Abstract
Background:
Infectious bronchitis virus (IBV) is the etiological agent of an acute and highly contagious disease. Infectious bronchitis (IB) affects chicken of all ages and poses major economic loses to the poultry industry worldwide. The continuous evolution of the spike protein (S1) of IBV is responsible for the prevalence of many serotypes/genotypes around the world. Multiple lineages of IBV strains have been detected in chicken flocks in India since 2003.
Aims:
To detect IBV genotypes prevalent in India.
Methods:
Organ samples from 20 IBV-positive flocks with variable clinical signs were used for the amplification of the S1 gene of IBV by reverse transcriptase-polymerase chain reaction (RT-PCR).
Results:
Positive PCR amplicons were sequenced. Sequence analysis showed that 14 field isolates belonged to the GI-1 genetic lineage (Mass 41 serotype), two field isolates belonged to the GI-13 (UK 4/91 variant IBV strain), one field isolate grouped with GIII, GV, and GVI genetic lineage and three belonged to a variant genotype unique to India (GI-24). Phylogenetic analysis also showed a similar type of grouping within the field isolates. Among the fourteen GI-1 isolates, 12 were isolated between 2003 and 2006 and only two were isolated between 2009 and 2011. The two field isolates belonging to GI-13 were isolated in 2007, another one belonging to GIII, GV, and GVI was isolated in 2010 and three field isolates were not close to any reference IBV sequences isolated in 2006 (IND-TN-168-06), 2010 (IND-TN-280-10) and 2011 (IND-TN-290-11).
Conclusion:
A unique variant of IBV is emerging in India (GI-24). Our findings will have important implications for future vaccine intervention.
Key Words: Infectious bronchitis virus, Phylogeny, Sequence, Spike protein 1 gene, Variant infectious bronchitis virus
Introduction
Infectious bronchitis (IB) is one of the most common diseases of chicken and difficult to control. It is caused by infectious bronchitis virus (IBV) which belongs to family Coronaviridae, genus Gamacoronavirus (Cavanagh and Naqi, 2003 ▶; International Committee on Toxonomy of Viruses). Infectious bronchitis virus is primarily a respiratory pathogen but it can also replicate in tissues such as the kidney, gonads and alimentary tract and is characterized by respiratory signs, nephritis and reduced egg production. The infectious bronchitis virus can spread through air or by mechanical transmission between birds, houses and farms. Infectious bronchitis virus exists in multiple serotypes due to frequent point mutations and recombinations (Cavanagh et al., 1992 ▶; Cavanagh et al., 1997 ▶). Immunizing chicken with one serotype has shown to induce partial or no protection against other serotypes. Hence, genotyping field IBV isolates is an important process to help recognize the circulating IBV genotype and evaluate current vaccination programs.
The genome of IBV consists of a single-stranded positive sense RNA of 27.6 kb (Boursnell et al., 1987 ▶) that codes for three major structural proteins, the spike (S), membrane (M), and the nucleocapsid (N) protein. The spike protein (S1) is post - translationally cleaved into two subunits, S1 and S2 (Cavanagh, 1983 ▶). Spike protein glycoprotein is responsible for generating neutralizing and serotype-specific antibodies (Koch et al., 1990 ▶; Kant et al., 1992 ▶).
Infectious bronchitis virus S1 gene sequences comprise of three hypervariable (HVR) regions, HVR I (amino acid residues 38-67), HVR II (amino acid residues 97-141), and HVR III (amino acid residues 274-387). It has been reported that the sequences of HVRs of the S1 gene correlate well with virus neutralization tests and can be used for genotyping IBV isolates (Kusters et al., 1989 ▶; Moore et al., 1997 ▶; Lee et al., 2003 ▶). Therefore, genotyping IBV field isolates based on HVRs of the S1 gene would be a useful tool in determining the epidemiology of field IBV and thus predicting the efficacy of vaccines against field isolates.
