Presence of Infectious Bronchitis Virus Strain CK/CH/LDL/97I in the Middle East

Infectious bronchitis virus (IBV) is a very dynamic and evolving virus, causing major economic losses to the global poultry industry. In early 2011, respiratory disease outbreaks were investigated in Iraq, Jordan, and Saudi Arabia. Five IBV isolates (JOA2, JOA4, Saudi-1, Saudi-2, and Iraqi IBV) were detected by diagnostic-nested nucleocapsid RT-PCR. Strain identification was characterised by sequencing and phylogenetic analysis of the amplified hypervariable region of the spike 1 (S1) gene. These five IBV isolates were found to be of the IBV strain CK/CH/LDL/97I. Nucleotide identity between these five IBV isolates ranged from 96.9% to 99.7%, and between these isolates and the CK/CH/LDL/97I strain in the range of 96.6–99.1%. The sequenced fragment of the S1 gene of the CK/CH/LDL/97I strain had less than 80% nucleotide identity to the IBV vaccine strains commonly used in the Middle East (M41 and H120). The presence of these CK/CH/LDL/97I-like strains may account for vaccination failure against IBV, since all IBV isolates were from vaccinated chickens. In this paper, we documented for the first time the presence of IBV strain CK/CH/LDL/97I in the Middle East. This strain is known to have originated in China and Taiwan.


Introduction
Infectious bronchitis virus (IBV) is a positive sense singlestranded RNA virus, belonging to the genus Coronavirus, group 3 (gamma coronaviruses) [1]. Infectious bronchitis virus is not only an important pathogen of the respiratory system, but also can be nephropathogenic and cause infection of the reproductive system [2][3][4]. IBV was first recognised as avian respiratory pathogen in 1930, after that many IBV vaccines were introduced to tackle this problem (H52, H120, M41, 4/91(793/B), and other strains). Recently, different IBV variants have emerged causing nephropathogenic and reproductive problems which require a dramatic change in vaccination programmes [5,6].
IBV strain CK/CH/LDL/97I was first reported in China in 1995 [7]. This strain was first implicated in proventriculitis in chickens [8], but in recent studies, this strain has been isolated from the trachea of infected chickens [9]. IBV CK/CH/LDL/97I strain accounts for 3.2% of total IBV strains found in China in the last 15 years [9]. The spike glycoprotein gene is the most variable gene in the IBV genome [10] and is composed of S1 and S2 subunits. Spike subunit 1 (S1) is about 1644 nt in length. The S1 protein is highly variable; it can differ from 20% to 25% and even up to 50% in the amino acid sequence among IBV serotypes [11]. This variability makes the S1 gene an ideal target in molecular assays to type IBV strains by RT-PCR and sequencing. The level of homology of the S1 subunit or part of it can predict cross-protection, that is, the higher the homology, the higher the chance of cross-protection [12,13], but this rule is not always fulfilled [3]. Heterologous protection against the CK/CH/LDL/97I strain has not been achieved, since the nucleotide and amino acid homology of the CK/CH/LDL/97I S1 gene is only around 79% compared to the IBV vaccine strains. Only homologous protection has been achieved against CK/CH/LDL/97I [7].
In the early months of 2011 we noticed IBV outbreaks in spite of massive vaccination, this was due to the presence of a new strain of IBV. In this study, we documented the presence of the IBV strain CK/CH/LDL/97I for the first time in three Middle Eastern countries.

Tissue
Samples and Viral RNA Extraction. Trachea, kidney, ovarian tissues, and cecal tonsils samples were collected from suspected IBV outbreaks in Iraq, Jordan, and Saudi Arabia. These samples were stored at −70 • C in RNAlater solution (Qiagen, Germany) until RNA extraction was performed. Homogenised tissues were subjected to viral RNA extraction using the Viral Gene-spin Viral DNA/RNA extraction kit (Intron Biotechnology, Korea). Viral RNA extraction was performed according to the manufacturer's instructions. Viral RNA was stored at −70 • C until analysis by RT-PCR.

RT-PCR (Diagnostic and Phylogenetic).
The reversetranscription (RT) step was performed using an RT system (Promega, USA). Briefly, viral RNA was denatured at 70 • C for 10 minutes, followed by the addition of a reaction mix including 4 µL MgCl 2 , 2 µL reverse transcription 10x buffer, 2 µL dNTP mixture (10 mM), 0.5 µL random primers, 0.75 µL AMV reverse transcriptase enzyme, 1 µg RNA, and nuclease-free water to a final volume of 20 µL. Then, the reaction was incubated at 42 • C for 60 minutes, then 94 • C for 5 minutes. The cDNA was diluted up to 100 µL with nuclease-free water for PCR amplification. Two RT-PCR assays were used. First, a diagnostic-nested RT-PCR assay based on the amplification of the nucleocapsid (N) gene. IBV-specific oligonucleotides for the N gene were obtained according to a published primers sequences [17]; the IBV primers sequences are presented in Table 1. The diagnostic N gene amplification was done in a final volume of 25 µL GoTaq Green master mix (Promega, USA) in which the final concentration of the N gene primers was 0.5 mmol. RT-PCR amplification was performed with a thermal profile of 94 • C for 45 sec, 60 • C (first step) and 53 • C (second step) for 1 min and 72 • C for 2 min for 40 cycles.
Next, a phylogenetic RT-PCR was performed using nested spike gene primers according to published protocols [4,18]. The amplification profile was the same as that used for N gene-nested RT-PCR, but the annealing temperature for both RT-PCR steps was 50 • C. Amplification was performed using the GenePro thermal cycler (Bioer, China). Nested S1 gene RT-PCR products were sequenced using an ABI Prism 310 genetic analyser at the Princess Haya Biotechnology Centre (Jordan University of Science and Technology, Jordan). Sequences were aligned using BioEdit (7.0.5.3) and MUSCLE (3.7) software. Aligned sequences were used for phylogenetic analysis. Maximum likelihood (ML) phylogenetic analysis with bootstrap values for n = 100 replicates was performed using the PhyML phylogenetic interface [19]. The nucleotide sequence identities were prepared using CLUSTAL W in the MEGALIGN Programme of the Lasergene software.

