
sample. 3- Sample of different organs tissue showed –ve result. 4- Negative
control sample of different organs tissue showed –ve result.
60
El - Shahidi, et al.,
Fig. 2: Sequence alignments and BLAST search indicated that the RT-PCR products
amplified from the quail sample was identical and had about 100% similarity to
the different published sequence of NDV strains.
DISCUSSION
The objective of the current
study is to isolate and identify the
NDV from naturally infected quails
and induction of such disease experimentally in commercial quails as
well as characterization of the isolated NDV quail strain pathotype and
studying the postmortem gross lesions and histopathological changes
in naturally and experimentally infected quails. The preliminary steps
towards confirmative characterization of NDV in quails are virus isolation followed by examination of
the antigenicity and pathogenicity of
isolated NDV quail strains and m-
olecular identification and sequencing of viral genome.
To confirm ND in naturally infected
quail farm, many methods were
carried out. The first one is proving
of seroconversion of NDV antibodies that was observed between acute and convalescent serum samples.
Results of ND virus isolation
showed that out of 20 pooled quail
samples, 15 samples were positive
for HA and the virus could be
successfully isolated from 3 cases
The same results obtained by
(Oladele et al., 2005). The virulence
and pathogenicity of the isolated
SCVMJ, IVX (2) 2009
strains were found to be intermediate
i. e. mesogeneic viruses. These
results coincide with the same obtained by (Islam et al., 1994; Lima et
al., 2004). The low HI antibody titer
of naturally infected quails recorded
in current study was below the protective level (Lima et al., 2004).
Results of pathological examination
showed that the isolated virus (quail
strain) can cause damage in tissue of
quails but of mild degree and inlittle
number of birds. The recorded changes agree with that found in previous studies in quails (Usman et al
2008 and Oladele et al. 2008), but
with slight differences with that
recorded in chikens (Okoye et al.
2002 and Oladele et al. 2005).
61
been established to identify the NDV
in allantoic fluid and in poultry
vaccine (Stauber et al., 1995). (Seal
et al., 1995; 1996) used RT-PCR to
obtain cDNA for direct nucleotide
sequencing of NDV for molecular
and epidemiological analysis. The
RT-PCR primers and RT-PCR conditions used to amplify a 202 bp DNA
sequence out of the tegument F gene
(fusion protein gene) was demonstrated as a specific and simple tool
for diagnosis and typing of the
APMV-1. Fusion protein gene corresponds to the cleavage site of the F0
precursor, which is a key determinant of NDV pathogenicity (Farkas
et al., 2009a; Tan et al., 2004). The
specificity of the primers to APMVND is caused by type 1 paramy- 1 was demonstrated by amplification
xovirus. The virus strains classified of a 202 bp product from all the
according to the pathogenicity, ded- lentogenic, mesogenic and velogenic
uced amino acid sequence of the cases tested, including pigeon PMV1 cases, and the absence of any amcleavage site of the fusion protein
into less virulent lentogenic, interm- plification product for all other eight
ediate virulence mesogenic and hig- serotypes of APMV2-9 and unrelahly virulent velogenic strains. The ted avian RNA viruses. Also the spehighly virulent velogenic strains are cificity of the RT-PCR was demonssuspected to arise from lentogen of trated by the negative RT-PCR reswild birds (Shengqing et al., 2002). ults obtained from five cases of susBased on genetic and antigenic ana- pect ND that were found to be negalysis of the isolates of ND obtained, tive by virus isolation (Creelan et
the existence of at least eight diff- al., 2002; Creelan & McCullough,
erent genotypes (I-VIII) has been 2006)
shown and host species associations The RT-PCR negative result of the
are often seen inside these groups different organs tissue can be exp(Alexander, 2000; Miller et al., 2007). lained by the presence of PCR inhiReverse transcription polymerase bitors (Abu Al-Soud & Radstrom,
chain reaction (RT-PCR) has already 2000; Al-Soud et al., 2000; Al-Soud
& Radstrom, 2001). This failure to
62
detect viral nucleic acid in all
samples from which viral isolates
were made indicates that, while this
RT-PCR protocol is a useful adjunct
to isolation in eggs, it cannot yet
replace this procedure completely.
Nucleotide sequence analysis of the
202-bp quail PMV-1 isolate amplification product demonstrated that
the virus detected was identical to
those from chicken, turkey and pigeon with lesions characteristic of
NDV infection reported by (Gohm
et al., 1999; Li et al., 2009).
In this study the amino acid sequence analysis of the obtained nucleotide sequence analysis showed that
the current quail isolate belongs to
the mesogenic/ velogenic strains
(Farkas et al., 2009a). As a result,
the precursor protein of virulent
isolates is more susceptible to cleavage by the ubiquitous proteases
present in different tissues of the
host (Collins et al., 1993). In summary, the virulence of NDV is highly
dependent on the cleavability of the
fusion glycoprotein precursor (F0)
by cellular proteases of the host
(Aldous et al., 2001). Another important conclusion from the sequence
analysis is that the mesogenic and
velogenic viruses cannot be differentiated on the basis of the nucleotide
sequence of the F0 proteolytic cleavage site, and the same is true for
asymptomatic enteric and lentogenic
viruses (Seal et al., 1995).
El - Shahidi, et al.,
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‫الملخص العربى‬
‫التوصيف الجزيئى لفيروس مرض النيوكاسل المعزول من السمان باستخدام اختبار‬
‫تفاعل سلسلة البلمرة‬
‫محمد الشهيدى*‪ ،‬مختار الطرابيلى*‪ ،‬متولى حمودة**‪ ،‬شهيرة عبد الوهاب*‪ ،‬محمد عبد الدايم*‪،‬‬
‫حمدى فتيح***‪ ،‬نيفين رمزى****‬
‫* قسم الفيرولوجيا – كلية الطب البيطرى – جامعة قناة السويس‬
‫** معهد بحوث صحة الحيوان بالدقى‬
‫*** قسم الباثولوجيا – كلية الطب البيطرى – جامعة قناة السويس‬
‫**** معهد بحوث صحة الحيوان – معمل االسماعيلية‪.‬‬
‫يعتبر السمان ذات اهمية انتاجية كبيرة فى المزارع والمنازل القروية وكذلك طائر معملى‬
‫للتجارب‪ ،‬ونظ را لتكرار اصابة الدواجن بفيروس النيوكاسل الخطير وما يسببه من خسائر‬
‫اقتصادية وانتقاله منها الى االنواع االخرى من الطيور ومنها السمان فقد اجريت هذه الدراسة‬
‫لمعرفة مدى قابلية السمان للعدوى بهذا الفيروس ودرجة شدة المرض وصورته فى هذا الطائر اما‬
‫بالعدوى الطبيعية اوالتجريبية‪ ،‬و لعمل التوصيف الجزيئى الكامل للفيروس‪ .‬وقد استخدم لهذ‬
‫الغرض اختبار تفاعل سلسلة البلمرة‪ ،‬وقد سجل التركيب والتسلسل الجينى الكامل لهذا الفيروس‬
‫(عترة السمان) ومضاهاتة بالعترات االخرى التى تصيب الطيور المختلفة ووجد تشابه بنسبة‬
‫‪ %011‬مع تركيب تلك ال عترات‪ ،‬ووجد ايضا ان الفيروس ذات ضراوة متوسطة‪ .‬وهذه الدراسة‬
‫تفيد الحقا فى تشخيص المرض ومحاولة انتاج مصل واقى ضده‪.‬‬