In India, until recently, the most common form of IBV was the respiratory form of Mass 41 (Sukumar and Prabakar, 1993 ▶; Kumanan et al., 2003 ▶; Sureshkumar et al., 2007 ▶). However, Elankumaran et al. (1999) ▶ reported serological evidence for the presence of the IBV variant 793/B, but failed to isolate the variant virus. Bayry et al. (2005) ▶ reported the presence of a single isolate of nephropathogenic IBV (PDRC/Pune/Ind/1/00) in India, isolated in 1999. Subsequently, Gaba et al. (2010) ▶ and Sumi et al. (2012) ▶ carried out virus isolation and genotyping and reported the presence of a single isolate of 793/B, a variant IBV. Recent studies showed that the field isolates belonged to the Mass 41 genotype (Patel et al., 2015 ▶; Parveen et al., 2017 ▶; Jakhesara et al., 2018 ▶). In India, in general, layer birds and broiler breeder birds are vaccinated with H120 vaccines during the 1st week, live attenuated vaccine (H120) in their 12th week, and IB killed (Mass 41) during their laying period. Commercial broiler birds are given a live attenuated vaccine (H120) during the first week of age. Despite the use of H120, IB problems are commonly found in vaccinated chickens. However, IBV genotyping studies using a large number of samples collected from chickens with different clinical manifestations and different geographical locations have not been carried out in India. This study highlights the results of IBV genotyping performed in India on samples collected during a 9-year period, from 2003-2011. Other Indian IBV S1 sequences available in the GenBank since 2011 were also analyzed in order to discover the molecular nature of the IBV prevalent in India.
Materials and Methods
Samples
Three hundred and eleven samples of trachea and kidneys of chickens from three major poultry housing states of India, Tamil Nadu, Andhra Pradesh and Karnataka, showing signs suggestive of IB were collected between 2003 and 2011 and used for this study. Among the 311 samples, 246 samples were kidney samples and 65 were tracheal samples. Most of the samples were from Tamil Nadu [181 - Kidneys (156) and Trachea (25)], followed by Andhra Pradesh [74 - Kidneys (52) and Trachea (22)] and Karnataka [56 - Kidneys (38) and Trachea (18)]. These samples were from vaccinated broiler birds (105 flocks), layers (180 flocks) and breeders (5 flocks). Details of 20 IBV positive samples are given in Table 1.
Table 1.
Details of IBV field isolates used in this study
| S. No. | Year of isolation | Isolate No. | Type of bird | Age in weeks | Type of sample collected | Clinical signs | Accession number |
|---|---|---|---|---|---|---|---|
| 1 | 2003 | Ind/113/03 | CB | 40 wks | Trachea | Respiratory distress | JX966392 |
| 2 | 2003 | Ind/114/03 | CB | 40 wks | Trachea | Respiratory distress | EF165593 |
| 3 | 2003 | Ind/TN/04/03 | CB | 4 wks | Kidney | Nephritis, pale kidneys | EF165596 |
| 4 | 2003 | Ind/TN/20/03 | CL | 36 wks | Kidney | Pale and Enlarged kidneys | EF165597 |
| 5 | 2003 | Ind/TN/92/03 | CL | 26 wks | Kidney | Nephritis | EF165598 |
| 6 | 2003 | Ind/TN/95/03 | CL | 31 wks | Trachea | Tracheal congestion, respiratory distress | EF165599 |
| 7 | 2003 | Ind/TN/97/03 | CL | 42 wks | Trachea and cloacal swabs | Drop in egg production | EF165600 |
| 8 | 2003 | Ind/TN/98/03 | CL | 36 wks | Kidneys | Pale kidneys with urate deposits | EF165601 |
| 9 | 2005 | Ind/AP/151/05 | BB | 3 wks | Kidneys | Pale kidneys | JX966393 |
| 10 | 2005 | Ind/KA/152/05 | BB | 3 wks | Kidneys | Nephritis, gout | EF165595 |
| 11 | 2006 | Ind/TN/162/06 | CL | 3 wks | Kidneys | Nephritis | JX966394 |
| 12 | 2006 | Ind/TN/163/06 | CL | 2 wks | Kidneys | Pale kidneys | JX966395 |
| 13 | 2006 | Ind/TN/168/06 | CL | 3 wks | Kidneys | Pale kidneys | JX966396 |
