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<pre>
Virology Journal
BioMed Central
Open Access
Research
S1 gene sequence analysis of a nephropathogenic strain of avian
infectious bronchitis virus in Egypt
Ahmed S Abdel-Moneim*1, Magdy F El-Kady2, Brian S Ladman3 and
Jack Gelb Jr3
Address: 1Department of Virology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt, 2Department of Poultry Diseases,
Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt and 3Department of Animal and Food Sciences, University of
Delaware, Newark, DE 19717, USA
Email: Ahmed S Abdel-Moneim* - [email protected]; Magdy F El-Kady - [email protected];
Brian S Ladman - [email protected]; Jack Gelb - [email protected]
* Corresponding author
Published: 20 September 2006
Virology Journal 2006, 3:78
doi:10.1186/1743-422X-3-78
Received: 11 August 2006
Accepted: 20 September 2006
This article is available from: http://www.virologyj.com/content/3/1/78
© 2006 Abdel-Moneim et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Infectious bronchitis is highly contagious and constitutes one of the most common
and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens.
It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of
vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the
respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and
the kidney.
Results: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype
was isolated from broiler chickens suffering from severe renal and respiratory distresses. The
isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then
genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV
strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain
reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe
renal and moderate respiratory distresses. Marked histopathological changes in both kidney and
trachea were observed in experimentally infected chickens. A protection study using the H120 live
attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%).
Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal
histopathology as well as IBV antigens detection by immunofluorescent antibody technique in
kidney sections.
Conclusion: Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently
recovered field isolates should be performed to ensure optimum control of IBV.
Background
Avian infectious bronchitis virus (IBV) is a highly contagious pathogen of chickens that replicates primarily in the
respiratory tract and also in some epithelial cells of the
gut, kidney and oviduct [1]. IBV is a virus member of
genus Coronavirus, family Coronaviridae, order Nidovirales [2]. The virus possesses a positive stranded RNA
genome that encodes phosphorylated nucleocapsid proPage 1 of 9
(page number not for citation purposes)
Virology Journal 2006, 3:78
http://www.virologyj.com/content/3/1/78
tein (N), membrane glycoprotein (M), spike glycoprotein
(S) and small membrane protein (E). The spike glycoprotein is post-translationally cleaved into two subunits, S1
and S2 [1,3]. The S1 protein forms the N-terminal portion
of the peplomer and contains antigenic epitopes mainly
within three HVRs [4-6]. Neutralizing and serotype specific epitopes are associated within the defined HVRs
[4,7,8].
Variation in S1 sequences [9-11], has been recently used
for distinguishing between different IBV serotypes. Diversity in S1 probably results from mutation, recombination
and strong positive selection in vivo [12]. Antigenically
different serotypes and newly emerged variants from field
chicken flocks sometimes cause vaccine breaks. The generation of genetic variants is thought to be resulted from few
amino acid changes in the spike (S) glycoprotein of IBV
[13,14].
In Egypt, isolates related to Massachusetts, D3128, D274,
D-08880, 4/91 and the novel genotype; Egypt/Beni-Suef/
01 were isolated from different poultry farms [15-18]. The
commonly used IBV attenuated vaccine is H120 while the
Mass 41 (M41) strain is commonly used in inactivated
vaccines.
In the present study, Egypt/F/03 was isolated from 25day-old broiler chickens in Fayoum Governorate, identified by Dot-ELISA, RT-PCR and sequenced to determine
its serotype. Pathogenicity test to 1-day-old chickens and
protection afforded by the commonly used H120 live
attenuated vaccine were also performed.
Results
Virus isolation and serological identification
The allantoic fluid of the first chicken embryo passage of
Egypt/F/03 was harvested at 48 h PI. Four additional egg
passages were performed. Five eggs of the 4th passage were
incubated till being 18-day-old and all of them (100%)
showed typical lesions of the IBV (stunting and dwarfing).
The virus identity was ascertained by performing DotELISA on the CAM homogenate (Fig. 1).
