Infectious bronchitis virus (IBV) is one of the major poultry pathogens of global importance. However, the prevalence of IBV strains in Malaysia is poorly characterized. The partial genomic sequences (6.8 kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of 11 Malaysian IBVs isolated in 2014 and 2015 were sequenced using next-generation sequencing technology. Phylogenetic and pairwise sequence comparison analysis showed that the isolated IBVs are divided into two groups. Group 1 (IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015) shared 90%–95% nucleotide and deduced amino acid similarities to the QX-like strain. Among these isolates, IBS142/2015 is the first IBV detected in Sarawak state located in East Malaysia (Borneo Island). Meanwhile, IBV isolates in Group 2 (IBS037A/2015, IBS037B/2015, IBS051/2015, and IBS180/2015) were 91.62% and 89.09% identical to Malaysian variant strain MH5365/95 (EU086600) at nucleotide and amino acid levels, respectively. In addition, all studied IBVs were distinctly separate from Massachusetts (70%–72% amino acid similarity) and European strains including 793/B, Italy-02, and D274 (68%–73% amino acid similarity). Viruses in Group 1 have the insertion of three amino acids at positions 23, 121, and 122 of the S1 protein and recombinant events detected at nucleotide position 4354–5864, with major parental sequence derived from QX-like (CK-CH-IBYZ-2011) and a minor parental sequence derived from Massachusetts vaccine strain (H120). This study demonstrated coexistence of the IBV Malaysian variant strain along with the QX-like strain in Malaysia.
Infectious bronchitis (IB) is an economically important, highly contagious, acute disease of chickens and other fowls caused by infectious bronchitis virus (IBV). IBV is a Gammacoronavirus and belongs to Nidovirales order, Coronaviridae family, Coronavirinae subfamily (11,23). IBV is an enveloped virus with a round to pleomorphic shape and has a diameter of approximately 120 nm with club-shaped surface spikes about 20 nm in length. The viral genome is a single-stranded, positive-sense RNA of approximately 27.6 kb in size which encodes the following structural proteins: spike glycoprotein (S), small membrane envelope protein (E), membrane glycoprotein (M), and nucleoprotein (N) located at the last one third of the genome. The genome also encodes nonstructural proteins such as the 3a, 3b proteins (encoded by gene 3) and 5a, 5b (encoded by gene 5) interspersed amongst the structural proteins. Hence, the genome of IBV based on the structural and nonstructural genes is in the following order: -S-3a-3b-E-M-5a-5b-N- (10).
During the virus replication cycle, the S undergoes posttranslational cleavage to form S1 and S2 subunits. Interestingly, the S1 protein is the most variable part of spike glycoprotein with up to 40% amino acid variability among some serotypes (8). Indeed, its complete nucleotide sequences or the hypervariable regions (HVR) are often used in the classification of IBV genotypes (26). In addition, the S1 protein carries neutralizing epitopes for induction of protective immunity in the infected hosts (26). The S2 protein anchors to the virion and contributes to the activities of fusion of S1 protein. The E and M genes encode for proteins involved in virus assembly and budding while the protein N contributes to virus replication, assembly, and induction of cell-mediated immunity (30). In addition to the structural proteins, 3a, 3b, 5a, and 5b encode for nonstructural proteins that are not essential for viral replication; however, they can serve as targets for attenuation of pathogenic IBVs (10,19).
The first IBV isolate was isolated in Massachusetts, United States in the 1930s, hence the name Massachusetts strain IBV (12). Currently, this strain has been reported in many countries across the globe that have major poultry production (21,36). In addition to the Massachusetts serotype, several other serotypes have emerged in different parts of the world (9,21). Among them, two economically important field strains of IBV, namely Italy-02 and QX viruses, were isolated in domestic chickens in Europe and in China, respectively (5,7). The QX serotype was first isolated almost 2 decades ago following an outbreak of the disease in poultry in China (39). However, at present the QX-like strains have become the most-widely distributed serotypes and have been accordingly reported in many European (2,6,14,15,18,37), African (1,16), and Asian countries (28,29,31,33). In addition, a unique virus type in Europe, namely 793/B (4/91), was first reported in 1991 (13). Since then, outbreaks of IBV caused by the 793/B strain have been reported in Eastern Europe, Russia, Turkey, the Middle East, China, Japan, Morocco, and South America (21). The live attenuated vaccines for IBV 793/B strain (4/91, CR88, and 1/96) have been developed and are commercially available in many countries (21).