| 14 | 2007 | Ind/TN/174/07 | CL | 14 wks | Kidneys | Gout | JX966397 |
| 15 | 2007 | Ind/TN/175/07 | CL | 25 wks | Kidneys | Gout | JX966398 |
| 16 | 2009 | Ind/TN/183/09 | CL | 32 wks | Trachea | Respiratory signs | JX966399 |
| 17 | 2009 | Ind/TN/270/09 | CL | 28 wks | Trachea | Respiratory signs | JX966400 |
| 18 | 2010 | Ind/TN/280/10 | CL | 3 wks | Kidneys | Nephritis | JX966401 |
| 19 | 2010 | Ind/TN/284/10 | CL | 3 wks | Kidneys | Nephritis | JX966402 |
| 20 | 2011 | Ind/TN/290/11 | CL | 3 wks | Kidneys | Nephritis | JX966403 |
CL: Commercial layers, CB: Commercial broilers, and BB: Broiler breeders
1998) which covers HVR regions I and II from nucleotide position 114 to 708 (Beaudette strain Accession number M95169). Reverse transcriptase-polymerase chain reaction was carried out in Eppendorf Thermal cycler using AmpliTaq Gold fast PCR master mix (ThermoFisher, USA) with the following cycling conditions: initial denaturation of 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s; annealing at 54°C for 30 s and extension at 72°C for 45 s and a final extension of 72°C for 10 min. The amplified RT-PCR products (600 bp) were analyzed by electrophoresis using 2% agarose gel and visualized using BioRad gel documentation system.
Sample preparation and isolation of IBV
Trachea and kidneys were homogenized using sterile Mortar and Pestle with liquid nitrogen and prepared in a 20% suspension in nuclease free phosphate buffered saline (PBS) (pH = 7.2) with antibiotics (10,000 IU/penicillin, 10,000 mg/ml streptomycin). Sample suspensions were centrifuged at 12,000 rpm (16128 × g) for 10 min at 4°C and the supernatant was filtered through 0.22 µm sterile syringe filters. Infectious bronchitis virus was isolated from these samples by inoculating into the allantoic cavity of 9-10 day-old specific pathogen free embryonated chicken eggs (SPF-ECE). After 5 to 6 passages in ECE, allantoic fluids were used to extract RNA to determine the presence of IBV by reverse transcriptase-polymerase chain reaction (RT-PCR). Allantoic fluids were confirmed negative for Newcastle disease virus (NDV) by RT-PCR and infectious laryngotracheitis virus by PCR.
RNA isolation and RT-PCR assay
Allantoic fluids of each sample were used to extract Total RNA using Trizol LS reagent (Invitrogen, USA) as per manufacturer’s instructions. RNA was converted into complementary DNA (cDNA) using a Revert-Aid cDNA synthesis kit (Fermentas, USA) with random hexa primers. The synthesized cDNA was used to amplify the HVR regions of S1 gene by RT-PCR using the forward primer 5´- TGG TTG GCA TTT ACA CGG GG-3´ (Wang and Tsai, 1996 ▶) and reverse primer 5´ -CTC GAA TTC C NGT RTT TRA YTG RCA-3´ (Keeler et al.,
Sequencing of S1 gene of IBV
Reverse transcriptase-polymerase chain reaction products were purified using AuPreP gel extraction kit (M/s. Life Technologies, USA) as per the manufacturer’s instruction. Sequencing of the PCR products with forward and reverse primers was done in the Center for Genomic Services (TCGS), New Delhi, India using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA).
S1 gene sequence analysis
The sequence data were aligned by MegAlign program (DNASTAR) using the Clustal W multiple sequence alignment algorithm. S1 gene sequences of major variant IBVs circulating in the world and other major vaccine strains were included in the multiple sequence alignment (Table 2), and nucleotide and amino acid similarities were determined using DNASTAR. Multiple sequence alignment of S1 gene sequences was also performed using the ClustalX program to construct a phylogenetic tree using the neighbor-joining (NJ) approach with bootstrap values of 1000 replicates and a p-distance model using, MEGA 7.0.
