Document Type : Research article
Author
Department of Microbiology and Parasitology, College of Veterinary Medicine, King Faisal University, Saudi Arabia, Fax: 009665896617; Bo-Box: 31982
Abstract
Keywords
Main Subjects
Assiut University web-site: www.aun.edu.eg
MOLECULAR CHARACTERIZATION OF AVIAN INFLUENZA VIRUS- H9N2 SUBTYPE FROM BROILER CHICKEN IN THE EASTERN REGION OF SAUDI ARABIA 2012 to 2014
ABDULLAH I.A. ALMUBARAK
Department of Microbiology and Parasitology, College of Veterinary Medicine,
King Faisal University, Saudi Arabia, Fax: 009665896617; Bo-Box: 31982
Received: 30 September 2018; Accepted: 28 October 2018
ABSTRACT
Avian Influenza Virus (AIV)-H9 subtype was reported to be endemic in Asia and Middle East. It induces considerable economic losses in poultry industry and was involved in human infection. In the present study, attempts were made to estimate the RT-PCR-based prevalence of Avian Influenza Virus-H9N2 subtype in the Eastern Region of Saudi Arabia during the period from January 2012 to March 2014. Tissue samples were collected from 115 flocks of broiler chicken from targeted region during the study period. Part of the Hemagglutinin (HA) gene was directly sequenced to determine circulating genotype. Samples from four flocks were positive to AIV-H9 subtype. Sequencing and phylogenetic analysis of the four detections showed high nucleotide identity to each other and to previous AIV-H9N2 isolates from Saudi Arabia, UAE and Israel. The four detections belong to G1 lineage of the H9N2 subtype. AIV-H9 subtype seems to be of low prevalence in broiler chicken of the Eastern region of Saudi Arabia. Further studies to determine biologic and pathologic characterizations of these detections and to determine prevalence of other AIV subtypes in Saudi Arabia are required to build control and prevention strategy and to minimize the threat it pose to public health.
Key words: Avian Influenza, H9N2, Eastern Saudi Arabia, Broiler Chicken, Phylogenetic Analysis.
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Corresponding author: Dr. ABDULLAH I.A. ALMUBARAK
E-mail address: aialsheikhmubarak@gmail.com
Present address: Department of Microbiology and Parasitology, College of Veterinary Medicine, King Faisal University, Saudi Arabia, Fax: 009665896617; Bo-Box: 31982
HU1c and HU2 primer (shown in table 1) that previously described by (Banks et al., 2000) and target HA gene of H9 subtype was used for detection
Primer sequence 5’ to 3’ |
Annealing temp. |
HU1c/ TATGGGGCATACAYCAYCC |
53°C/1min |
HU2/ TCTATGAACCCWGCWGCWATTGCT |
|
During the period from January 2012 to March 2014, a total of 115 flocks of broiler chicken were sampled. Four flocks (3.5%) were positive to AIV-H9 subtype using RT-PCR that targets HA gene. Sequencing and phylogenetic analysis of these four detections showed that it belongs to the G1 lineage as in figure 1. A/Chicken/SA/AH21/13 and A/Chicken/SA/AH29/13 were almost identical (99.8% nucleotide similarity). These two isolates
share, respectively, 98.8% and 99.1% nucleotide similarity with the third isolate A/Chicken/ SA/AC72/13. All of these isolates showed a lower degree of similarity with the fourth isolate A/Chicken/SA/AC9/13 as shown in table 2. Blast search relabeled that the old Saudi isolate A/CK/SA/C-36362/2010 (GB# JX273557.1) is the most similar to A/Chicken/SA/AC9/13with 99.1% nucleotide identity. On the other hand, A/Chicken/ Saudi Arabia/16/2015 (GB# MG051077.1) was the most similar to AH29/13 and AC72/13 with 97.9% nucleotide identities and to AH21/13 with 97.7% nucleotide identity.
Figure 1: Phylogenetic tree, based on partial HA gene sequence, showing relatedness of the present Saudi AIV-H9 detections (tagged with black square) and reference sequences from lineages of H9N2 subtype including locally and regionally circulating strains. Genbank access numbers are shown after strain name.
Table 2: Nucleotide identity of partial HA gene sequence of the present four Saudi AIV-H9N2 detections together with local/regional reference sequences from lineages of H9N2 subtype. Genbank access numbers are shown in brackets.