In Malaysia, there are a limited number of studies on the genetic characterization of IBV. Furthermore, those studies only characterized the partial S1 protein which corresponds to the hypervariable region of the protein. However, there are overwhelming reports that indicate high sequence variation and possible recombination events are not only limited to the S1 gene alone, but other genes such as 3a (3); 5a, 5b, and N (41) may equally be affected. With the advantage of high throughput next-generation sequencing (NGS) technology, it is possible to sequence a large DNA fragment with incredible accuracy and considerable cost using a small amount of purified DNA as a starting material. Indeed, this sequencing technology has dramatically improved the understanding of the genetic diversity of several avian pathogens such as Newcastle disease virus (35), avian influenza virus (24), avian picornbirnavirus (24), avian picornavirus (24), and avian gamma-coronavirus (24). Therefore, in the present study a robust NGS technology was used to obtain the partial genomic sequences (6.8Kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of IBVs isolated in Malaysia between 2014 and 2015. The obtained sequences were analyzed for phylogenetic diversity and evidence of genetic recombination.
MATERIALS AND METHODS
Detection of infectious bronchitis virus by real-time polymerase chain reaction (rt-PCR). Between 2014 and 2015, a total of 135 suspected IB cases from commercial poultry farms at different states in Malaysia were screened for rt-PCR detection of IBV using conserved primers (Table 1) and iQ SYBR Green Supermix (Bio-Rad, Philadelphia, PA).
Primers employed for screening and amplification of the partial genomic sequences of IBVs.
Virus isolation. Clinical samples from the outbreaks of chicken farms positive for IBV (using rt-PCR) were homogenized and used for virus isolation as previously described by the World Health Organisation (OIE, 2013) and Yu et al., (2001) (32,40). Briefly, tissue homogenates (200 μl) were inoculated into the allantoic cavity of 9-day-old embryonated specific-pathogen-free (SPF) eggs. The inoculated eggs were candled daily to check the status of embryos and mortality. Early-death embryos within 24 hr postinoculation (PI) were discarded. Allantoic fluid was harvested on day 3 PI while changes of embryos such as stunting, curling, or both, with feather dystrophy, were recorded on day 7 PI. Then the allantoic fluids of the eggs were pooled and used for further passage into another set of eggs for up to 3–5 passages. Following each passage, the allantoic fluids were used for viral RNA extraction and tested for the presence of IBV nucleic acid by rt-PCR.
Primer designs. Two sets of gene-specific primers were designed using NCBI Primer3 and BLAST tools (National Center for Biotechnology Information [NCBI], https://blast.ncbi.nlm.nih.gov/Blast.cgi; Table 1). The specificity of the designed primers was evaluated by aligning the primers with 56 aligned IBV complete genome sequences including QX, QX-like, 793/B, and other local strains from China, Korea, Taiwan, United States, and Australia using MEGA 6.06 software (http://www.megasoftware.net/).