Table 2.
Reference sequences used in this study
| Strain | GenBank accession number | Strain | GenBank accession number |
|---|---|---|---|
| Australia | AY775779 | IND-HBL1310 | AB861538 |
| Australia-VicS | KF931628 | IND-IB257 | KF809781 |
| Belgium-B1648 | KR231009 | IND-IB422 | KF809791 |
| China-AH3 | JQ900137 | IND-MZ1 | KX832218 |
| China-HC1312 | KX107748 | IND-PDRC | AY091551 |
| China-JL1312 | KX107768 | IND-IB236 | KF809778 |
| China-LX4 | AY189157 | IND-IB238 | KF809779 |
| China-NTP | KX107792 | IND-IBV470 | KF809795 |
| China-NX1401 | KX107803 | IND-MZ2 | KX832219 |
| China-SCMS4 | JF951377 | IND-MZ3 | KX832220 |
| China-SCZJ2 | KX721498 | IND-21-B-2008 | KF360981 |
| Egypt-F03 | DQ487085 | IND-V24 | KF757450 |
| Europe-H120 | KR605489 | IND-V39 | KF757456 |
| Israel-Variant | EU780077 | IND-23-B-2008 | KF360983 |
| Italy-IT02 | AJ457137 | IND-IBV422 | KF809791 |
| Europe-D274 | X15832 | IND-415 | KF809790 |
| Pak | KX013102 | IND-IB-MZ-1 | KX832218 |
| Taiwan | DQ646405 | IND-IBV238 | KF809779 |
| UK-4-91 | JN192154 | IND-IBV236 | KF809778 |
| USA-Ark99 | L10384 | IND-IB-MZ-3 | KX832220 |
| USA-Cal99 | AY942738 | IND-IB-MZ-2 | KX832219 |
| USA-Connecticut | L18990 | IND-HBL-IB13 | AB861538 |
| USA-M41 | AY561711 | IND-IBV257 | KF809781 |
| IND-IB-MZ6 | KX832223 | IND-IBV506 | KF809796 |
| IND-V17 | KF757449 | IND-V57 | KF757464 |
| IND-V6 | KF757440 | IND-IBV398 | KF809789 |
| IND-V7 | KF757441 | IND-IBV270 | KF809783 |
| IND-V62 | KF757469 | IND-GE1 | KF757465 |
| IND-V65 | KF757472 | IND-GE2 | KF757466 |
| IND-V64 | KF757471 | IND-IB-MZ5 | KX832222 |
| IND-V63 | KF757470 | IND-IB-MZ4 | KX832221 |
| IND-V28 | KF757454 | IND-HBL-13 | AB86260 |
| IND-V27 | KF757453 |
Accession numbers of nucleotide sequences
The nucleotide sequence data from the HVR region of 20 IBV isolates reported here were submitted to GenBank, and were assigned the accession numbers shown in Table 1.
Results
Isolation and identification of IBV
Two hundred and ninety flocks of poultry suspected for IBV were tested for the presence of IBV by RT-PCR from 2003 to 2011. Most of these cases were from commercial layer birds with a history of nephritis and very few with respiratory problems. The epidemiological information regarding the field isolates used in this study is presented in Table 1. All layer birds and broiler breeder birds were vaccinated with two doses of live attenuated Mass 41 vaccine with or without Newcastle disease (ND) vaccine. Commercial broiler birds received a single vaccine. Successful isolation of IBV required at least 5 passages. The embryonic lesions suggestive of IBV such as dwarfing and curling of toes were not observed for all the isolates. Regardless of embryonic lesions, allontoic fluids from all the isolates were tested for IBV by RT-PCR.