Control +v e
Tested sample
Control -ve
Figure 1
membrane homogenate) and control tested sample
Dot-ELISA shows positive reaction in positive(chorioallantoic
Dot-ELISA shows positive reaction in tested (chorioallantoic
membrane homogenate) and control positive sample.
Polymerase chain reaction and S1 gene cycle sequencing
RT-PCR of Egypt/F/03 resulted in a product of 1600 base
pairs using S1 primers OLIGO 5' and OLIGO 3'. Egypt/F/
03 is closely related to the Beaudette US reference strain;
98% nucleotide identity and 96% amino acid identity
(Table 1 and Fig. 2). It showed 97% similarities both in
nucleotides and amino acids to H120 and 98% nucleotide
and 96% amino acid homology to M41(Table 1). Egypt/
F/03 showed 34 point mutations from H120; 20 silent
and 14 non silent mutations. On the other hand, it
showed 30 point mutations; 10 silent and 20 non silent
mutations from M41 (Fig. 3, 4). Sixteen potential glycosylation sites were found in Egypt/F/03 while 17 were
found in H120 and M41 (Fig. 4). All potential glycosylation sites found in Egypt/F/03 were shared with those
found in H120 and M41(Fig. 4).
Virulence test
Chickens inoculated with Egypt/F/03 exhibited snicking
and rales in approximately 50% of infected birds at 3rd day
of inoculation. Conjunctivitis was observed in 20/30 at
3rd day PI that was elevated to 22/30 by the 5th day PI of
infected birds whereas no birds exhibited watery eyes. Six
birds were dead after Egypt/F/03 experimental infection; 4
birds in the 7th day PI and 2 birds in the 8th day PI. Postmortem examination of dead birds, revealed petechial
haemorrhages in larynx and thymus, severe congestion of
liver, spleen and lungs as well as renal haemorrhages.
These changes appeared but in milder form in birds sacrificed at 5 days PI. Histopathological examination of sacrificed birds at 5 days PI and freshly dead birds at 7 days PI
revealed mucus, marked loss of cilia, desquamation,
mononuclear infiltration, epithelial hyperplasia and vascular congestion of the trachea (Fig. 5). Kidneys showed
severe changes including haemorrhages, degenerative
changes in renal tubules and hypercellularity of the renal
glomeruli as well as focal lymphocytic infiltration (Fig. 5).
In general the tracheal and renal histopathological lesions
were more severe in dead birds (Fig. 5b, d) than birds sacrificed at 5 days PI (Fig. 5a, c).
Vaccination trial
Chickens vaccinated with H120 (Group A) showed 58.3%
protection (7/12) by virus reisolation procedure and
66.6% (8/12) protection by histopathology after challenge with Egypt\F/03 while all control unvaccinated
birds (Group B) were not protected (5/5) (Table 2). On
day four PI, the kidneys of (3/12) birds in group A (vaccinated and challenged with IBV) showed focal lymphocytic infiltration, urates deposition and degenerative
changes in renal tubules while birds of group B (unvaccinated and challenged with IBV) showed multifocal lymphocytic infiltration, urates deposition and degenerative
changes in renal tubules in 4/5 birds, while only focal
lymphocytic infiltration, urates deposition and degenera-
Page 2 of 9
(page number not for citation purposes)
Virology Journal 2006, 3:78
http://www.virologyj.com/content/3/1/78
Table 1: Nucleotide and amino acid identities of Egypt/F/03 with selected IBV sequences
Nucleotide identity (%)
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
71
74
97
96
96
99
70
90
74
74
72
72
68
71
97
71
1
2
74
74
71
70
71
71
69
70
75
75
73
72
97
68
71
77
2
3
73
73
73
72
73
74
75
72
98
94
81
72
73
72
73
83
3
4
97
74