RNA extraction and reverse-transcription PCR (RT-PCR). Allantoic fluid of IBV-infected SPF eggs at passage 3 to 5, depending on the IBV isolates, was used for viral RNA extraction and sequencing. Indeed, viral RNA was extracted from 400 μl allantoic fluid of infected SPF eggs using Trizol® Reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA) as described by the manufacturer's instructions. RT-PCR was carried out to amplify the partial genomic sequence of IBVs using two set of primers (Table 1). First-strand cDNA was synthesized using an MMLV reverse transcriptase 1st-strand cDNA synthesis kit (Epicentre, Madison, WI). In brief, a 12.5-μl total reaction volume including 5.5 μl of RNase-free water, 5 μl of extracted RNA sample, and 2 μl of 10 μM R2 reverse primer was prepared in a tube and incubated at 65 C for 2 min in a thermocycler with a heated lid. After incubation, the mixture was chilled on ice for 1 min, and 2 μl of MMLV RT 10× reaction buffer, 2 μl of 100 mM DTT, 2 μl of dNTP Premix, 0.5 μl of RiboGuard RNase inhibitor (Epicentre), and 1 μl of MMLV reverse transcriptase were prepared in a total volume of 20 μl. The reaction was incubated at 37 C for 60 min followed by heating at 85 C for 5 min and chilled on ice for at least 1 min. The synthesis cDNA was immediately used for PCR amplification.
The PCR was performed by using KAPA HiFi HotStart ReadyMix kit (KAPA Biosystems, Wilmington, MA) according to the manufacturer's instructions. In brief, 22 μl of master mix including 7.5 μl of nuclease free water, 12.5 μl of 2x KAPA HiFi Hotstart ReadyMix, and 1 μl of 10 μM forward and reverse primers were combined with 3 μl of cDNA to reach a total 25-μl reaction. Subsequently, PCRs were performed with the following protocol: 1 cycle of initial denaturation at 95 C for 3 min, a sequence of 35 cycles followed by denaturation at 98 C for 20 sec, annealing at 56 C for 20 sec, extension at 72 C for 2 min, and 1 cycle of final extension at 72 C for 5 min. The PCR products were detected on 1% (w/v) agarose gel electrophoresis and the gel picture was captured by using an ultraviolet light transilluminator (Bio-Rad).
Next-generation sequencing (NGS). PCR products were purified using the MEGAquickspin™ total fragment DNA purification kit (iNtRON Biotechnology, South Korea) according to the manufacturer's instructions. The purified PCR products were used for tagmentation of genomic DNA, PCR amplification, and PCR clean up according to the NGS library preparation protocol using Nextera XT DNA sample Prep kit (Illumina Inc., San Diego, CA) as described by the manufacturer's instructions. DNA fragment size and concentration of the NGS libraries were measured by using Agilent high sensitivity DNA assay kit (Agilent Technologies, Germany) and Qubit™ dsDNA HS Assay kit (Invitrogen), respectively. Normalization of each NGS library into 4nM was performed by adding 0.1% Tween® 20 diluted in nuclease-free water as described by the manufacturer's instructions. An equal volume of normalized libraries was pooled, denatured, and hybridized before sequencing. The pooled library was loaded into the designated well in an MiSeq® cartridge (Illumina Inc.). The flow cell and cartridge were loaded into the MiSeq system (Illumina Inc) and preceded to sequence with the read length of 300 bp.
Sequencing and phylogenetic analysis. Raw data (fastq files) containing the overlapping paired-end reads of libraries were filtered on a Phred quality score (Q30) and analyzed by using CLC genomics workbench 7.5.1 (CLC bio, Aarhus, Denmark). The genetic similarity of a partial genomic sequence of the IBV isolates was compared with reference IBV strains retrieved from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The nucleotide and amino acid sequences of the partial genomic sequence of 11 isolates were aligned and compared with those of other reference IBV strains using the ClustalW multiple alignment method in BioEdit 7.2.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Mutations and insertions were determined according to the results of pairwise comparisons between IBV isolates and reference IBV strains consisting of H120 (EU822341), H52 (EU817497), Mass41 (FJ904713), and LX4 (AY338732). Phylogenetic analyses of partial genomic sequence and S1 gene were conducted using MEGA 6 and the trees were constructed using the maximum likelihood method based on the Tamura-Nei parameter model with 1000 bootstrap replicates.