Sequence analysis of S1 gene of IBV
Of the 311 samples analyzed, 20 were positive (6.4%) by RT-PCR and all the RT-PCR products were sequenced. Sequences of 20 field isolates were compared with the reference S1 gene sequences from GenBank (Table 2). Infectious bronchitis virus sequence genetic lineage for field isolates were assigned as suggested by Valastro et al. (2016) ▶. Nucleotide sequence and amino acid sequence similarities of field isolates are given with the reference sequences in Table 3. Sequence analysis showed that 14 isolates belonged to the GI-1 lineage (Mass 41 genotype). The nucleotide sequence identity percentage of field isolates was between 85.0% to 96.2%, for the Mass 41 reference sequence (AY561711). For the H120 vaccine, these 14 field isolates had nucleotide sequence identities between 85.8% to 100%. The field isolate, IND-TN-183-09 had 100% nucleotide and amino acid identities with the H120 vaccine sequence. Two field isolates, IND-TN-174-07 and IND-TND-175-07 had 95.6% and 97.4% nucleotide sequence identities with the UK 4/91 variant IBV strain (GI-13 lineage) and IND-TN-284-10 had a 92% nucleotide sequence identity with the Indian nephropathogenic IBV (AY091151). Three field isolates, IND-TN-168-06, IND-TN-270-09 and IND-TN-290-11 had nucleotide sequence identities from 65.7% to 77.8% with Mass 41 genotype (AY561711) and nucleotide sequence identities of 70.9% to 79.6% with the H120 vaccine. The analysis of IBV genetic lineage and year of isolation showed that between 2003 and 2006, 12 out of 14 GI-1s were detected, whereas all six variant IBVs were detected from 2006 to 2011 (Table 4) (Fig. 1).
Table 3.
Nucleotide and amino acid identity percentage of S1 gene sequences of Indian isolates with the reference IBV strains. First line of each row represents nucleotide identities and second line represents amino acid identities of the field isolate with the corresponding reference sequence
| Reference virus/Field virus | IND-113-03 | IND-114-03 | IND-TN-04-03 | IND-TN-20-03 | IND-TN-92-03 | IND-TN-95-03 | IND-TN-97-03 | IND-TN-98-03 | IND-AP-151-05 | IND-KA-152-05 | IND-TN-162-06 | IND-TN-163-06 | IND-TN-168-06 | IND-TN-174-07 | IND-TN-175-07 | IND-TN-183-09 | IND-TN-270-09 | IND-TN-280-10 | IND-TN-284-10 | IND-TN-290-11 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Aus-Vic KF931628 |
68.3 60.3 |
67.9 59.6 |
67.8 53.4 |
67.6 58.6 |
68.8 59.3 |
67.4 56.9 |
68.5 59.6 |
68.5 59.6 |
68.1 57.6 |
68.1 57.6 |
61.5 62.5 |
67.9 60.0 |
76.9 68.1 |