Sequences (Genbank access number) |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
|
1 |
A/Chicken/SA/AH21/13(MK123386) |
|||||||||||||
2 |
A/Chicken/SA/AH29/13(MK123387) |
99.8 |
||||||||||||
3 |
A/Chicken/SA/AC72/13(MK123384) |
98.8 |
99.1 |
|||||||||||
4 |
A/Chicken/SA/AC9/13(MK123385) |
96.3 |
96.5 |
97.0 |
||||||||||
5 |
A/Chicken/Saudi_Arabia/16/2015(MG051077) |
97.7 |
97.9 |
97.9 |
95.4 |
|||||||||
6 |
A/chicken/Israel/1163/11(JQ973659.1) |
97.2 |
97.5 |
97.5 |
97.2 |
96.3 |
||||||||
7 |
A/avian/Saudi_Arabia/910135/2006(GU050287.1) |
93.5 |
93.8 |
94.2 |
95.4 |
92.6 |
94.0 |
|||||||
8 |
A/chicken/Saudi_Arabia/D-36363/2010(JX273558.1) |
96.5 |
96.8 |
96.8 |
98.4 |
95.6 |
97.5 |
95.1 |
||||||
9 |
A/avian/Saudi_Arabia/910134/2006(GU050279.1) |
93.3 |
93.5 |
94.0 |
95.1 |
92.4 |
94.2 |
97.5 |
94.9 |
|||||
10 |
A/pheasant/UAE/D1521/2011(KC555089.1) |
96.8 |
97.0 |
97.0 |
96.8 |
95.8 |
97.7 |
93.5 |
97.0 |
94.2 |
||||
11 |
A/chicken/Saudi_Arabia/C-36362/10(JX273557) |
96.3 |
96.5 |
97.0 |
99.1 |
95.4 |
97.2 |
95.4 |
98.4 |
95.1 |
96.8 |
|||
12 |
A/Quail/Hong_Kong/G1/97(AF156378) |
86.3 |
86.6 |
86.8 |
87.7 |
85.6 |
86.8 |
90.5 |
87.5 |
90.3 |
86.1 |
88.2 |
||
13 |
A/Duck/Hong_Kong/Y280/97(AF156376) |
83.8 |
84.0 |
84.0 |
84.5 |
82.9 |
84.0 |
87.3 |
84.5 |
87.7 |
83.6 |
85.0 |
89.6 |
|
14 |
A/Duck/Hong_Kong/Y439/97(AF156377) |
80.3 |
80.6 |
80.3 |
80.3 |
79.9 |
80.3 |
81.7 |
80.8 |
81.9 |
81.0 |
80.8 |
83.8 |
83.1 |
Analysis of deduced amino acid
Sequenced region of the HA gene from the present Saudi detections were trimmed to 432 nucleotides (nt. 571 to 1002 according to HA gene of the strain A/Quail/Hong Kong/G1/97, GenBank access # AF156378.1). A segment of 144 amino acids (191-334) were deduced. This region contains part of the receptor binding site and relevant glycosylation sites. The four detections were completely identical over deduced amino acids except one difference at position 282, where Serine (S) present in A/Chicken /SA/AC9/13 while Asparagine (N) present in the other three detections. When compared with A/Quail /Hong Kong/G1/97, G1 prototype of the AIV-H9 subtype, 18 amino acid substitutions were found in this segment with no insertion/deletion mutation.
Analysis of the amino acids at Receptor Binding Site (RBS)
Amino acid residues at receptor binding site of the present detections were compared with that of A/Quail/Hong Kong/G1/97 as shown in table 3.
Residues at position 191, 197, 202, 203, 232, 234, 236 and 237 remain conserved, while change from Glutamic acid to Alanine at position 198, from Aspartic acid to Glycine at position 233 and from Glutamine to Isoleucine at position 235 were occurred.
Extending the comparison to previous sequences retrieved from gulf area showed that other substitutions Threonine, Isoleucine, Valine have occurred at position 198. Similarly, other substitutions occurred at position 235 including Methionine, Leucine, Threonine, Phenylalanine as shown in table 3.