Recombination analysis. Nucleotide sequences of the studied IBV isolates and reference IBVs were analyzed for recombination events by using Simplot v3.5 (http://sray.med.som.jhmi.edu/SCRoftware/simplot/) and recombinant program v4.70 (RDP4 for Windows, Microsoft, Redmond, WA). Both programs were applied to identify putative recombination breakpoints and parental isolates of recombinants. The Simplot program performed based on a sliding window method. Nucleotide identity was calculated using the Kimura 2-parameter method with a transition–transversion ratio of 2, and the window width and step size, respectively, were 200 bp and 20 bp. Bootscan analysis was also carried out using a signal of 70% of observed permuted tree. In the RDP4 program, various potential recombination methods including RDP, Geneconv, Bootscan, Maxchi, Chimaera, Siscan, and Topal DSS were used to identify the recombination events and parental isolates of recombinants with significance set at P-values < 0.05.
GenBank accession numbers of reference strains (strain_accession number).
IBV isolates characterized in this study. IBS037A/2014_KU949737, IBS037B/2014_KU949738, IBS051/2014_KU949739, IBS124/2015_KU949740, IBS125/2015_KU949741, IBS126/2015_KU949742, IBS130/2015_KU949743, IBS131/2015_KU949744, IBS138/2015_KU949745, IBS142/2015_KU949746, IBS180/2015_KU949747.
Massachusetts. EP3_DQ001338, P65_DQ001339, Mass41_AY851295, M41_DQ834384, H52_EU817497, Mass41 (Vaccine)_GQ504725, H120_EU822341 (Taiwan), H120_EU822341 (China), H120_FJ888351 (Netherlands), B17_KT203557, Mass41-2006_FJ904713 (U. S. A.), Mass_GQ504724, Ind-TN92-03_KR902510, Beaudette_IBACGB, CK-CH-LHB-111232_KJ425490 (China), IBASPMNCF_KB8523, CK-CH-IBWF-2007_KF696629, CK-CH-LDL-110931_KJ425485, H120-Vaccine-China_FJ807652, KM067901-M41-UPM2013-Malaysia.
QX and QX-like. LX4_AY338732, ITA-90254-2005_FN430414, Sczy3_JF732903, SDZB0808_KF853202, P100_KM586818, CK-ZA-3665-11_KP662631, CK-CH-LDL-091022_JX195175, CK CH-LJL-110302_KC136209, CK-CH-SD-121220_KJ128295, DY07_HM245923, YX10_JX840411, SDIB821-2012_KF574761, CK-SWE-0658946-10_JQ088078, CK-CH-IBYZ-2011_KF663561, THA300252_GQ885136, THA310252_GQ885137, THA351052_GU111581, THA361052_GU111582.
Taiwan. TW2575-98_DQ646405, 3382-06_GQ229232, 3374-05_EU822337, Taiwan-2992-02_EU822340, 3071-03_EU822339, TW2296-95_DQ646404, SP-3263-04_EU822338.
United States. Grey_GU393334, JMK_GU393338, Arkansas-DPI_GQ504720, ArkDPI11_EU418976, ArkDPI101_EU418975, Arkansas (vaccine)_GQ504721.
Australia. V1-71_DQ490214, V2-71_DQ490216, N2-75_DQ490208, Q1-73_DQ490209, Q1-99_DQ490211, S_DQ490213, VicS-V_KF460437, Vic_DQ490221, VicS-del_JN983807, VicS-del_KF931628.
793/B. CK-CH-LHB-140532_KP118887, CK-CH-LBJ-140413_KP118881, CK-CH-LSD-110857_KP118885, CK-CH-LBJ-140402_KP118882, CK-CH-LSD-110851_KP118884, CK-CH-LHB-130630_KP036504, CK-CH-LLN-130102_KP118888, 4/91 (vaccine)_KF377577, CK-CH-LHLJ-111246_KP118891, CK-CH-LHB-130927_KP118880.