73
96
96
97
70
91
74
73
72
71
68
69
96
71
4
5
98
74
77
97
95
96
69
90
72
72
71
70
68
68
93
70
5
6
98
74
73
97
97
96
71
89
74
73
72
71
68
70
96
71
6
7
99
75
74
97
94
98
71
90
74
74
72
72
69
71
97
72
7
8
76
74
72
76
76
76
76
68
75
76
77
71
66
69
70
76
8
9
95
73
71
94
95
94
95
75
73
74
70
69
67
67
84
69
9
10
75
76
99
75
79
75
74
74
73
94
82
73
73
73
73
83
10
11
68
76
97
68
75
68
69
69
67
91
83
73
73
73
73
85
11
12
72
77
82
73
81
73
73
73
72
82
83
72
69
71
72
77
12
13
77
75
70
77
77
77
78
78
76
73
67
70
71
88
72
73
13
14
74
98
76
73
73
73
75
74
72
76
76
76
75
69
70
78
14
15
76
73
76
75
75
75
76
73
73
76
76
76
93
73
71
73
15
16
98
75
78
97
96
97
98
75
91
78
78
79
77
74
76
72
16
17
78
79
84
78
77
78
79
78
76
84
84
81
76
79
76
78
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Egypt/F/03
Egypt/Beni-Suef/01
Egypt/D/89
H120
M41
Beaudette.US
GX1-98.China
B1648.Belgium
Connecticut
D274
D3896
Vic.S
UK/4/91
Israel/720/99
IS/188/96/Var.1
IS/385/97
IS/585/98/Var.2
17
Amino acid identity (%)
tive changes in renal tubules were observed in 1/5 birds.
Birds in group C (vaccinated unchallenged group) did not
show any abnormalities (0/12) either in tracheae or kidneys. Immunofluorescenct assay on kidneys of challenged
and unchallenged groups showed that all challenged
unvaccinated and 3/12 of challenged vaccinated groups
possessed kidney immunofluorescence. None of control
unchallenged groups possessed kidney immunofluorescence (Table 2).
Discussion
In this study, an Egyptian IBV strain; Egypt\F/03 was isolated from a tissue pool of kidney and trachea from unvaccinated broiler flock with a history of respiratory and renal
disease. The strain produced typical lesions of IBV in inoculated embryos and identified as IBV by Dot-ELISA and
RT-PCR. The isolate was found to be devoid of major concomitant viruses; avian influenza virus, Newcastle disease
virus, infectious laryngotracheitis virus, reovirus and adenovirus (data not shown).
Figure
strains 2
netic tree of Egypt/F/03 isolate and expressed as a phylogeIBV S1 gene sequence relationships selected IBV reference
IBV S1 gene sequence relationships expressed as a phylogenetic tree of Egypt/F/03 isolate and selected IBV reference
strains.
S1 sequence analysis of Egypt/F/03 revealed its close relatedness to Mass serotype. It showed high nucleotide similarities to GX1-98.China (99% nucleotide and amino acid
identities), Beaudette-US (98% nucleotide and 96%
amino acid identities), IS/385/97 (98% nucleotide and
97% amino acid identities), H120 (97% nucleotide and
amino acid identities) and M41 (98% nucleotide and
96% amino acid identities).
Page 3 of 9
(page number not for citation purposes)
Virology Journal 2006, 3:78
http://www.virologyj.com/content/3/1/78
Egypt/F/03
H120
M41
1 ----------------------------------------------------------------------------------ATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTG
1 ----------------------------------------------------------------------------------......................................
1 ACGCCAGTTGTTAATTTGAAAACTGAACAAAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGGTAAGAG......................................
Egypt/F/03
H120
M41
39 TGCACTATGTAGTGCTGTTTTGTATGACAGTAGTTCTTACGTGTACTACTATCAAAGTGCCTTCAGACCACCTAATGGTTGGCATTTACATGGGGGTGCTTATGCGGTAGTTAATATTTC
39 .................C.................................C.....................G.........................G........T...........
121 ..T..............C........................T........C...........T..........................C.............................