36.3 20.5 |
37.0 19.2 |
71.1 63.0 |
76.4 54.5 |
42.5 30.1 |
72.5 59.1 |
71.6 58.3 |
| Belg-B1648 KR231009 |
36.8 21.3 |
37.7 22.0 |
34.6 20.0 |
35.3 21.3 |
37.3 22.6 |
36.6 21.7 |
37.7 22.0 |
37.7 22.0 |
39.1 23.0 |
39.1 23.0 |
36.3 22.7 |
37.6 21.0 |
34.8 21.6 |
84.8 83.8 |
85.1 83.9 |
36.7 21.3 |
37.8 27.8 |
42.9 30.1 |
70.5 57.6 |
69.9 58.1 |
| China-LX4 AY189157 |
73.2 63.8 |
73.9 64.9 |
67.8 58.6 |
72.4 63.8 |
74.2 64.4 |
72.8 62.1 |
74.5 64.9 |
74.5 64.9 |
74.1 64.4 |
74.1 64.4 |
66.8 65.0 |
72.2 67.3 |
71.6 61.1 |
38.6 20.5 |
38.9 16.4 |
75.3 67.1 |
77.0 64.6 |
41.6 28.8 |
74.5 66.7 |
69.0 55.6 |
| Europe-D274 X15832 |
70.0 58.6 |
69.6 57.9 |
66.1 55.2 |
68.1 56.9 |
69.9 57.6 |
70.7 55.2 |
70.2 57.9 |
70.2 57.9 |
70.3 59.3 |
70.3 59.3 |
63.7 62.5 |
68.5 60.0 |
72.1 62.5 |
38.3 17.8 |
37.7 23.3 |
73.2 64.4 |
76.1 64.6 |
46.1 27.4 |
74.0 69.5 |
70.0 60.0 |
| Europe-H120 KR605489 |
96.2
91.4 |
96.2
89.5 |
91.3
84.5 |
93.5
87.9 |
96.8
91.5 |
95.7
86.2 |
96.7
89.5 |
96.7
89.5 |
97.3
93.2 |
97.3
93.2 |
96.3
92.5 |
96.3
92.7 |
79.6 75.0 |
36.3 19.2 |
36.9 19.2 |
100
100 |
98.3
88.3 |
43.4 37.0 |
74.0 71.0 |
73.5 66.7 |
| IND-PDRC AY091151 |
74.0 66.1 |
73.3 63.8 |
71.3 62.1 |
73.9 63.8 |
75.9 66.1 |
75.0 61.0 |
76.1 63.8 |
76.1 63.8 |
76.3 66.1 |
76.3 66.1 |
41.8 45.2 |
76.8 66.1 |
39.3 12.5 |
38.2 20.5 |
38.6 21.9 |
79.5 75.3 |
74.4 62.0 |
47.9 43.8 |
92.0
84.8 |
34.7 16.4 |
| Israel-Variant EU780077 |
71.3 62.1 |
69.3 61.4 |
62.9 58.6 |
72.7 65.3 |
71.8 62.7 |
71.0 60.3 |
71.6 61.4 |
71.6 61.4 |
71.8 62.7 |
71.8 62.7 |
74.7 70.0 |
72.1 63.6 |
36.1 18.9 |
33.6 20.5 |
34.9 21.9 |
74.0 65.8 |
79.9 63.3 |
42.9 31.5 |
73.0 65.2 |
58.1 56.9 |
| Italy-IT02 AJ457137 |
68.4 62.1 |
66.5 61.4 |
63.5 58.6 |
70.5 63.9 |
72.7 68.1 |
68.8 60.3 |
68.8 61.4 |
68.8 61.4 |
75.7 62.7 |
75.7 62.7 |
71.9 70.0 |
69.7 63.6 |
36.6 16.2 |
85.2 86.6 |
86.0 87.4 |
70.8 64.4 |
72.8 60.8 |
44.3 30.1 |
69.7 63.6 |
62.9 61.1 |
| Taiwan DQ646405 |
76.0 73.7 |
76.6 73.2 |
72.1 64.9 |
74.1 76.4 |
76.3 71.8 |
75.5 70.2 |
77.2 75.0 |
77.2 75.0 |
76.8 74.1 |
76.8 74.1 |
68.6 43.1 |
74.3 75.9 |
76.9 73.2 |
35.8 19.4 |
36.6 19.2 |
77.5 76.4 |
76.1 64.6 |
46.6 23.0 |
73.5 68.2 |
71.2 64.8 |
| UK-4-91 JN192154 |
36.8 21.3 |
37.7 20.3 |
34.6 18.3 |
35.3 19.7 |
37.3 22.6 |
36.6 20.0 |
37.7 20.3 |
37.7 20.3 |
38.6 19.7 |
38.6 19.7 |
35.8 22.7 |
35.5 17.7 |
37.5 23.0 |
95.6
93.0 |
97.4
94.4 |
36.3 18.7 |
78.4 64.6 |
37.7 27.4 |
74.0 66.7 |
59.5 50.5 |
| USA-Ark99 L10384 |
63.9 58.6 |
64.1 56.1 |
60.7 53.4 |
