Transcript Isolation and Molecular Characterization of Infectious Bronchitis

Ziad W Jaradat1 and Mustafa M Ababneh2
1 Fatima
College Of Health Sciences, Al Ain, UAE;
2 Jordan University of Science and Technology, Irbid, Jordan
Avian infectious bronchitis virus (IBV)
 It is a highly contagious viral respiratory pathogen of chicken
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


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mainly.
Produce severe economic losses due to mortality, decrease in egg
production and affect both internal and external egg quality.
The disease occur worldwide.
Transmission: Airborne, Direct contact, Indirect by contaminated
equipment's.
Incubation period: 24-48hrs (average = 36 hrs)
IBV affects mainly 3 systems in chicken:
Respiratory
Kidneys
Oviduct
Avian Infectious Bronchitis Virus
Classification
 Genus: Coronavirus
 Family: Coronaviridae
 Order: Nidovirales
Characteristics of Coronaviruses:

Enveloped

Non-segmented

Positive-sense

have Single-stranded RNA genome
Structural proteins of IBV
 All coronaviruses contain a
common set of four structural
proteins, spike (S), envelope
(E), membrane (M) and
Nucleocapsid (N).
 Some of the group 2
coronaviruses also contain a
fifth structural protein in the
form of the haemagglutininesterase (HE) protein.
Newcastle disease Virus (NDV)
 Newcastle disease (ND) is a highly contagious viral
disease and is a continuous threat to the poultry
industry worldwide
 Newcastle disease virus, is a negative-sense, single-
stranded RNA virus.
 NDV strains can be classified as velogenic, mesogenic
or lentogenic
 Phylogenetic classification
 Order: Mononegavirales
 Family: Paramyxoviridae
 Genus: Avulavirus
Genome organization of NDV, a
Paramyxovirus
Diagnosis of IBV and NDV
IBV and NDV diagnosis is performed based on:
 Clinical history, Signs and lesions.
 Serology (ELISA, Hemagglutination [HA] and
Hemagglutinatioin inhibition [HI], Virus Neutralization [VN])
 Isolation using specific pathogen free (SPF) embryonated
chicken
 Detection by RT-PCR or Real-Time PCR
 DNA Sequencing
Diagnosis of IBV should include, if possible, identification
of the serotype or genotype of the virus because of the
great antigenic variation exhibited by IBV strains and the
availability of vaccines designed for different serotypes.
Previous IB studies in Jordan
1. In one study serum samples were collected from 20 chicken flocks
with respiratory disease; 14 broiler. 5 broiler breeder and only one layer
flock. Samples were tested by ELISA at initial signs and repeated
samples at 10-14 days later. ELISA titer increased in 70% of flocks
after 10-14 days of clinical signs. Results indicated an exposure of
some flocks to different serotypes such as Ark, DE-072 and Mass like
serotypes (Gharaibeh, 2007).
2. In another study about 175 tracheal swabs were obtained from
broiler flocks at acute phase of respiratory disease and tested by RTPCR. Results indicated 60% of samples were positive for IB. Among
the positive results, serotype Mass accounted for 35.2%, serotype 4/91
accounted for 31.4%, while serotype D274 accounted for only 8.6%
(Roussan et al, 2008).
Previous studies cont…
3. A third study conducted by Roussan et al, 2009 performed
on 51 chicken flocks; 25 broiler, 15 layer and 11 broiler
breeder suffering from respiratory disease. Samples were
tested for IBV using RT-PCR. About 64% of the positive
samples were broilers while 53% and 54% of the layers and
the breeders where positive respectively.
Previous studies Cont….
Recently 5 IBV isolates (JOA2, JOA4, Saudi-1, Saudi-2 and Iraqi)
were detected by Nested RT-PCR. Strain identification was
characterized by sequencing and phylogenetic analysis of the
amplified hypervariable region of the spike 1 (S1) gene. The 5
isolates were found CK/CH/LDL/971.
Similarity between the 5 isolates ranged from 96.9% - 99.7%, and
between these isolates and the CK/CH/ LDL/971 strain was ranged
from 96.6 to 99.1%. The sequenced fragment of the S1 gene of the
CK/CH/ LDL/971 had less than 80% nucleotide identity to IBV
vaccine strains commonly used in the Middle East (M41 and H120).
(Ababneh, et al., 2012).
NDV in Jordan