Egypt/F/03
H120
M41
159 TAGCGAATCTAATAATGCAGGCTCTTCATCTGGGTGTACTGTTGGTACTATTCATGGTGATCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCACCGTCATCAGGTATGGCTTGGTC
159 ...T...........................................T...........G............................................................
241 ............................C.........T....................G............................................................
Egypt/F/03
H120
M41
279 TAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTGTTTGTTACACATTGTTATAAACATGGTGGGTGTCCTATAACTGGCATGCTTCAACAGCATTTTATACGTGT
279 ......................T...........................................C.......T...................................C.........
361 ......................................................................T...A............................A..A.....T.......
Egypt/F/03
H120
M41
399 TTCTGCTATGAAAAATGGCCAGTTTTTCTATAATTTAACAGTTAGTGTAGCTAAGTACCCTACTTTTAAATCATTTCAGTGTGTTAATAATTTAACATCCGTATATTTAAATGGTGATCT
399 ......................C....T............................................................................................
481 ......................C.................................................................................................
Egypt/F/03
H120
M41
519 TGTTTACACCTCTAATGAGACCACAGATGTTACATCTGCAGGTGTTTATTTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTATTTTGTTAATGG
519 ...............................................................................................G........................
601 ........................................................................................A...............................
Egypt/F/03
H120
M41
639 TACTGCACAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTAGCATGCCAGTATAATACTGGCAATTTTTCAGATGGCTTTTATCCTTTTATTAATAGTAGTTTAGTTAAGCA
639 ..............................G...................................................................C.....................
721 ........................................................................................................................
Egypt/F/03
H120
M41
759 GAAGTTTATAGTCTATCGTGAAAATAGTGTTAATACTACTTTTACGTTACACTATTTCAGTTTTCATAATGAGACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAATATTCAAACTTA
759 .........T..........................................A......C...........................A..............C.................
841 .........T..........................................A......C.....................................................T......
Egypt/F/03
H120
M41
879 CCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCCTTTCTGAGTAGTTTTGTTTATAAGGAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGTAATTTTAGACT
879 ........................................................................................................................
961 ........................................................................................................................
Egypt/F/03 999 AGAAACTATTAATAATGGCTTGTGGTTTAATTCACTTTCAGTTTCAATTGCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAACTTGTTGTTATGCTTA
H120
999 ..................T......................................................................................C..............
M41
1081 ........................................................................................................................
Egypt/F/03 1119 TTCATATGAAGGTCCTTTGCTTTGTAAAGGTGTTTATTCGGGTGAGTTAGACCATAATTTTGAATGTGGACTGTTAGTTTATGTTACTAAGAGCGGTGGCTCTCGTATACAAACAGCCAC
H120
1119 C.......G............G.................A...........T....................................................................
M41
1201 ........G........C...G.................A...........T.T..................................................................
Egypt/F/03 1239 TGAACCGCCAGTTATAACTCAACACAATTATAATAATATTACTTTAAATACTTGTGTTGATTATAATATATATGGCAGAACTGGCCAAGGTTTTATTACTAATGTAACCGACTCAGCTGT
H120
1239 ........................................................................................................................
M41
1321 ....................G...................................................................................................
Egypt/F/03 1359 TAGTTATAATTATCTAGCAGACGCAGGTTTGGCTATTTTAGATACATCTGGTTCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTAATTATTATAAGGTTAATCCTTGCGAAGA
H120
1359 .....................................................................C......A...............................C...........
M41
1441 ............................................................................................C...............C...........
Egypt/F/03 1479 TGTCAACCAGCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTTACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGAACCAGTTTTACATCAAAATCACTAATGGAACACG
H120
1479 ........................................................................C..............T................................
M41
1561 ............................................................ ......................................T....................
Egypt/F/03 1599 TC
H120
1599 ..
M41
1681 ..
Figure 3 identities of Egypt/F/03 with commonly used vaccine strains sequences
Nucleotides
Nucleotides identities of Egypt/F/03 with commonly used vaccine strains sequences. Dots indicate residues identical to Egypt/F/03. Bold letters denotes codon areas. Shaded letters denote sites of differences.
