MOLECULAR ANALYSIS OF AVIAN INFECTIOUS BRONCHITIS VIRUS IN QALYOUBIA GOVERNORATE DURING 2017-2018

Assiut University web-site: www.aun.edu.eg

MOLECULAR ANALYSIS OF AVIAN INFECTIOUS BRONCHITIS VIRUS IN QALYOUBIA GOVERNORATE DURING 2017-2018

HEMAT S. EL-SAYED ¹ and ALI M. ZANATY ²

¹ Poultry DiseasesDepartment, Animal Health Research Institute, Benha Branch, Benha, Egypt

² Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research

 Institute, Dokki, Giza, Egypt

Received: 31 March 2019;     Accepted: 30 April 2019

INTRODUCTION

Avian Infectious Bronchitis Virus (IBV) is a member of genus Gammacoronavirus, family Coronaviridae, order Nidovirales (De Groot et al., 2011 and King et al., 2011). Avian infectious bronchitis is one of highly contagious infectious diseases affect chicken with significant economic impact on poultry industry. Commonly, Respiratory manifestation, renal disorders, drop in egg production and both poor egg quality and quantity were seen and associated with IBV infection in chicken (Cavanagh, 2007). Some strains showing special tropism to proventriculus (Yu et al., 2001) and this feature was recorded in strain called Egypt/Qal/014p isolated from 14-day-old broilers from Egypt (EL-Nahas et al., 2017).

The  IBV genome harbors of  a single  positive-stranded    RNA    of   approximately  27.6 kb in size

Corresponding author: Dr. Hemat S. EL-Sayed

E-mail address: hemat.elsayed@yahoo.com

Present address: Poultry DiseasesDepartment, Animal Health Research Institute, Benha Branch, Benha, Egypt

and encodes for four main structural proteins including, the phosphorylated nucleoprotien (N) protein, the membrane (M) glycoprotein, small envelope (E) protein and the spike (S) glycoprotein. The spike (S) glycoprotein, an integral membrane protein, is a major structural protein; it is cleaved post translationally into the S1 (N terminal part) and S2 fragments (Masters, 2006). The S1 subunit contains the receptor binding site and thus plays a pivotal role in tissue tropism and inducing protective immunity (Wickramasinghe et al., 2011; Belouzard et al., 2012). In the S1 gene, three HVRs located within amino acids 38­67 (HVR­1), 91­141 (HVR­2) and 274­387 (HVR­3) that contribute in  induction of both  neutralizing and serotype specific antibodies (Cavanagh et al., 1988 and Moore et al., 1997). Genotyping of IBV based on S1 gene sequencing, especially the HVRs, is the most reliable way to classify IBV isolates. Usually IBV serotypes have a wide range of genetic variations in the S1 gene ranged from 2% to 25% (Cavanagh et al., 1992; Kingham et al., 2000). In Egypt, many strains of live attenuated and inactivated vaccines were used to control IBV. However, the outbreaks of the disease have continued to cause severe infections (Abd El Rahman et al., 2015). Different types of mutation including insertions, deletions, point mutations, substitutions and /or recombination of S1 gene are the main factors for emergence of new IBV variants (Alvarado et al., 2005 and Carstens,  2009).

IBV strains have been reported in chickens worldwide (Jackwood, 2012). Since the first report of isolation of IBV in Egypt in 1950 (Ahmed, (1954), ongoing scientific attention was directed in several publications for isolation and/ or detection of IBV strains from poultry farms in Egypt. Earlier IBV genotypes related to Massachusetts, D3128, D274, D08880 and 4/91 were circulating  in poultry farms with nephropathy (Abdel-Moneim et al., 2006 and Jackwood et al., 2012) and with the continuing detection of IBV strains, different variants closely related to the Israeli variant strain were identified including Egyptian variant I (Egypt/Beni-Seuf/01) in 2001 (Abdel-Moneim et al., 2002) and Egyptian variant II (CK/Eg/BSU-2,3/2011) in 2012 (Abdel-Moneim et al., 2012). Moreover, IS/885/00 and IS/1494/06 were dominant variants circulating in different Egyptian poultry farms causing respiratory and renal disorders  (Meir et al., 2004; El-Mahdy    et al., 2012; Selim et al., 2013). The continous circulation of different variants of IBV in majority of farms all over Egypt even in vaccinated ones with different strains (H120, MA5 and 4/91) was dramatically complicated the situation in poultry sector and unfortunately, allow to emerge new mutants with risk of IBV vaccination failure (Zanaty et al., 2016). Our study was a contribution in IBV surveillance in Qalyoubia which represent one of the provinces that characterized by the intensity of poultry production in Egypt for updating and monitoring the most important molecular changes in currently circulating IBV isolated form poultry farms through  the partial sequence of S1 gene during a period between 2017-2018.

