Phylogenetic analysis of avian infectious bronchitis virus circulating in Egypt during 2021-2022

Phylogenetic analysis of avian infectious bronchitis virus circulating in Egypt during 2021-2022

Hamdi Mohamed Sallam¹, Mohamed Rady², Zienab Mosaad³, Mostafa Mohamed Saleh⁴, Ahlam E. Yonis¹, Ali Mahmoud Zanaty³

¹Reference Laboratory for Veterinary Quality Control on Poultry Production, Damanhour Branch, Animal Health Research Institute, Agricultural Research Center.²Reference Laboratory for Veterinary Quality Control on Poultry Production, Fayoum Branch, Animal Health Research Institute, Agricultural Research Center.

³Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agricultural Research Center.

⁴Animal Health Research Institute, MansouraBranch, Agricultural Research Center.

ABSTRACT:

Avian infectious bronchitis virus (IBV) is still the cause of huge economic losses among poultry farms despite extensive vaccination. During this study, clinical samples were collected from four Egyptian governorates in the period 2021-2022. Samples with positive IBV real-time RT-PCR results were subjected to virus isolation on chicken embryos. IBV was identified by RT-PCR that was directed to the S1 gene. Partial sequencing of S1 gene revealed seven strains (HS1-7) that were submitted to GenBank under the accession numbers (OQ349378-OQ349384). Phylogenetic analysis revealed clustering of five strains (HS1-5) with genotype I- lineage 23(GI-23), whereas the other two strains (HS6 and HS7) were clustered with genotype I- lineage 1 (GI-1). Strains HS1-3 were found to have a unique amino acid insertion in hypervariable region 1 (HVR1). GI-23-related (variant) strains shared less than 86.2% and 88.4% nucleotide and amino acid identities, respectively with the commonly used vaccine strains. The closest vaccine strain (EG/1212B) showed 9-15 amino acid substitutions within HVR1 and 2. Although GI-1-related strains shared close relation to Mass like vaccine strains, 5-13 amino acid substitutions were observed within HVR1 and 2. This study reports the identification of genetically distinct IBV strains in different Egyptian governorates with probable impact on the protection attracted by commercial vaccines.

Keywords: Avian infection bronchitis, Phylogenetic, Egypt

INTRODUCTION:

Avian infectious bronchitis virus (IBV), the gammacoronavirus, is the cause of highly contagious economically important viral disease of poultry (Moharam et al., 2020 and Marandino et al., 2022). IBV-infected chickens show respiratory signs (sneezing, coughing, tracheal coarse crackles), pro-ventriculitis, air sacculitis, enteritis, nephritis, and reduction in feed intake (Yu et al., 2001; Cook et al., 2012; Al-Jalladet al.,2020 and Wu et al., 2022).

IBV which belongs to family Coronaviridae, is an enveloped virus with positive sense single-stranded RNA of approximately 27.6 kb in size. This RNA encodes four structural proteins [namely; nucleocapsid protein (N), small envelope protein (E), membrane glycoprotein (M), and spike glycoprotein (S)] (Cavanagh, 2003 and Su et al., 2011).

The S protein is cleaved into amino-terminal (S1) and carboxyl-terminal (S2) glycopolypeptides that are involved in both virus attachment and activation of fusion between viral and host cell membranes in order to viral RNA release (Cavanagh et al., 1988 and Cavanagh, 2007). In addition, to be involved in virus infectivity, S1 protein contains three antigenic epitopes that are associated with three hypervariable regions (HVRs) (encompass aa 38–67, 91–141, and 274–387) that are the targets for neutralizing antibodies and have the highest nucleotide heterogeneities among IBV serotypes and can be used to determine genetic relationship between field strains, vaccines and novel variants (Cavanagh, 2005; Gelb et al., 2005 and Al-Jalladet al., 2020). Moreover, heterogeneities of S1 became the base for genotypic classification of IBV. Emerging new antigenic variant strains can occur due to genetic changes as a result of deletions, insertions, point mutations, or RNA recombination, especially in the S1 gene. In addition, the similarity of S1 gene between the used vaccine and field strains determines the efficacy of vaccination against IBV (Lian et al., 2021). Thus, new outbreaks in vaccinated chicken flocks often occur due to emerging of new variants (Jia et al., 1995; Abdel-Moneim, et al., 2012; Valastro et al., 2016 and Lian et al., 2021). Accordingly, continuous monitoring of genetic changes in S1 gene is important for determination of these new variants and essential for disease control via vaccination. Therefore, this study was carried out to identify the circulating IBV in four Egyptian governorates and to determine its genetic relation with the commonly used vaccine strains.