It is known that the most severe clinical response of IBV
appears in very young chickens and severity is alleviated in
older chickens [19,20]. This fact explains the high mortality rate observed in 1-day-old chickens that experimentally inoculated with Egypt/F/03 compared to mortality
pattern in the original flock (25-day-old chickens). The
presence of acute interstitial nephritis on days 5 and 7
post infection indicated that Egypt\F\03 is a nephrogenic
IBV. The microscopic findings of the renal tubules
matched the general findings recorded with nephrogenic
IBV strains [21,22]. The microscopic findings in tracheal
sections appeared similar to those recorded by [19,21]
including: loss of cilia, degenerative changes of the tracheal mucosa, irregular loss of epithelium, desquamation
of the sloughed epithelium in the tracheal lumen and
lymphocytic infiltration that ranged from focal aggregation to diffuse massive infiltration. Severe renal haemorrhages observed grossly and in hisopathological sections
of birds dead after experimental infection with Egypt/F/03
denote that deaths resulted from acute renal failure. Our
finding regarding the presence of petechial haemorrhages
in larynx and thymus as well as severe congestion of liver,
spleen and lungs in birds dead after IBV experimental
infection is in agreement with [23,24] who confirmed the
presence of IBV viral antigens in such organs.
Evaluation of the immune response to IBV vaccination is
based on several criteria including: clinical signs, tracheal
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Egypt/F/03
H120
M41
http://www.virologyj.com/content/3/1/78
1 MLVTPLLLVTLLCALCSAVLYDSSSYVYYYQSAFRPPNGWHLHGGAYAVVNISSESNNAGSSSGCTVGTIHGDRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKHGGC
1 ..................A..................D..............................I...G...........................Y................V..
1 .............V....A...........................................P..I......G...........................................YD..
Egypt/F/03 121 PITGMLQQHFIRVSAMKNGQFFYNLTVSVAKYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKVMREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSD
H120
121 .........S..........L...............................................................R...................................
M41
121 .......KN.L.........L.............................................................K.....................................
Egypt/F/3
H120
M41
241 GFYPFINSSLVKQKFIVYRENSVNTTFTLHYFSFHNETGANPNPSGVQNIQTYQTQTAQSGYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIAYGPLQGGCKQ
241 .....T........................N.T.......................................................................................
241 ..............................N.T.................L.....................................................................
Egypt/F/03 361 SVFSGRATCCYAYSYEGPLLCKGVYSGELDHNFECGLLVYVTKSGGSRIQTATEPPVITQHNYNNITLNTCVDYNIYGRTGQGFITNVTDSAVSYNYLADAGLAILDTSGSIDIFVVQGE
H120
361 ...............G......................................................................................................S.
M41
361 ...............G..S...........L............................R............................................................
Egypt/F/03 481 YGLNYYKVNPCEDVNQQFVVSGGKLVGILTSRNETGSQLLENQFYIKITNGTR
H120
481 .....................................................
M41
481 ...T.................................................
Figure 4
Amino acid identities of Egypt/F/03 with commonly used vaccine strains sequences
Amino acid identities of Egypt/F/03 with commonly used vaccine strains sequences. Dots indicate residues identical to Egypt/F/03. Potential glycosylation sites (NXS or NXT, except where X = P) are underlined. Shaded letters denote sites
of differences. A:Alanine, C:Cysteine, D:Aspartic acid, E:Glutamic acid F:Pheny-lalanine, G:Glycine, H:Histidine, I:Isoleucine,
K:Lysine, L:Leucine, M:Methionine, N:Asparagine, P:Proline, Q:Glutamine, R:Arginine, S:Serine, T:Threonine, V:Valine, W:Tryptophan, Y:tyrosine.
histological lesion, virus neutralization, virus re-isolation
from trachea, antigen detection in trachea and/or kidney
by immunofluorescent or immunoperoxidase techniques
[25-29]. In this study we used tracheal histological lesion
and virus re-isolation as parameters for tracheal protection. Kidney histopathology and antigen detection by IFA
were used as indicators of kidney protection. Complete
protection is expected upon using closely related vaccine
strain as the degree of cross protection among IBV strains
generally reflects the similarities between the S proteins
[12,30]. The re-isolation of Egypt/F/03 from the trachea of
vaccinated birds and the presence of tracheal and renal
microscopic lesions as well as viral antigen in kidneys (by
IFA) in H120 vaccinated birds denote lack of complete
protection afforded by H120 vaccination.