Virus isolation. A total of 17 cases that were detected IBV-positive by rt-PCR were used for virus isolation by using 9-day-old embryonated SPF eggs. Samples from these cases also caused mortality and lesions in the embryo such as curling and stunting with feet deformed, compressed over the head, and feather dystrophy after the third to fifth passage (data not shown). Out of these 17 cases, IBV isolates from 11 cases of different locations were considered for sequence analysis of the partial genomic sequence (6.8 kb; Table 2).
History of IBV-positive cases and the accession numbers of the partial genomic sequences of IBV isolates.
Characterization of partial genomic sequence. The results of sequence analysis showed that the length of partial genomic sequence of IBV isolates IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, and IBS138/2015 were 6795 bp while isolates IBS037A/2014, IBS037B/2014, IBS051/2014, and IBS142/2015 were 6789 bp which encompassed the four structural proteins (S, E, M and N), four accessory proteins (3a, 3b, 5a and 5b), and the intergenic region. The start and stop codons of each gene of the sequenced IBV isolates are summarized in Table 3, where the IBV isolates can be grouped into three different groups.
Length and position of structural and nonstructural protein genes and the intergenic region of IBV isolates.A
Sequence analysis of individual genes revealed that the length of 3a, E, M, 5a, 5b, and N genes of all the IBV isolates was 174 bp, 327 bp, 678 bp, 198 bp, 249 bp, and 1230 bp, respectively. However, variations were detected in S, 3b genes, and the intergenic region of the isolates. Amino acid sequence analysis of the spike protein revealed that an insertion of three amino acids occurred at the positions 23, 121, and 122 in isolates IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, IBS142/2015, and IBS180/2015 when compared to the vaccine strains (H120_EU822341 and H52_EU817497) and the virulent strain (Mass41_FJ904713). In addition, the insertion of an amino acid at position 26 was detected in the sequences of these strains when compared with the QX strain (LX4_AY338732). Deletion at amino acid position 24 and insertions of spike protein at two amino acid positions, 121 and 122, were detected in IBS037A/2014, IBS037B/2014, and IBS051/2014 isolates when compared to the Massachusetts strains. The analysis also showed that isolates IBS037A/2014, IBS037B/2014, and IBS051/2014 were shorter than isolates IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, and IBS138/2015 by two amino acids and with QX (LX4-China) by one amino acid (Table 4). In addition, deletion of an amino acid residue at position 61 in IBS037A/2014, IBS037B/2014, IBS051/2014, IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS180/2015 was detected in gene 3b when compared with vaccine strains (H52 and H120) and the classic strain of Massachusetts (Mass 41; Table 5). Interestingly, the length of the intergenic region sequence of IBS142/2015 isolate was shorter than other isolates by six nucleotides (Table 3). Nucleotide sequence analysis of the intergenic region between all isolates and IBV reference strains showed that the deletion of six nucleotides in the intergenic region of IBS142/2016 was similar to IBV strains from China such as SC021202 (EU714029), GX-YL9 (HQ850618), CK-JS-YZ07-2008 (FJ807653), and CK-CH-IBWF-2007 (KF663560; data not shown). In addition, Table 5 shows the substitution, deletion, and inversion mutations detected in gene 3b of the IBS142/2015 isolate at amino acid 55, 56, and 62 and 63, respectively.
Amino acid sequence analysis of the S1 protein.A
Amino acid sequence analysis of the 3b protein.A
Amino acid sequence analysis of the characterized IBV isolates also revealed that the most-common spike glycoprotein cleavage motif was 536H-R-R-R-R540, representing six isolates: IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, and IBS138/2015. Another motif consisting of 534R-R-V-R-R538 was detected in four isolates, IBS037A/2014, IBS037B/2014, IBS051/2014, and IBS180/2015, and 536R-R-Y-R-R540 was detected in isolate IBS142/2015.