 NDV is endemic in Jordan with multiple outbreaks in
different parts of Jordan.
 In 2011, an outbreak of NDV was characterized and
partial sequence of fusion gene was published.
 The causative virus found to be of lineage 5d and closely
related to the Chinese strain of NDV.
Objectives of this study:
To survey all operating layer farms in Northern Jordan
for the presence of IBV.
To characterize the isolated IBV strains using RT-PCR
and sequencing assays targeting spike and nucleocapsid
genes.
To characterize the NDV isolates from broiler farms in
Jordan.
Materials and Methods
 This study was done in 40 operating layer farms for the IBV
and on 40 broiler farms for the NDV.
 From each flock 10 birds were randomly selected and
euthanized, 10 tracheas were collected under minimal
contamination conditions.
 Tracheas where stored at (–70°C) for virus isolation until used.
Tissue Samples and Viral RNA Extraction:
 Viral RNA was isolated from Tracheal samples which
were stored at −70◦C in RNAlater® (Qiagen,
Germany).
 Homogenized tissues were subjected to viral RNA
extraction using the Viral Gene-spin Viral DNA/RNA
extraction kit (Intron Biotechnology, Korea), as per the
manufacturer’s instructions.
 Extracted viral RNA was stored at −70◦C until tested
by RT-PCR.
RT-PCR (Diagnostic and Phylogenetic).
The reverse transcription (RT) step was performed using
an RT system (Promega, USA).
Briefly, viral RNA was denatured at 70oC for 10
minutes, followed by the addition of a reaction mix
including 4μL MgCl2, 2μL reverse transcription 10x
buffer, 2μL dNTP mixture (10mM), 0.5 μL random
primers, 0.75μL AMV reverse transcriptase enzyme,
1μg RNA, and nuclease-free water to a final volume of
20μL.
 Then, the reaction was incubated at 42oC for 60
minutes followed by 94oC for 5 minutes.
 The cDNA was diluted up to 100 μL with nuclease-
free water for PCR amplification.
RT-PCR Assay
Two PCR assays were used for IBV.
1. A diagnostic-nested RT-PCR assay based on the
amplification of the neucleocapsid (N) gene
2. A phylogenetic-nested assay for spike (S) gene.
 IBV-specific primers for the N gene were obtained
according to published primers sequences (Farsang, et al 2002).
 The phylogenetic PCR was performed using nested spike
gene primers according as per published protocols (Jones
2005).
 For NDV , a nested PCR was employed for the partial
amplification of Fusion gene following protocol by
Nanthakumar, et al (2000).
Observed Clinical Signs for IBV
Respiratory Signs
Nasal discharge
Depression
Eggshell deformity
Tracheal Hemorrhage
Caseous Plugs
Oophoritis
Oviduct atrophy
Table 1; Tracheal samples tested by PCR for Infectious
Bronchitis N and S genes
Sample
Total
No. of +ve
Number for N gene
% + ve for
N gene
No. of +ve
S gene
% +ve S
Farms
40
9
22.5
7
17.5
Houses
58
38
65.5
8
13.79
Samples
400
38
9.5
8
2
Diagnostic N gene RT-PCR assay
 Figure 1: Electrophoresis (2% agarose gel) for infectious bronchitis virus
(IBV) detection by the N gene of primers. Lane M = 100 bp DNA ladder
(Promega Corporation, Madison, Wisconsin, USA). Lane 1 = IBV positive
control (positive; band at 383 bp). Lane 2 = Negative control for IBV (no
band). Lanes 3–9 = Positive flocks (positive; band at 383 bp).
Phylogenetic S gene RT-PCR assay
 Figure 2: Electrophoresis (2% agarose gel) for infectious bronchitis virus
(IBV) detection by the S gene of primers. Lane M = 100 bp DNA ladder
(Promega Corporation, Madison, Wisconsin, USA). Lane + = IBV positive
control (positive; band at 390 bp). Lane NTC = Negative control for IBV
(no band). Lanes 1-3 = Positive flocks (positive; band at 390 bp). Lanes 4-5
= negative flocks.
NDV fusion gene nested PCR
 Figure 3: the gel electrophoresis image of tested samples from
1-37. The positive samples are showing the 216 bp band.
 36 samples out of 40 were positive in the nested PCR
DNA Sequencing
 Amplified partial PCR products of S gene on gel were
sliced then these PCR products were submitted to the
Macrogen Co. Seoul, Korea for sequencing using big
dye terminator technology.
 Sequence analysis procedure was carried out using
lasergene Software.
 Sequence alignment was conducted using reference