H120 is a mild vaccine and it is possible that the challenge
virus was too virulent for the level of immunity that the
vaccine produced in these young chickens. Other possible
consideration includes that baby chickens are not fully
immunocompetent at one-day of age, the time that they
were vaccinated for the protection study experiment.
However, commercial broiler chickens possess maternally
derived antibodies, are routinely vaccinated at one-day of
age [31,32] without apparent interference by the maternal
derived antibodies in the development of active immunity, at least in the respiratory tract that measured by challenge [33]. On the other hand, variable results were
recorded regarding homologous protection of IBV. Cavanagh et al. [12] inoculated groups of 10 chickens with the
virulent UK/6/82 isolate and challenged with isolates that
differed by up to 4% of S1 amino acids. Challenge with
two variants (98% S1 identity with UK/6/82) resulted in
challenge scores virtually the same as with the homologous challenge however, challenge with two others iso-
lates (96% and 98% S1 identity, respectively), resulted in
less cross-protection, although the numbers were not statistically significantly different.
In the S1 subunit, three HVRs are located within amino
acids 38–67, 91–141 and 274–387 [4-6]. HVR1 and
HVR2 contain sequences that have been associated with
specific IBV serotypes [34,35] as well as serotype specific
neutralizing epitopes [4,5,14]. IBV serotypes commonly
differ by 20 to 25% in S1 [11,36] but some serotypes differ
in S1 by as little as 2% [13]. Although H120 showed 97%
amino acid and nucleotide identity to Egypt/F/03, it possesses 34 different nucleotides that resulted in 14 amino
acid substitutions. Among such amino acids, one is
located in HVR1, four in HVR2 and one in HVR3 (Fig.4).
The region between amino acid residues 123–152 has
been previously identified as a possible region involved in
the differing pathogenicity of Gray and non-virulent JMK
strains [37]. Egypt/F/03 possesses different amino acid;
phenylalanine within this region at positions 130 and 141
instead of serine and leucine in H120 respectively. It is apt
to mention that some serotypes differ in S1 by as little as
10 amino acids [13], suggesting that only a few epitopes
may induce most of the VN antibody [38].
Conclusion
Egypt/F/03 is a nephropathogenic IBV strain closely
related to Mass serotype. Vaccination by H120 did not
provide satisfactory protection against challenge with
Egypt/F/03. Complete protection of trachea against the
Egypt/F/03 and consequently efficient prevention of kidney infection may be quite feasible upon development of
safe attenuated vaccines based on indigenous field strain.
Preparation of live and inactivated vaccines from indigenous isolates should parallel periodic evaluation of cross-
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a
http://www.virologyj.com/content/3/1/78
b
arate rooms and used in both virulence test and protection study.
Rabbit anti-IBV
Rabbit anti-IBV polyclonal antiserum raised against vero
adapted H120 vaccine was prepared previously in our lab
[17] and used for detection of IBV antigens in both DotELISA and indirect immunofluorescent antibody technique.