Phylogenetic and pairwise sequence comparison (PASC) analysis of partial genomic sequence. Phylogenetic relationships between the sequences of 11 IBV isolates and 71 reference IBV strains were assessed based on partial genomic sequence at position 1-6795. The results showed that isolates IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015 clustered into Group 1 (97% identity within the group) while isolates IBS037A/2014, IBS037B/2014, IBS051/2014, and IBS180/2015 clustered into Group 2 (99% identity within the group). According to the phylogenetic tree (Fig. 1), isolates in Group 1 have a close relationship to the QX-like group (approximately 94% similarity). In addition, isolates in Group 2 were distinctly separated from reference IBV strains (QX-like, Massachusetts, 793/B, USA, Korea, China, Taiwan, Australia) with a similarity of about 82%–90%.
Phylogenetic and PASC analysis of S1 protein gene. Similar to the phylogenetic analysis of partial genomic sequence, the phylogenetic relationship of the S1 protein gene between 11 IBV isolates and 63 reference IBV strains showed that studied IBV isolates can be clustered into two groups (Fig. 2). Group 1 shared 94.56% nucleotide similarity with the QX-like strain (YX10_JX840411; Table 7). Group 2 shared 91.62% nucleotide similarity with the Malaysian variant strain (MH5365/95_EU086600; Table 6). Similarly, analysis of the amino acid sequences of S1 protein between 11 IBV isolates and reference IBV strains revealed that Group 1 has a high identity with QX-like (95.02%) and distinctly separated from Malaysian variant strain MH5365/95 (74%; Table 7). In addition, Group 2 has a high identity with the Malaysian variant strain MH5365/95 (89.09%) and is distinctly separated from the QX-like strain (76%; Table 6). Moreover, IBV isolates in both groups distinctly separated from Massachusetts (69%–74% nucleotide similarity and 70%–71% amino acid similarity), 793/B (68%–70% nucleotide similarity and 69%–73% amino acid similarity), D274 (73% nucleotide similarity and 68%–71% amino acid similarity), Italy-02 (72%–74% nucleotide similarity and 69%–73% amino acid similarity), and other unique geographic strains (67%–83; Tables 6, 7).
Nucleotide and amino acid similarities of partial genomic sequence and individual genes between IBS130/2015 isolate and reference IBV strains.A
Nucleotide and amino acid similarities of partial genomic sequence and individual genes between IBS037A/2014 isolate and reference IBV strains.A
PASC analysis of S2, 3a, 3b, E, M, 5a, 5b, and N protein genes. Previous sections described the molecular characterization of IBV isolates based on a partial genomic sequence and S1 proteins. Due to the high homology of IBV isolates in Group 1 and Group 2 (97% and 99%, respectively), IBS130/2015 and IBS037A/2014 isolates from Group 1 and 2, respectively, were selected to perform PASC analysis of genes S2, 3a, 3b, E, M, 5a, 5b, and N (Tables 6, 7). Similar to the results of PASC analysis of the partial genomic sequence and the S1 gene, nucleotide and amino acid sequences of S2, 3a, 3b, E, 5b, and N genes of IBS130/2015 isolate (Group 1 viruses) have a close relationship with the QX-like strain (shared 86%–99% and 84%–99% similarity, respectively). Interestingly, PASC analysis of structural protein and nonstructural protein genes of IBS142/2015 and reference IBV strains revealed that the nucleotide and amino acid sequences of most genes shared a close relationship with the QX-like strain excluding gene 3b (shared only 74.64% and 61.30%, respectively; data not shown). The aligned nucleotide sequences of 11 IBV isolates and reference IBV strains revealed that the E, M, and 5b genes of IBV isolates were the most conservable regions (Tables 6, 7).
PASC analysis between isolate IBS130/2015 and the QX-like strain showed that both isolates have a very close relationship when compared based on the S1, S2, 3b, and E genes (shared 95%–99% nucleotide and amino acid similarity); however, they only shared around 88%–90% nucleotide and 71%–91% amino acid similarities when compared based on the M, 5a, 5b, and N genes. In contrast, PASC analysis between isolate IBS130/2015 and the vaccine strain (H120) revealed that the nucleotide and amino acid similarity of genes S1, S2, 3a, and 3b were 69%–82% and 53%–88%, respectively, while the nucleotide and amino acid similarities of genes M, 5a, and 5b increased to 90%–95% and 87%–95%, respectively (Table 7).