Materials and methods:

2.1. Samples:

Samples were collected from 30 broiler and five Broiler breeder farms in four Egyptian governorates (Giza, Sharkia, Behera, and Gharbia) during 2021-2022. These chickens suffered from nasal discharge coughing, gasping, sneezing, conjunctivitis, and watery whitish diarrhea. Tracheal swabs, trachea, kidney, and cecal tonsils were collected. The swabs specimens were suspended in 0.5 mL of phosphate-buffered saline (PBS) containing streptomycin and penicillin then vortexed for 20 min. Tissue samples were homogenized in PBS (a suspension of 20% w/v) containing also streptomycin (100μg/mL) and penicillin (100 U/mL)(Lian et al., 2021). Samples were stored at -80 ⁰C until to be examined with RT-PCR.

2.2. Detection of viral RNA by real-time - reverse transcription PCR (rRT-PCR):

RNA of IBV was extracted from samples using QiaAmp viral RNA mini kit (Qiagen, Valencia, Calif., USA), according to the manufacturer's instructions. Extracted RNA was reverse transcribed and amplified using Quantitect probe RT-PCR kit (Qiagen, Valencia, Calif., USA) following the manufacturer's instructions.Briefly, 12.5 µl of 2x Quantitect probe RT-PCR buffer, 4.5 µl of RNase - free water, 0.125 µl of Quantitect probe RT enzyme, 2.25 μl from each primer and 1μl of probe (table 1) were added to 5 µl of RNA template. Real-time PCR machine (Stratagen, MX 3005P, USA) was used to accomplish the reaction with the following thermal profile: 50 °C for 30 min (one cycle), 95 °C for 15 min (one cycle), 94 °C for 15 sec and 60 °C for 40 sec (40 cycles).

Table 1. Primers and probe were used in this study.

2.3. Virus isolation:

Samples with rRT-PCR positive results were inoculated onto chorioallantoic sac of ten-day-old SPF (specific pathogen-free) embryonated chicken eggs. Embryos were checked for seven days post inoculation (PI) and that died within 24 hrs (PI) were discarded. The allantoic fluid was harvested for viral RNA identification and sequencing (Momayez et al., 2002).

2.4. Identification of IB viral RNA by RT-PCR:

Verso 1-Step RT-PCR ReddyMix Kit (Thermo Fisher Scientific, USA) was used for partial amplification of S1 gene of IBV according to the manufacturer's instructions. Briefly, 25 μl of 1-Step PCR ReddyMix buffer, 2.5 µL of RT enhancer, 1 μl of Verso Enzyme Mix, 13 μl of Nuclease-free water, and 2 µl of each primer (table 1), were mixed to amplify 5 µl of RNA template (extracted from allantoic fluid of each isolate)in Applied biosystem 2720 thermal cycler (Germany). The following thermal profile was used: 50 °C for 15 min (one cycle), 95 °C for 2 min (one cycle), 95 °C for 20 sec, 52 °C for 30 sec and 72 °C for one min (40 cycles) then final extension at 72°C for 10 min. On 1.5% agarose gel containing ethidium bromide, PCR products were visualized with 100 bp DNA ladder (Fermentas, Thermo, Germany). 

2.5. Partial sequencing of S1 gene:

QIAquick Gel Extraction Kit (Qiagen Inc. Valencia, CA, USA)was used for purification of DNA fragments that obtained by RT-PCR according to the manufacturer’s instructions. Partial sequencing reaction was performed withsequencing kit (Big dye Terminator V3.1 cycle, Perkin-Elmer, Foster city, CA). Sequence reaction was purified using spin column (Centrisep).  ABI automated sequencer(Applied Biosystems 3130 genetic analyzer, USA) was used for sequencing.

2.6. Phylogenetic analysis:

The obtained nucleotide sequences of S1 gene were subjected to basic local alignment search tool (BLAST) within GenBank database and aligned with reference and vaccine strains using CLUSTALW tool of BioEditsoftware (version 7.2.5). The pairwise distance was determined and Neighbor-joining (NJ) tree was constructed using maximum composite likelihood of MEGA11 software with 1000 bootstrap replications (Tamura et al., 2021).

Table 2. Epidemiological data of the seven IBV isolates.

Table 3. Nucleotide and amino acid identities of the seven tested IBV strains in relation to some vaccine and reference strains.

Table 4. Amino acid substitutions within HVR1 and 2 of the strains of variant groupin relation to some IBVreference and vaccine strains.

Numbers of amino acids are indicated according to strain Mass41 except No. 41, 118 and 119 that are amino acid insertions.

Figure 1. Neighbor-Joining tree based on partial amino acid sequences of S1 gene of the seven tested IBV strains (indicated with red circles) and other vaccine and reference strains.

Figure 2. Unique deduced amino acid substitutions identified in the partially sequenced S1 gene. Green rectangulars indicate amino acid substitutions within HVR1 and 2. Orange rectangulars indicate amino acid substitutions out of HVR1 and 2. Red rectangular indicates amino acid insertion in three strains.