c
d
Figure
infection and kidney histopathology Egypt/F/03
Trachea5of 1-day old chickens withfollowing experimental
Trachea and kidney histopathology following experimental infection of 1-day old chickens with Egypt/F/
03. Trachea and kidney stained with H & E: a. Trachea of
chickens 5 d P.I with Egypt/F/03 showed hyperplasia, lymphcytic infiltration and oedema (40 ×). b. Trachea of chickens 7
d P.I with Egypt/F/03 showed diffuse lymphocytic aggregation,
degeneration of the epithelium mucus, and haemorrhages (20
×). c. Kidney of chickens 5 d P.I with Egypt/F/03 showed focal
lymphocytic aggregation in the interstitium and in the
glomeruli, as well as degenerative changes in tubular epithelium (40 ×). d. Kidney of chickens 7 d P.I with Egypt/F/03
showed massive renal haemorrhages and degeneration renal
tubular epithelium (20 ×).
protective capabilities of such vaccine(s) versus recently
recovered field isolates in order to ensure optimum control of IBV.
Methods
Embryonated chicken eggs
SPF ECE obtained from Nile SPF (Koom Oshiem, Fayoum, Egypt) were used for isolation of the field isolate,
serial passages, titration of the seed stocks of Egypt/F/03
and vaccine strain (H120), as well as virus re-isolation
attempts following challenge in the protection study.
Chickens
Sixty nine commercial 1-day-old chickens (El-Waddi Co,
Egypt) were reared under strict hygienic conditions in sep-
Clinical history
Infectious bronchitis was diagnosed during Augest 2003
in Fayoum Governorate, Egypt. The outbreak occurred in
25-day-old commercial broiler farm with no previous IBV
vaccination. The flock was vaccinated against Newcastle
disease and infectious bursal disease viruses at 14 and 18
days of age respectively. The total flock density was 3000
birds. The first signs were depression and respiratory distresses including sneezing, coughing and rales. Other
signs included conjunctivitis and watery eyes. Within a
period of 10 days after the appearance of the disease, the
mortality rate increased to 10% of the flock density. Postmortem examination of dead birds revealed increased tracheal mucus, severe renal congestion, urates filled ureters
as well as congestion in liver and spleen.
Virus isolation and passage in SPF ECE
Egypt/F/03 was isolated from 25-day-old broiler chickens
suffering from both respiratory and renal distresses from
Fayoum Governorate in 2003. A kidney homogenate
(10% in sterile PBS) and a tracheal scraping suspension
were pooled, centrifuged at 500 × g for 10 min. The supernatant fluid was inoculated into chorioallantoic sac of 10day-old SPF ECE. Allantoic fluid was harvested after 48 h
and was used for re-passage into ECE. Five eggs of the 4th
egg passage were incubated till being 18-day-old and
examined for typical lesions of IBV (stunting, curling and
urates deposition in ureters).
Dot-ELISA for virus identification
A Dot-ELISA was performed according to [39]. Briefly,
NCM of convenient size was cut, marked with waterproof
ink for identification and then soaked for 10 min. in distilled water. NCM was laid on absorbent paper and airdried for 5 min. Three μl of CAM homogenate of the 4th
virus passage of Egypt/F/03, positive control (CAM
homogenate 48 h after inoculation of H120 vaccine) and
negative control (normal CAM homogenate) samples
were applied as small spots on the membrane. The dotted
membrane was allowed to air dry for 15 min then blocked
for 30 min. in Tris buffer (20 mM Tris base, 500 mM NaCl
pH 7.5) containing 0.5% Tween 20 then rinsed for 5 min.
in Tris buffer. NCM was then incubated for 1 h with rabbit
anti-IBV (prepared previously in our lab.) predilluted to
1:10 with diluents buffer (Tris buffer containing 0.05%
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Table 2: Protection of chickens following vaccination with IBV H120 strain and challenged with Egypt/F/03
Tracheal Protection
GroupA
nB
Vaccina Challen
tion
geC
Virus ReisolationD
Kidney Protection
Tracheal
HistopathologyE
Kidney histopathology
Immunoflourscence
Positive
A
B
C
12
5
12
+
+
+
+
-
Negativ
e
Positive
Negati
ve
Normal
Focal
Multifocal
Diffuse
Positive
Negative
IntensityF
5
5
0
7
0
12
4
5
0
8
0
12
9
0
12
3
3
0