IBV and NDV strains obtained from GenBank.
Phylogenetic analysis
IS-1464.seq
IBV-23 RC.seq
IBV-44 RC.seq
IBV-46 RC.seq
IBV-37 RC.seq
IBV-64 RC.seq
IBV-47 RC.seq
IBV-48 RC.seq
Variant 2.seq
IS-885.seq
Sul-01-09.seq
M41.seq
IBV-40 RC.seq
H120.seq
IS-1366.seq
Variant 1.seq
4-91.seq
IS-1201.seq
QXIBV.seq
J2 .seq
19.9
18
16
14
12
10
8
Nucleotide Substitutions (x100)
6
4
2
0
Figure 4: Phylogenetic tree analysis for the 8 Jordanian IBV isolates and the
reference IBV strains. 7 IBV were close to the Israeli IB strain IS-1464, while one
isolate fall within M41 cluster.
Table 2: Nucleotide similarity matrix showing the 8 isolated Jordanian IBV isolates and
their relation to the reference IBV strains. The IBV isolates (23, 37, 44,46,47,48 and 64)
are very similar to the IS-1464 strain with a range of (95.4-98.6). One IBV isolate (40)
has a 98.5% nucleotide similarity to the strain M41.
Sequence analysis for NDV
Table 3: Nucleotide similarity percentages for the partial fusion gene
sequence.. NDV isolates 1,6,7,10,11,22,24,37,42 belong to the same lineage
(lineage 2). And the nucleotide similarity percentages range between 98 to
100%. While isolate 5 belong to lineage 5d and has the highest nucleotide
similarity percentage to the ampv-Chicken-Jordan-2o11/ with a percentage of
88.9%.
Table 4 ; cleavage sequence, strain and lineage designation of NDV
viruses analyzed in the study.
Number of
samples
1
6
7
10
11
22
24
37
5
Jordanian
sample
2011
Fusion gene cleavage motif
Virus strain
lineage
GGRQGR
GGRQGR
GGRQGR
GGRQGR
GGRQGR
GGRQGR
GGRQGR
GGRQGR
RGRQRR
RRRQKR
Lentogenic
Lentogenic
Lentogenic
Lentogenic
Lentogenic
Lentogenic
Lentogenic
Lentogenic
Virulent
Virulent
2
2
2
2
2
2
2
2
5d
5d
The change in the cleavage cite is an indicator of virus virulence
Most APMV-1 viruses that are pathogenic for
chickens have the sequence 112R/K-R-Q/K/R-K/RR116 at the C-terminus of the F2 protein and F
(phenylalanine) at residue 117, the N-terminus of
the F1 protein, (Choi et al., 2010; Kim et al., 2008a).
Viruses of low virulence have sequences in the
same region of 112G/E-K/R-Q-G/E-R116 and L
(leucine) at residue 117. (Meulemans et al., 2002).
Thus, there appears to be the requirement of at
least one pair of basic amino acids at residues
116 and 115 plus a phenylalanine at residue 117
and a basic amino acid (R) at 113 if the virus is to
show virulence for chickens.
Discussion
 The numbers of positive results in the highly
conserved N gene PCR were more than the positive
results for the more variable S gene PCR.
For Example, 9 out of 40 farms were positive for the N
gene (22.5%), while 7 farms (17.5%) were positive for
S gene).
 It is known that the N gene is more stable and
abundant than the S gene (S. Youn, 2003, J. You et al.,
2005, D.Y. Zhang et al., 2005).
Eight samples were sequenced, 7 of them (87.5%) were
similar to the IBV strain of IS/1464/06. This strain was
reported in:
 Israel and known as variant II –like strain (Meir, R.
and Maharat, O. Unpublished data).
 Egypt (Mansoura) (El-Mahdy, S. S., El-Hady, M. M.,
Soliman, Y. A., 2010).
 Turkey: EU780077 (IS/ 1464/06) IBV strain that was
named as IS/1464/06 in turkey (Kahya, S., Coven, F.,
Temelli, S., Eyigor, A., Carli, K. T. 2013).
 One sample was found by partial S sequencing to be
similar to IBV strain M41.
 This pathogenic M41 strain was isolated in Jordan
earlier, Roussan et al, 2009 reported the overall sero-
prevalence for M41 (92.9%), 4/91 (90%), and D274
(61.4%), among 40 broiler, 18 layer and 12 broiler
breeder. However, they reported (58.8%) overall PCR
positive for IBV without identifying the strain.
 For NDV, 36 farms were positive for the presence of
NDV virus by nested PCR. Nine samples were
selected randomly for sequencing
 Upon sequencing, 8 samples were identified as
lineage 2 and having a sequence of GGRQGR in the
cleavage site indicating the lentogenic nature of the
virus. While one isolate (No. 5) has a RGRQRR
indicating the velogenic nature of this isolate and
belong to the genus 5d.
Conclusion
 Most of IBV isolates in Jordan are closely related to
the IS-1464 strain.
 Most of NDV isolates are of lineage 2. and one
sample was of lineage 5d.
Thank you