Recombination analysis. The possible recombinant events in partial genomic sequences of the isolated IBVs were characterized by using a standard similarity plot (Simplot) and recombinant detection programme (RDP). Figure 3 shows that recombinant events occurred in IBV isolates in Group 1 (IBS130/2015), which have a major parental sequence derived from the QX-like strain (CK-CH-IBYZ-2011 [KF663561]) and a minor parental sequence derived from Massachusetts vaccine strain (H120 ]EU822341]). The potential recombinant region breakpoints were detected at nucleotide positions 1–4353 and 5865–6895 from major parent and 4354–5864 from minor parent by using RDP with a P-value equal to RDP (1.408 × 10−38), Geneconv (5.091 × 10−24), Bootscan (1.481 × 10−36), Maxchi (1.183 × 10−20, 3.629 × 10−15), Chimaera (1.401 × 10−17, 2.259 × 10−12), and Siscan and Topal DSS (2.975 × 10−12). In addition, comparative analysis of partial genomic sequences of IBS130/2015, CK-CH-IBYZ-2011, and H120 were carried out by using bootscan (Simplot) to confirm the recombination event (Fig. 4). The results showed the recombinant region at nucleotide position 4354–5864 which covered the complete or a part of M, 5a, 5b, and N genes (Figs 3, 4).
Infectious bronchitis is an economically important disease with worldwide distribution affecting major poultry industries. However, the genetic diversity of IBV strains found in Malaysia is poorly studied. Besides the detection of IBV vaccine strains, limited studies indicated the detection of local variants such as MH5365/95 and V9/04 that were isolated in 1994 and 2004 (4,42). Furthermore, most studies focus on the sequence analysis of S1 and N genes. In this study, we characterized 11 IBV isolates detected in different states in Malaysia in the year 2014 and 2015 based on partial genomic sequences.
The phylogenetic analysis and PASC of both partial genomic sequences and S1 sequences revealed that IBV isolates in Group 1 have a close relationship to the QX-like strain, with nucleotide homology reached up to 94.56% (Table 7). The PASC of S2, 3a, 3b, E, M, 5a, 5b, and N also revealed the close relationship between these IBV isolates and the QX-like strain (shared 88%–99% nucleotide similarity and 82%–99% amino acid similarity; Table 7). The alignment of deduced amino acid sequences of IBV isolates in Group 1 showed that all isolates (excluding IBS142/2015) had the cleavage motif H-R-R-R-R, which has been widely reported in QX or QX-like strains (1,16,38,41). In addition, amino acid sequence analysis of S1 and 3b proteins between these isolates and reference IBV strains revealed the insertions of three amino acids at positions 23, 121, and 122 of the S1 protein, a deletion of an amino acid residue at position 61 of the 3b nonstructural protein (comparing to vaccine strain H120), and the insertion of an amino acid at position 26 of the S1 protein (compared to LX4). According to Sanjuan (in 2012), mutation rate determines molecular evolution across all types of viruses, where size of viruses is identified as a major determinant of mutation rates (34). Moreover, the presence of QX-like IBV isolates in Malaysia might be due to a recombination event between the QX-like strain (CK-CH-IBYZ-2011) and a vaccine strain (H120). Recombinant analysis showed the potential recombination region located at nucleotides 4354–5864 which encompassed the M, 5a, 5b, N genes, and intergenic region (Figs. 3, 4). However, the intergenic region is a noncoding region and is not essential for viral replication, but it does play an important role in transcription of subgenomic mRNAs (25). The intergenic region might also serve as recombination ‘hotspots' due to its conserved region which assisted as a template switching site for the viral-encoded polymerase (27). This result is consistent with a previous report that recombination usually occurs in a region of genes 5a, N, and 5b (41) and the intergenic region (27). Previous reports showed that recombination is one of the important IBV evolution mechanisms in the generation of new variant viruses in the field (20,27), and the use of live attenuated vaccines could produce new variant viruses by recombination with field strains resulting from the spread of vaccine strains (17). Hence, the presence of QX-like IBV strains in Malaysia, as detected in other Asian countries such as China, Japan, Thailand, and Korea (28,29,31,33), might be caused by mutations of the S1 and 3b proteins associated with a recombination event between the QX-like strain (CK-CH-IBYZ-2011) and the vaccine strain (H120).
