Thirty-three field isolates of avian infectious bronchitis virus (IBV) were recovered from commercial chicken flocks in Korea between 2003 and 2006 and were characterized phylogenetically by nucleotide sequence analysis of the IBV S1 gene hyper-variable region. Our phylogenetic analysis revealed that recent field isolates of IBV formed at least three distinct phylogenetic types, including K-I, K-II, and K-III. K-I type IBV consisted of indigenous, 13 IBV isolates which evolved from the Kr-EJ/95 strain and then separated into the lineages of type K-Ia and type K-Ib. K-II type IBV isolates (n = 19) were closely related to nephropathogenic IBV variants from China and Japan. The K-III type isolate (Kr/D064/05), first identified by this study, was closely related to enteric IBV variants from the Chinese strains that cause proventriculitis. Sequence comparisons showed amino acid differences of >27.5% between IBV types. The molecular epidemiologic characteristics of IBV field isolates are briefly discussed.
Infectious bronchitis (IB), caused by the avian infectious bronchitis virus (IBV), is a globally distributed, highly infectious, and highly contagious viral disease of chickens that is characterized by respiratory signs, nephritis, and reduced egg reproduction. Avian infectious bronchitis virus (IBV) belongs to Group 3 of the genus Coronavirus in the family Coronaviridae.
In Korea, avian IB was first recognized in a laying flock exhibiting reduced egg production in 1986 10. Vaccination programs based on vaccines of the Massachusetts (Mass) type strain (e.g., H120), or local strain KM91, have been implemented for several decades as a preventive measure. Despite extensive vaccination, IB is still a continual problem in the poultry industry. Epizootiology of IB in the republic of Korea is not well documented, although some studies of the virus have been underway since the 1990s.
The genetic typing of IBV field isolates is very important, not only for the study of virus evolution, but also for the development of effective vaccines against IBV. Classical methods such as cross haemagglutination inhibition (HI) and virus neutralization (VN) tests using serotype-specific antiserum panel can be used for identification of IBV serotypes, but they are labor-intensive and time-consuming. Recently, reverse transcription-polymerase chain reaction (RT-PCR) has been successfully applied for quick differentiation of IBV types, based on the sequences of the S1 gene region 13, because the S1 glycoprotein of the coronavirus plays an important role in inducing protection against virulent challenge 3,4,11 and is highly variable 1,2.
The purpose of this study was to extend our knowledge of the molecular epidemiology and evolution of the IBV field isolates recently circulating in Korea, knowledge that is important for efficient vaccination programs.
Materials and Methods
Thirty-three field isolates (Table 1) of IBV from various tissue samples of chickens in different parts of Korea between 2003 and 2006 were used. All isolates were propagated using specified pathogen-free (SPF) embryonated eggs and kept at −70 C at the Avian Virology Laboratory of the Avian Disease Division, National Veterinary Research and Quarantine Service, Amyang, Korea, until use.
RT-PCR and Sequencing
Viral genomic RNA (0.5–1.0 µg per reaction) used in the RT-PCR was extracted from virus containing allantoic fluid using an RNeasy minikit (Qiagen, Valencia, CA, U.S.A.) according to the manufacturer's instructions. cDNA fragments of the N-terminal region of the S1 glycoprotein subunit gene were amplified by RT-PCR using a previously described primer set (IBV-S1, 5′AGGAATGGTAAGTTRCTRGTWAGAG-3′ and IBV-S2,5′-GCCCAGTACCRTTRAYAAAATAAGC-3′) 8. The amplified DNA products of approximately 620 base pairs (bp) to 642 bp were purified using the Qiagen Gel extraction kit (Qiagen) for direct nucleotide sequencing with an ABI PRISM version 377 DNA autosequencer (PE Applied Biosystems Inc., Foster City, CA U.S.A.).
Sequence Analysis of S1 Protein Genes
The sequence data were aligned by the MegAlign program in the Lasergene package (DNASTAR Inc., Madison, WI, U.S.A.) using the Clustal W multiple alignment algorithm. The phylogenetic tree was obtained using the neighbor-joining method within Clustal X ( http://bips.u-strasbg.fr/en/documentation/ClustalX) with 1000 bootstrapping replicates 9, and TreeView ( http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) was used to display phylogenetic relationships among field isolates of IBV 12.
Accession Numbers of Nucleotide Sequences
The nucleotide sequence data reported here have been submitted to GenBank and were assigned the accession numbers shown in Table 1. The GenBank accession numbers for S1 nucleotide sequences of 21 previously reported IBV strains are as follows: EF621369 (KM91), EF621370 (Kr/EJ95), AY257060 (K142-02), AY257061 (K069-01), AY257062 (K281-01), AY257063 (K446-01), AY257064 (K507-01), AY257065 (K774-02), AY257066 (K161-02), and AY257067 (K203-02) for Korean IBV strains; AF193423 (Ch/QXIBV), AF286303 (Ch/J2), AY277632 (Ch/LD3), and DQ068701 (CK/Ch/LDL/97I) for Chinese IBV strains; AB120640 (JP/Fukui/00) and AB120645 (JP/Aichi/00) for Japanese IBV strains; AY606323 (TW/2292/02) and DQ402364 (TW/3374/05) for Taiwanese IBV strains; and AY561711 (M41), L18990 (Connecticut), M21970 (H120), and X02342 (Beaudette) for other representative IBV strains.