MATERIALS AND METHODS

Samples collection

In the present study, samples were collected from broiler flocks range from 18 to 36-day-old and suffering from severe respiratory signs from twenty different farms located in AL-Qalyoubia Governorate during the period from January 2017 till December 2018. The chickens were vaccinated with H120 vaccine against IBV at one day of age. Chickens showed respiratory signs such as gasping, coughing, sneezing and tracheal rales with white diarrhea in some cases. Necropsy showed mild to severe tracheitis with congested lung. In addition, birds were suffering from kidney lesions such as enlargement and congestion. The samples were collected as pooled homogenate from trachea and kidneys representing 20 farms were collected from diseased birds and freshly dead birds, stored in sterile Falcon tubes containing transport media with antibiotics. The samples were transported to the Reference Laboratory for veterinary Quality control on Poultry production (RLQP), Animal Health Research Institute, Dokki, Giza, Egypt., in ice box and stored at -80 °C until use.

Virus detection and isolation

Viral RNA was extracted directly from the samples by using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the kit manufacturer’s instructions. The virus identification was confirmed by real­time reverse transcription­polymerase chain reaction (rRT-PCR) for the presence of the neucloprotein (NP) gene of the IBV using Quantitect probe RT­PCR kit (Qiagen, Hilden, Germany), with specific primers and probe targeting the NP gene (Callison et al., 2001).

For Viral isolation, samples from trachea and kidneys were prepared as 10% tissue suspensions in transport medium (phosphate-buffered saline) (pH 7.4), after three cycles of freezing and thawing the suspension was clarified by centrifugation at 3000 rpm for 10 min and filtered through 0.45 micron membrane filters (Millipore) before inoculation in 10 day-old embryonated SPF eggs via allantoic sac route. Three passages were performed and characteristic embryo changes such as dwarfing, stunting, curling of the embryos, subcutaneous hemorrhage and ureate deposition in the mesonephros were observed between post-inoculation days 2 and 7 (OIE, 2013). The harvested allantoic fluids from each sample were screened using rRT-PCR for further confirmation.

Sequencing of the S1 gene

Screening was further tested using a specific primer set for the amplifications of the HVR-3 of the S1 gene using Qiagen one­step RT­PCR (Qiagen, Hilden, Germany), according to (Adzhar et al., 1997). Amplificates of 400 bp were excised and purified from gels using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The purified RT­PCR products were sequenced using Bigdye Terminator V3.1 cycle sequencing kit (Perkin­Elmer, Foster City, CA) and Applied Biosystems 3130 genetic analyzer (ABI, United States).

Phylogenetic analysis

 Sequences similarities and relationships of the HVR-3 of the S1 gene of positive samples in the present study were compared with previously published IBV vaccine and reference strains available in the public database (NCBI, United States). Amino acids phylogenetic tree was drawn for the sequenced isolates along with other vaccine and reference strains available in the GenBank database using MEGA program version 6 (Tamura et al., 2013). A comparative analysis of deduced amino acids and nucleotide sequences of the HVR­3 was created using the CLUSTAL W Multiple Sequence Alignment Program, version 1.83 of MegAlign module of Lasergene DNAStar software (Ziegler et al., 2002). Sequences generated in the frame of this study were submitted to the GenBank database with accession numbers showed in Table 1.

RESULTS

Clinical reports of examined farms:

The main complaints of the majority of examined farms were summarized in the following signs, high mortalities up to 30%, ruffled feathers, depression, loss of appetite, mild to severe respiratory signs in form of difficult breathing, tracheal rales, gasping, sneezing and coughing. White diarrhea in some cases was also observed. The most prominent findings at necropsy were mild to severe tracheitis with congested lung, in addition to swollen and pale kidneys and in some cases the tubules and ureters filled with ureate.

Virus detection and isolation

IBV was detected in examined samples in 12 out of 20 farms with percentage of (60%) using rRT-PCR (Fig. 1). Virus isolation was obtained from the homogenate pool of the trachea and kidneys from each flock. The allantoic fluid from the 3rd passage of each sample was further confirmed positive using rRT-PCR.

Genetic and phylogenetic characterization

The 400 bp amplified PCR products of the HVR-3 of S1 gene were obtained from the 5 positive selected samples (Fig. 2), and then the partial sequencing of HVR­3 and sequence analysis was conducted. Phylogenetic tree was constructed from the amino acid sequences of HVR­3 of the S1 protein. The results indicated that Egyptian IBV viruses in our study found to be closely related to the variant II isolates and were clustered within the Egy/Var­Ⅱsubgroup (IBV-Eg-12120s-2012 and IBV/IS/885­00) and other Egyptian related strains in the GenBank (Fig. 3).