The evolution of IBV variant strains in Malaysia was illustrated by the presence of IBV isolates in Group 2 (IBS037A/2014, IBS037B/2014, IBS051/2014, and IBS180/2015). Firstly, the cleavage recognition motif R-R-V-R-R was detected in IBV isolates in Group 2. These cleavage motifs have not been reported before. Jackwood et al. (21) reported that the cleavage motifs of IBV strains may have geographic relationships. The unique cleavage motifs of T-R-R-R-R (V18/91, N1/88), R-R-S-G-R (N2/75), and H-R-R-G-R (Q3/88) presented only in Australia, and a unique cleavage motif, H-R-F-R-R, was recorded for IBVs in California (CAV 56b, CAV1686) (22). In the present study, R-R-V-R-R is probably the native cleavage motif of IBV variant strains in Malaysia. This result is consistent with a previous study by Zulperi et al. (42) that detected the cleavage recognition motif R-R-V-R-R in a V9/04 isolate. Secondly, the phylogenetic and PASC analysis of the S1 sequence also revealed that IBV isolates in Group 2 were clustered into Malaysian variant strains (Fig. 2) and shared 91.62% nucleotide similarity and 89.09% amino acid similarity to the Malaysian variant strain (Table 6). Moreover, they were distinctly separated from other reference strains (shared 68%–76% nucleotide similarity and 67%–77% amino acid similarity; Table 6). Unlike other isolates in Group 1, the mutations in gene 3b and the deletion of six nucleotides in the intergenic region were detected in the IBS142/2015 isolate. In addition, the IBS142/2015 isolate owned a unique cleavage motif, R-R-Y-R-R, which had not been reported before. Moreover, the IBS142/2015 isolate is the first IBV sequenced that originated from Sarawak state located at East Malaysia (Borneo Island), indicating that the IBS142/2015 isolate is probably a local QX strain found in East Malaysia. This study has provided information on the evolution of IBV in five different states in Malaysia since the first molecular detection of two variant IBV isolates in 1995 and 2004. Further studies on these isolates will able to provide information on the effectiveness of the current vaccines against these isolates, as Massachusetts-based vaccines are unable to give full protection against IBV QX or QX-like strains.
In conclusion, the analysis of nucleotide and amino acid sequences of partial genomic sequence illustrated an insertion mutation at the S1 and 3b proteins, and viral recombinations detected in genes 5a, 5b, and a part of genes M and N, thus contributing to occurrence of the IBV QX-like genotype in Malaysia. This study has provided valuable information on the evolution of IBV in Malaysia.
E = small membrane envelope protein; IB = infectious bronchitis; IBV = infectious bronchitis virus; M = membrane glycoprotein; N = nucleoprotein; NGS = next-generation sequencing; PASC = pairwise sequence comparison; PI = postinoculation; rt-PCR = real-time polymerase chain reaction; RT-PCR = reverse-transcription PCR; S = spike glycoprotein; SPF = specific-pathogen-free
This study was supported by an Institute of Bioscience, Higher Institution Centre of Excellence grant (IBS HICoE 6369101) from the Ministry of Higher Education, Government of Malaysia. N.P. K. was supported by the PhD program under VietNam International Education Development, Ministry of Education and Training in VietNam.