65.7 55.2 |
65.1 59.3 |
63.6 53.4 |
64.7 56.1 |
64.7 56.1 |
66.5 59.3 |
66.5 59.3 |
68.7 60.0 |
64.2 58.2 |
70.2 65.3 |
34.5 21.9 |
34.8 21.9 |
69.6 64.4 |
71.1 57.0 |
42.0 28.8 |
68.0 57.6 |
66.1 58.3 |
| USA-Cal99 AY942738 |
66.7 62.1 |
66.8 61.4 |
62.8 58.6 |
65.4 60.3 |
67.7 62.7 |
66.3 58.6 |
67.4 61.4 |
67.4 61.4 |
69.7 62.7 |
69.7 62.7 |
71.4 62.5 |
68.4 63.6 |
72.0 63.9 |
35.0 20.5 |
36.6 21.9 |
71.4 67.1 |
68.9 58.2 |
42.5 27.4 |
70.0 60.6 |
70.3 56.9 |
| USA-M41 AY561711 |
96.2
89.7 |
95.7
87.7 |
90.7
82.8 |
94.1
86.2 |
96.2
88.1 |
96.2
84.5 |
96.2
87.7 |
96.2
87.7 |
95.7
89.8 |
95.7
89.8 |
95.992.5 |
95.2
90.9 |
77.8 68.1 |
34.6 19.2 |
38.6 17.8 |
95.6
91.8 |
95.3
81.8 |
43.8 13.7 |
71.5 60.6 |
69.3 59.7 |
Table 4.
Genotyping of Indian isolates based on S1 gene sequences
| Genotype | Year of isolation | Isolates |
|---|---|---|
| Mass 41 IBV | 2003 | IND-113-03 |
| 2003 | IND-114-03 | |
| 2003 | IND-TN-04-03 | |
| 2003 | IND-TN-20-03 | |
| 2003 | IND-TN-92-03 | |
| 2003 | IND-TN-95-03 | |
| 2003 | IND-TN-97-03 | |
| 2003 | IND-TN-98-03 | |
| 2005 | IND-AP-151-05 | |
| 2005 | IND-KA-152-05 | |
| 2006 | IND-TN-162-06 | |
| 2006 | IND-TN-163-06 | |
| 2009 | IND-TN-270-09 | |
| 2009 | IND-TN-183-09 | |
| UK 4/91 IBV | 2007 | IND-TN-174-07 |
| 2007 | IND-TN-175-07 | |
| Indian nephropathogenic IBV | 2010 | IND-TN-284-10 |
| Variant IBV | 2006 | IND-TN-168-06 |
| 2010 | IND-TN-280-10 | |
| 2011 | IND-TN-290-11 |
Fig. 1.
Distribution of IBV genotypes during the period of 2003-2011. M: Mass 41 IBV, U: UK 4/91 IBV, I: Indian nephropathogenic IBV, and V: Variant IBV
Phylogenetic tree analysis of 20 field isolates showed that 14 field isolates, IND-113-03, IND-114-03, IND-TN-04-03, IND-TN-20-03, IND-TN-92-03, IND-TN-95-03, IND-TN-97-03, IND-TN-98-03, IND-AP-151-05, IND-KA-152-05, IND-TN-162-06, IND-TN-163-06, IND-TN-270-09, IND-TN-183-09 belonged to GI-1 genetic lineage (Mass 41 genotype), two field isolates, IND-TN-174-07 and IND-TN-175-07 belonged to GI-13 genetic lineage (UK 4/91 genotype), one field isolate, IND-284-10 belonged to GIII-1, GV-1, and GVI-1 genetic lineage (Indian nephropathogenic IBV) and three field isolates, IND-TN-168-06, IND-TN-280-10, IND-TN-290-11 belonged to GI-24 lineage (Fig. 2). A phylogenetic tree was also constructed using the S1 gene sequences of IBV isolates from India, isolated after 2011. Most of these isolates belonged to the GI-24 genetic lineage and indicated an emergence of a unique variant IBV in India (GI-24) (Fig. 2).
Fig. 2.