Glycosylation motifs
Over sequenced region, two glycosylation N-X-T/S motifs (Where N=Aspragine, X = any amino acid except Proline, T= Threonine, S= Serine) were found at positions 298-300 and 305-307 while two motifs at positions 206-208 and 218-220 were lost. The former lost due to substitution of Asparagine to Threonine at position 206, while the latter lost due to substitution of Asparagine to Aspartic acid at position 218.
Table 3: Comparison of some HA amino acids between the present Saudi detections and earlier detections reported by previous studies from gulf area. Only amino acids relevant to receptor binding are shown.
Name of isolate |
Genbank access # |
Receptor Binding Site |
Receptor left pocket |
Glycosylation motifs |
||||||||
Number of amino acid according to A/Quail/Hong Kong/G1/97, Genbank access # AF156378 |
191 |
197 |
198 |
201 |
202 |
203 |
232-237 |
206-208 |
218-220 |
298-300 |
305-307 |
|
H3 numbering according to Kandeil and others (2014) |
183 |
189 |
190 |
193 |
194 |
295 |
224-229 |
198-200 |
210-212 |
290-292 |
297-299 |
|
A/Quail/Hong Kong/G1/97 |
AF156378 |
H |
T |
E |
N |
L |
Y |
NDLQGR |
NDT |
NRT |
NST |
NIS |
A/chicken/Saudi Arabia/CP7/1998 |
CY081264.1 |
. |
. |
. |
S |
. |
. |
NGQQGR |
TDT |
NRI |
NST |
NIS |
A/chicken/United Arab Emirates/AG537/99 |
AJ781824.1 |
. |
. |
A |
. |
. |
. |
NGQQGR |
TDT |
NRT |
NST |
NIS |
A/chicken/Saudi Arabia/AG516/2000 |
AJ781826.1 |
. |
. |
A |
. |
. |
. |
NGLQGR |
TDT |
NRI |
NST |
NIS |
A/quail/Dubai/301/2000 |
EF063510.1 |
. |
. |
. |
. |
. |
. |
NGLQGR |
TDT |
NRT |
NST |
NIS |
A/quail/Dubai/303/2000 |
EF063512.1 |
. |
. |
A |
D |
. |
. |
NGQQGR |
TDT |
NRT |
NST |
NIS |
A/chicken/Dubai/339/2001 |
KF188352.1 |
. |
. |
A |
. |
. |
. |
NGLMGR |
TDT |
NRT |
NST |
NIS |
A/chicken/Emirates/R66/2002 |
CY076723.1 |
. |
. |
. |
. |
. |
. |
NGQLGR |
TDT |
NRT |
NST |
NIS |
A/chicken/Saudi Arabia/EPD-22-01/2002 |
GU050554.1 |
. |
. |
A |
. |
. |
. |
NGLQGR |
TDT |
NMI |
NST |
NIS |
A/chicken/Dubai/463/2003 |
EF063516.1 |
. |
. |
A |
. |
. |
. |
NGLLGR |
TDT |
NRT |
NST |
NIS |
A/chicken/Kuwait/9/2004 |
JX273545.1 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/stone curlew/United Arab Emirates/1147/2005 |
KF188337.1 |
. |
. |
T |
. |
. |
. |
NGQIGR |
TDT |
DRT |
NST |
NVS |
A/stone curlew/United Arab Emirates/1127.3/2005 |
KF188371.1 |
. |
. |
V |
. |
. |
. |
NGQIGR |
TNT |
DRT |
NST |
NVS |
A/white bellied bustard/ United Arab Emirates/ 1036/2005 |
KF188236.1 |
. |
. |
A |
. |
. |
. |
NGQTGR |
TNT |
DRT |
NST |
NVS |
A/white bellied bustard/ United Arab Emirates /1127.1/2005 |
KF188244.1 |
. |
. |
A |
. |
. |
. |
NGQIGR |
TDT |
DRT |
NST |
NVS |
A/white bellied bustard/ United Arab Emirates/1019/ 2005 |
KF188258.1 |
. |
. |
A |
. |
. |
. |
NGQTGR |
TDT |
DRT |
NST |
NVS |
A/quail/United Arab Emirates /1136/2005 |
KF188254.1 |
. |
. |
A |
. |
. |
. |
NDQTGR |
NDT |
DRT |
NST |
NVS |
A/chicken/Saudi Arabia/ 582/ 2005 |
JX273556.1 |
. |
. |