Alignment report of the S1­HVR3 for both nucleotide and deduced amino acid were performed and compared with the previously published reference and vaccine IBV strains commonly used in the field (H120, Ma5, D274, 4/91,1/96 and CR88121) as well as original Egyptian viruses of Egy/Var­Ⅰ and Egy/Var­Ⅱ. In comparison to vaccine strains used in Egypt, Egy/ Var­Ⅱ strains isolated in this study showed identity from 75% to 80% for vaccinal strains H120, Ma5, 4/91, CR88 and D274 (Table, 2). In the meantime, strains shared 90% to 98% amino acid with each other according to sequence of HVR3 (Table, 2)

Table 1: Data  including time of collection, ages of birds and accession no. of Infectious bronchits virus in the present study.

Fig. 1: Amplification curve for IBV (N) gene rRT- PCR.

Fig. 2: Gel electrophoresis shows the result of PCR products of five selected isolates.

                  1 - Ladder

2 - Negative control

3 - Sample 1

4 - Sample 2

5 - Sample 3

6 - Sample 4

7 - Sample 5

Fig. 3: Phylogenetic tree representing the partial amino acid sequences of the S1 gene for 5 avian infectious bronchitis virus isolates (marked with black circle) with other related infectious bronchitis virus and vaccine strains.

Majority                        IVYRENSVNTTLVLTNFTFTNESNASPNTGGVQTIQLYQTQTAQSGYYNFNFSFLSSFVYKPSDFMYGSYHPSCNFRPETINNGLWFNSLSVSLAYGPLQ

                    ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+

10        20        30        40        50        60        70        80        90        100

                    ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+

IBV-IB-VAR2-06-                 VVYRETSVNTTLVLTNFTFTNVSNASPNTGGVNTINIYQTQTAQSGYYNFNFSFLSSFVYKQSDFMYGSYHPKCDFRPETINNGLWFNSLSVSLAYGPLQ       298

IBV-attenuated-IS-1494-06-spike ....................................................................................................       298

Avian-IBV-variant-1-S1          I....SGT....K....S.......P..S...D.FQL...H...D......L.........P..........H.N....N..............T...I.       298

IBV-EGY-F-03                    I....N.....FT.HY.S.H.ETG.N..PS..QN.QT........................E.N........S.N..L...............I......       298

IBV-Eg-CLEVB-1-IBV-012          ....PK.T...IS.F..L..................................................................................       298

Avian-IBV-IS-885-S1-spike       .....N.F...........Y.E......V.......L...H........Y.L....G....A......................................       298

IBV-(strain-D207)-              I....S......E......S.....T......Q..QL...S........L........I..A..Y.......S.K.KL................G...I.       298

IBV-Ma5-SP1                     I....N.....FT.H....H.ETG.N..PS..QN.QT........................E.N........S.N..L...............I......       298

IBV-Connecticut                 I....N.I....K.H....H.ETG.N..LS..QN.QT...................G.F..E.N........S.N..................I......       298

IBV-M41                         I....N.....FT.H....H.ETG.N..PS..QN.QT........................E.N........S.N..L...............I......       298

IBV-D274                        I....S......E......S.....T......Q..QL...S........L........I..A..Y.......S.K..L................G...I.       298

IBV-QXIBV-SP1                   I....S......A............Q..S.....FHL..............L....Q....A..........S.S.......S...........T.....       298

IBV-UK-4-91                     I....S.T....E...............S...D.FQL...H...D......L.........P..........N.N....N..............T...I.       298

IBVCR88121                      I....S.T....K....S..........S...D.FQL...S...D......L.........P..........H.K....N..............T...I.       298

IBV-H120-                       I....N.....FT.H....H.ETG.N..PS..QN.QT........................E.N........S.N..L...............I......       298

IBV-EGY/QAL-1-2017             .....N......G......Y.E...H..N...H..RL...H...........TLM..V...P..Y.......Q...........................        298

IBV-EGY/QAL-2-2017             .....N......G...Y..H.E...H..N...H..SL...H.......K...TL.......P..L.......Q.N....................F....        298

IBV-EGY/QAL-3-2018             .....N......G...Y..Y.E...H..N...H..SL...H.......K...TL.......P..L.......Q.N.........................        298

IBV-EGY/QAL-4-2018             .....N......G..K...Y.E...H..N...H..RL...H...........TL.......P..N.......Q...........................        298

IBV-EGY/QAL-5-2018             .....N.........K...H.E...H..N...H..SL...H....................P..N.......Q......................F....        298

Fig. 4: Sequence alignment for the amino acids of S1 gene for the 5 isolates of the study; vaccine strains and the reference strains.