Phylogenetic tree constructed by the neighbor-joining method with 1000 boot strap values (Mega 7.0) for the field isolates collected during the period of 2003-2011 (•) and for the field isolates from India available in GenBank during the period of 2011-2016 (∆)
Discussion
In this report, we analyzed 20 field isolates of IBV from India from 2003 to 2011 using RT-PCR and sequencing HVR I and HVR II of the S1 gene. Based on the following observations from the present study, from the 20 isolates studied, 3 can be considered as new IBV genotypes. First, nucleotide sequence analysis of HVR regions of S1 gene sequences of field isolates with reference strains showed that IND-TN-168-06, IND-TN-280-10, and IND-TN-290-11 isolates did not belong to the variants reported previously (Bande et al., 2017 ▶) but rather belonged to the GI-24 genetic lineage (Valastro et al., 2016 ▶), which is unique to India. Second, phylogenetic analysis confirmed that these three isolates did not group with the vaccine sequences or variant IBVs. Third, subsequent reports of GenBank nucleotide sequences of IBV from India clearly showed an emergence of a unique IBV variant in India (GI-24).
For majority of poultry flocks in India, IB control strategy is based mainly on the use of attenuated live Mass 41 strain, H120 vaccine. However, despite extensive vaccination, the number of IBV-suspected cases was large. Most gout cases, even in chickens less than a week old were suspected to be IB; however,, such cases were negative upon analysis, indicating other causes (Sályi, 1999) for these birds.
To date, only stray reports on the presence of IBV variants are seen in the Indian literature (Bayry et al., 2005 ▶; Gaba et al., 2010 ▶; Sumi et al., 2012 ▶). Although the number of genotyped samples is few, a clear pattern seems to be emerging. IBV in the early years of 2003-2006 was predominantly Mass 41 type. Earlier reports of IBV isolations in these years also revealed only the Mass type (Kumanan et al., 2003 ▶; Suresh Kumar et al., 2005 ▶). As seen in this study (n=7) and reports by Gaba et al. (2010) ▶ (n=1) and Sumi et al. (2012) ▶ (n=2), variant viruses seem to be emerging in India since 2006.
These observations confirm that one or more genotypes of variant IBV are circulating in Indian farms and are more likely to diversify further. This could likely result in vaccination failures if only Mass type vaccines are used. The emergence of new IBV genotypes or serotypes (Ducatez et al., 2009 ▶; Lim et al., 2011 ▶; Mahmooda et al., 2011 ▶) along with the poor cross-protection observed among IBV serotypes have complicated IB control programs (Cavanagh, 2007 ▶). Moreover, the prevalence of IBV in Asian countries shows the presence of genotypes like QX, LX4, Mass 41, 4/91, IS1494, D274 and some local and regional IBV genotypes (Bande et al., 2017 ▶; Lin and Chen, 2017 ▶; Sadri et al., 2018 ▶).
The IBV isolates used in this study were isolated from the major poultry rearing areas of India where outbreaks of IBV have been increasing annually. Our results support the idea that the vaccine presently in use is not protecting the birds against these field variant IBVs. Since the S1 of IBV is related to host range and cell tropism (Kuo et al., 2000 ▶; Casais et al., 2003 ▶), alterations in the pathogenic and antigenic characteristics of IBV are expected (Wang et al., 1994 ▶). Moreover, the use of live attenuated vaccines could produce new variant IBVs by recombining with field isolates resulting from the spread of vaccine strains (Farsang et al., 2002 ▶; Chen et al., 2009 ▶).
The present study indicates an emergence of a unique variant IBV in India (GI-24). Moreover, the observations indicate that these variant IBVs might increase the genetic diversity of IBV and hamper IB control in India. Hence a continuous IBV isolation and genotyping program is recommended, not only for the study of virus evolution but also for effective modifications of vaccination programs (Cavanagh et al., 2005 ▶). It may also be necessary to ponder the need for inclusion of new variant IBV strains for vaccine preparation to ensure a broader spectrum of protection against IBV infection.
Acknowledgements
The authors thank the World Poultry Science Association, Indian Branch, for providing a research grant to carry out part of this work and the ICAR-Niche area of excellence on Animal Biotechnology project for carrying out part of this work. The authors also thank the Tamil Nadu Veterinary and Animal Sciences University for providing facilities to carry out the work.
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