Results
Five IBV isolates were obtained in early 2011. Two IBV isolates were from Jordan (JOA2 and JOA4), two were from Saudi Arabia (Saudi-1 and Saudi-2), and one isolate was from Iraq (Iraqi IBV). The Jordanian isolates were isolated from layer farms at their peak of egg production, the Saudi strains were obtained from broilers and the Iraqi IBV isolate was obtained from a breeder farm as shown in Table 2. According to the clinical and gross examination coupled with the RT-PCR results from the different tissues (trachea, kidney, ovarian tissues, and cecal tonsils), these isolates had an extensive tissue tropism, different than the CK/CH/LDL/97I strain, which has tropism only for the respiratory system. The clinical signs ranged from respiratory to reproductive symptoms; two isolates (JOA2, Iraqi IBV) were implicated in kidney pathology in affected birds.
All five IBV isolates (JOA2, JOA4, Saudi-1, Saudi-2, and Iraqi IBV) were found to be of the IBV strain CK/CH/LDL/97I and therefore they are CK/CH/LDL/97Ilike strains. The CK/CH/LDL/97I strain is known to be endemic in China and Taiwan. The nucleotide identity between these five IBV isolates ranged from 96.9 to 99.7% with the Saudi-2 isolate being the most divergent. The identity of relatedness between the isolated IBV strains, CK/ CH/LDL/97I, and the related strain CK/CH/SCYA/10I [20] is shown in      identity to the CK/CH/LDL/97I and CK/CH/SCYA/10I strains, respectively.
A phylogenetic tree was constructed based on the alignment of the partial S1 sequence. The five IBV isolates were aligned with other reference and related IBV strains as shown in Figure 1. It showed that all five IBV isolates were grouped with CK/CH/LDL/97I, CK/CH/SCYA/10I, J2, and Q1 (both J2 and Q1 were isolated from the proventriculus) [8] in one group. The nucleotide identity of these five isolates to the IBV vaccine strains in the three countries (M41, H120, and 4/91) were in the range of 78-82.1%, to variant 1 IBV strains in the range of 78.9-81.5%, to variant 2 IBV strains in the range of 81.7-83.6%, and to the IS/885 and Sul/01/09 strains in the range of 78.3-80.9% (data not shown).
Also, these IBV isolates represent a group distinct from the LX4 IBV type and from QXIBV and IS/1201 (a Middle Eastern strain) related to it. These strains share only 79% nucleotide sequence identity. The lowest nucleotide identity was seen with the DEL072 strain (58%) and with the N1/88 strains (63%) from Australia.

Discussion
In this study, the CK/CH/LDL/97I-like strains were isolated from three different Middle Eastern countries (Jordan, Saudi Arabia, and Iraq) in 2011. The CK/CH/LDL/97I-like strains were isolated from broilers in Saudi Arabia, layers in Jordan, and from breeders in Iraq. The CK/CH/LDL/97I-like strain was first isolated in China in 1995 [7]. This strain was isolated from the tracheas of infected chickens [9]. The IBV CK/CH/LDL/97I strains showed slight variability in the partial S1 sequence among IBV isolates from these three countries. The mode transmission of this strain from China to the Middle East is not clear; one reason might be that Chinese poultry vaccines and products have recently been registered and used in the Middle East.
All isolates came from flocks vaccinated with Masstype H120, and 4/91 vaccines, which implies insufficient protection against these isolates. The poor relationship in the partial S1 sequence between the five IBV isolates and the vaccine strains (average nucleotide identity of 80%) could explain the failure of the Mass 41, H120, and 4/91 vaccination programmes to control IBV in these flocks [5]. There is no solution to tackle infection coming from these five IBV isolates except to make a homologous vaccine against this strain. Homologous vaccine was made for the IBV, CK/CH/LDL/97I strain found in China, for IBV QX strain and a nephropathogenic IBV strain in Korea [7,21,22].
In summary, this is the first paper indicting the presence of the IBV CK/CH/LDL/97I strain outside mainland China and Taiwan (in three Middle Eastern countries). In the near future, we expect that this strain might represent a serious problem for the poultry industry and there will be an urgent need to develop a homologous vaccine against this strain.