3. RESULTS:

3.1. Clinical examination:

The prevalent clinical signs were nasal discharge, coughing, gasping, sneezing, conjunctivitis, and watery whitish diarrhea.

3.2. Viral Identification and phylogenetic analysis:

Six isolates of IBV (HS1-5 and HS7) were identified in the samples collected from broiler farms in three Egyptian governorates (Giza, Sharkia and Behera) during 2021 and 2022. Additionally, one isolate (HS6) was identified in broiler breeder farm in Gharbia governorate during 2022. All these farms were vaccinated by different types of IBV vaccines (table 2). These isolates shared 70.6%-99.2% nucleotide identities and 61.2%-97.7% amino acid identities. Strains HS1-5 were found to be closely related and clustered within distict subclade which distantly related to the subclade of both HS6 and HS7 that were close to each others (table 3 and figure 1). Nucleotide and amino acid identities of 60.6%-97.2% and 53.45%-95.5%, respectively were identified with the commonly used vaccine strains (table 3).

A unique amino acid insertion was found in HVR1 of strains HS1-3. Moreover, HS1, HS6 and HS7 had an amino acid substitution within the same region. Additionally, within HVR2, a unique amino acid substitution was located in strains HS2 and HS4 and two unique amino acid substitutions in the strain HS7 (table 4 and figure 2).

4. DISCUSSION:

In Egypt, IBVs are usually identified and continue to circulate in commercial poultry production although of vaccination programs that are adopted with several viral strains in many poultry farms. It was suggested that the using of homologous vaccine strains can achieve the best protection against IBV challenge (Ali et al., 2018; Sultan et al., 2019; Erfanmanesh et al., 2020; Houta et al., 2021). Therefore, virus identification from infected chickens should be continuously undertaken in order to accomplish disease control. IBV strains were classified into seven genogroups (GI-GVII) with at least 32 distinct lineages, from which 29 lineages were confined to GI (Houta et al., 2021 and Kariithi et al., 2022). The genotype I- lineage 1 (GI-1) which represents the old Massachusetts (Mass) like strains, is widely distributed in all endemic countries and therefore is most commonly used for vaccine production. On the other hand, the genotype I- lineage 23 (GI-23) that includes IBV variant strains, continues to spread and circulate in the Middle East posing a major challenge the disease control (Houta et al., 2021).

During 2021-2022, samples were collected from chickens that suffered from respiratory signs and/or diarrhea in four Egyptian governorates (Giza, Sharkia, Behera and Gharbia) for identification and molecular characterization of IBV. Phylogenetic analysis that was based on partial sequences of S1 gene (including HVR1 and 2), revealed dividing of the isolated IBV strains into two groups; one (included HS1-5) was clustered with variant strains (GI-23), while the other one (included HS6 and HS7) was clustered with Mass like strains (GI-1). The two groups shared 70.6%-73.2% and 61.2%-66.4% nucleotide and amino acid identities, respectively (table 3). The strains within each group shared high homology of nucleotide and amino acid identities that were interpreted in the amino acid-based neighbor-Joining tree by clustering of these strains together within distinct two monophyletic groups (figure 1). The variant group was clustered with IBV/CK/EG/QENA-18/2018 that was also assigned under a distinct subclade on the bases of sequencing of full-length S1 gene (Sabraet al., 2020 and Houta et al., 2021).

Nucleotide and amino acid identities of 65.1%-86.2% and 57.9%-88.4%, respectively, were estimated between the strains of the variant group and the commonly used vaccine strains, from which EGY/VAR II (EG/1212B) was the closest one. However, 9-15 amino acid substitutions were found between EG/1212B and the strains of the variant group (table 4). Moreover, Strains HS1-3 that were identified from three different governorates (Giza, Sharkia and Behera) during 2021, were found to have the same unique amino acid insertion (leucine) within HVR1 in relation to vaccine strains (figure 2). Meanwhile, Mass like group shared wider ranges of nucleotide and amino acid identities (60.6%-97.2% and 53.4%-95.5%, respectively) with the commonly used vaccine strains, from which Mass like vaccine strains were the closest ones (table 3). However, 5-13 amino acid substitutions were observed between Mass like group and the closely related vaccine strains. Eight and six amino acid substitutions were identified in H120 in relation to HS6 and HS7, respectively, from which five were found within HVR1 and 2 that are the targets for neutralizing antibodies (Cavanagh, 2005; Gelb et al., 2005 Al-Jalladet al., 2020), and that might explain vaccination failure although of close relationship with the identified strain (figure 2).

5- Conclusion:

Collectively, this study indicated the circulation of both variant and Mass like strains of IBV in four Egyptian governorates. These strains were found to have some genetic variations from previously identified strains as well as commonly used vaccine strains. More investigations are needed to study the antigenicity of the tested IBV in relation to currently used vaccine strains to determine the proper protectotype.