T |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/avian/Saudi Arabia/ 910135/ 2006 |
GU050287.1 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/avian/Saudi Arabia/ 910136/ 2006 |
GU050295.1 |
. |
. |
T |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/chicken/Saudi Arabia/E-36364/2006 |
JX273559.1 |
. |
. |
V |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/houbara/United Arab Emirates/78/2006 |
KF188329.1 |
. |
. |
T |
. |
. |
. |
NGLFGR |
TDT |
DRT |
NST |
NIS |
A/falcon/United Arab Emirates/897/2007 |
KF188240.1 |
. |
S |
I |
D |
. |
. |
NGQIGR |
NDT |
DRT |
NST |
NVS |
A/chicken/United Arab Emirates/F1P7/2011 |
JX273562.1 |
. |
. |
A |
. |
. |
. |
NGLLGR |
TDT |
DRT |
NST |
NIS |
A/white bellied bustard/ United Arab Emirates/ D1520/2011 |
KC555081.1 |
. |
. |
V |
. |
. |
. |
NGLFGR |
TDT |
DRT |
NST |
NIS |
A/quail/United Arab Emirates /D1556/2011 |
KC555097.1 |
. |
S |
I |
Q |
. |
. |
NGQFGR |
NDT |
DRT |
NST |
NVS |
A/Chicken/SA/AH21/13 |
MK123386 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/Chicken/SA/AH29/13 |
MK123387 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/Chicken/SA/AC72/13 |
MK123384 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
A/Chicken/SA/AC9/13 |
MK123385 |
. |
. |
A |
. |
. |
. |
NGLIGR |
TDT |
DRT |
NST |
NIS |
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التوصيف الحيوي الجزيئى لفيروس انفلونزا الطيور- النمط الفرعي H9 من قطعان الدجاج اللاحم في المنطقة الشرقية بالمملکة العربية السعودية خلال الفترة2012 الى 2014
عبد الله إبراهيم ال الشيخ مبارک
قسم الاحياء الدقيقة والطفيليات - کلية الطب البيطري - جامعة الملک فيصل - المملکة العربية السعودية
Email: aialsheikhmubarak@gmail.com Assiut University web-site: www.aun.edu.eg
تشير الدراسات الحديثة الى ان فيروس أنفلونزا الطيور - النمط الفرعي AIV-H9 مستوطن في فطعان الدواجن في قارة آسيا والشرق الأوسط ويسبب خسائر اقتصادية لصناعة الدواجن اضافة الى خطره على الصحة العامة. هدفت هذه الدراسة الى تقدير مدى انتشار هذا النمط في قطعان الدواجن اللاحمة في المنطقة الشرقية من المملکة العربية السعودية باستخدام تفاعل البلمرة المتسلسل. لتحقيق هذا الهدف تم جمع عينات نسيجية من 115 قطيع من قطعان الدواجن اللاحمة خلال الفترة من يناير 2012 الى مارس 2014. تم تحديد الشفرة الوراثية لجزء من جين الملزن الدمويHemagglutinin (HA) للتعرف على النمط الجيني المنتشر في المنطقة المستهدفة. وجدت الدراسة ان العينات الماخوذة من اربعة قطعان کانت ايجابية للنمط الفرعي AIV-H9، فيما کشف تحليل الشفرة الوراثية لفيرسات هذه العينات وجود درجة عالية من التشابه فيما بينها وکذلک تشابهها مع العزلات السابقة من المملکة العربية السعودية والامارات العربية المتحدة واسرائيل وانتمائها الى السلالة G1 لهذا النمط. خلصة الدراسة الى ان النمط الفرعي AIV-H9 ذو انتشار محدود في الدجاج اللاحم في المنطقة المستهدفة. توصى الدراسة باجراء المزيد من الأبحاث لتوصيف الخواص الحيوية والامراضية هذا الفيرس ولتحديد مدى انتشار الانماط الفرعية الاخرى لفيرس انفلونزا الطيور في المنطقة الشرقية وبقية مناطق المملکة العربية السعودية لما لذلک من اهمية في بناء برامج السيطرة على هذه الفيروسات والحد من اثرها على صناعة الدواجن وعلى الصحة العامة.