Table 2: Nucleotide and deduced amino acid identities of infectious bronchitis virus Egyptian isolates with other selected references and vaccine strains from different serotypes.

DISCUSSION

IBV, one of the important viruses that infect poultry flocks worldwide and recently the new variants topped the scene in Egypt and putting poultry policy makers in the face of a catastrophic crisis that represented in huge economic losses and failure of vaccination. Our study was a contribution to follow up and identify the IBV genotypes circulated in the poultry flocks in Qalyoubia locality. Herein, we examined twenty broilers farms distributed in different centers followed the Qalyoubia governorate, the majority of complaints of examined farms were high mortalities up to 30%, with the inspection of affected birds the most common signs were seen in form of respiratory manifestation including, difficult breathing, tracheal rales, sneezing and coughing. On necropsy, the frequently PM findings were seen in form of mild to severe tracheitis with caseous exudate in some cases, air-sacculitis and swollen, congested and pale kidney with distended ureter with urates.  Similar signs and PM findings were observed in previous study isolated IBV from poultry flocks in Delta region in Egypt during 2014-2015 (Sultan et al., 2017).

The confirm and on-time diagnosis was urgent need specially after increasing incidence of mixed infection in commercial Egyptian poultry farms nowadays. RRT-PCR was rapid, sensitive and specific molecular diagnostic tool for detection and screening of IBV suspect samples (Naguib et al., 2017). In our study the screening conducted on 20 different farms for the presence of the neucloprotein (N) gene sequence of the IBV using rRT-PCR confirmed IBV in 12 farms with percentage of 60%.

Recently IBV variants are widely distributed in many countries all over the world with more than 20 different serotype (Sjaak de Wit et al., 2011). In Egypt, from the available molecular data reported during the few years ago, the IBVs were evolved into two groups classic (GI-1) and variant (GI-23), the last one were subdivided into two subgroups (Egy/Var-Ⅰand Egy/Var-Ⅱ) according to the sequence of the HVR-3 (Abdel-Moneim et al., 2012 and Ganapathy et al., 2015). The viruses clustered to the subgroup Egy/Var-Ⅱ were shown to be widely spread in Egypt in last years (Abo-Elkhair et al., 2012 and Valastro et al., 2016).

The phylogenetic analysis conducted on the selected five IBVs showed that these viruses were related to each other (90-98% identity) according to sequence of HVR-3 and found to be closely related to the variant isolates and were clustered within the Egy/Var­Ⅱsubgroup (IBV-Eg-12120s-2012 and IBV/IS/885­00) and other Egyptian related strains deposited in the GenBank database. These findings referred to the circulation of strains belongs to Egy/Var­Ⅱ in broiler flocks in Qalyoubia during 2017-2018, also our findings were agree with Abdel-Moneim et al., 2012; Sultan et al., 2015; Tatar-Kis et al., 2015; Zanaty et al., 2016 and EL Samadony   et al., 2017) who reported that Egyptian variant II strains were the most dominant strains circulated in Egyptian poultry flocks recently. 

One of our study aims is to compare between the isolated variants circulated in poultry flocks located in Qalyoubia governorate and the vaccine strains currently used for IBV vaccination (H120, Ma5, 4/91, CR88, and D274). Herein, the comparison showed that there was 75% to 80% identity between our isolates and the mentioned vaccine strains which indicate to the wide gap of difference between the isolated viruses and the existing vaccines types which resulted in emerging of new antigenic types that increase the risk of vaccine failure. Similar observation were recorded in previous studies (Samir et al., 2014 and bdel-ELGhany et al., 2015). The small changes detected along the S1 protein lead to genetic alteration and recombination and subsequently evolve new field strains (liu and Kong, 2004), these changes arises from immunological pressure caused by the massive use of live vaccines in presence of infection with field strain (Zanaty     et al., 2016), so it is necessary to evaluate the  protective capabilities of the commonly used vaccine in Egypt as well as preparation of vaccine from indigenous isolates for achieving the successful control of IBV in Egypt (EL-Nahas et al., 2017).

CONCLUSION

The present study confirmedthe detection and isolation of IBVs from broilers flocks in Qalyoubia governorate during 2017-2018, the Egypt/Qal strains were closely related to Egy/Var-Ⅱthat dominant circulated in last years in commercial poultry farms in Egypt. Also the phylogenetic analysis showed the dissimilarities between our isolates and the currently used vaccines for IBV in Egypt. The continuous follow up through surveillance activities and preparation of vaccines from indigenous isolates were necessary issues for successful control of IBV in Egypt.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ACKNOWLEDGEMENT

The authors are grateful to members of Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute, Dokki, Giza, Egypt, for their help in viral isolation and sequencing process.

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