Phylogenetic Relationships among Field Isolates of IBV
PCR products of 620 bp to 642 bp in length were successfully amplified from all IBV field isolates used in this study. Nucleotide and predicted amino acid sequences of partial S1 genes (nt 1–621) of the IBV isolates were determined and compared with sequences of published IBV strains. Based on the phylogenetic analysis, the 33 IBV samples isolated in this study were classified into at least three distinct phylogenetic types, including K-I, K-II, and K-III (Fig. 1). Thirteen field isolates were assigned to the first cluster, designated K-I type, and these isolates were further grouped into the two sublineages of K-Ia and K-Ib (Fig. 2). Type K-1a includes 11 IBV isolates (Kr/107/04, Kr/154/04, Kr/180/04, Kr/D9/05, Kr/D20/05, Kr/Q22/05, Kr/Q45/05, Kr/D21/06, Kr/D39/06, Kr/D63/06, and Kr/D85/06), while type K-Ib included the remaining three IBV samples (Kr/Q8/05, Kr/48-4/05, and Kr/EJ95). The second cluster, designated K-II type, contained 19 IBV isolates (Kr/17/03, Kr/31/03, Kr/242/03, Kr/342/03, Kr/343/03, Kr/354/03, Kr/379/03, Kr/10/04, Kr/162/04, Kr/D31/05, Kr/D42/05, Kr/D075/05, Kr/D79/05, Kr/Q35/06, Kr/Q39/06, Kr/Q43/06, Kr/D62-3/06, Kr/D62/06, and Kr/D63/06). The IBV isolate Kr/D64/05 was included in the third cluster and designated as K-III type. No IBV related to either the previously reported Mass type strain or the K161-02 strain 6 was isolated in this study.
Comparison of Amino Acid Sequences in the Hypervariable Regions Of S1
The nucleotide and predicted amino acid sequences of the N-terminus of the S1 glycoprotein (aa1–aa207) from 33 IBV field isolates were determined and compared. The IBV isolates examined in this study shared 69.9% to 72.5% amino acid similarity to S1 from the Mass type vaccine strain H120, which has been widely used for vaccine production in Korea for decades. All IBV field isolates, except for the type K-II isolates (90.9% to 93.6% similarity), possessed low amino acid identity (66.8% to 68.8%) to the KM91 strain which has been used as a K-II type local vaccine strain in Korea. The type K-I IBV isolates had 84.5% to 99.5% (94.4% to 99.5% within sublineage K-Ia, 97.7% within sublineage K-Ib, and 82.3%–86.3% between both sublineages) similarity within same cluster and 66.2% to 69.4% similarity to isolates from other clusters. Type K-II IBV isolates had 91.3% to 99.5% identity within same cluster and 65.1% to 69.2% identity with other clusters. The type K-III IBV isolate had 67.3% to 78.1% identity with isolates from other clusters.
In this study, we examined the genetic diversity and genetic evolution of field isolates of IBV in Korea from 2003 to 2006, using sequence analysis of the S1 gene. Our results revealed that at least three distinct phylogenetic types, K-I, K-II, and K-III, are responsible for the IB outbreaks in Korea since 2003. The type K-I isolates were further divided into sublineages K-Ia and K-Ib because the type K-Ia IBV isolates had relatively high similarity (82.3% to 86.3%) to K-Ib IBV isolates.
Among them, types K-Ia and K-II were the major types of IBV circulating in Korea. Since their first recognition in 2001 6, the IBV type K-Ia isolates were recovered from unvaccinated broiler chickens with severe respiratory symptoms, while IB outbreaks caused by type K-Ib occurred in chickens vaccinated with the Mass type vaccine and were first recognized in this study. This implies that the emergence of type K-Ib viruses might be an IBV variant arising by immune pressure due to vaccination in the field, when considering that IBVs have a high mutation frequency in the S1 gene. Unlike K-Ia field isolates, some of K-II IBV isolates were recovered from chickens with nephritis, as shown in Table 1. This is supported by the results that K-II IBV isolates are closely related to the nephropathogenic Chinese IBV strains (e.g., ch/QXIBV strain). The type K-III IBV (isolate Kr/D64/05) was first found in Korea in this study. Notably, the IBV isolate Kr/D64/05 was closely related to Chinese IBV variants from enlarged proventriculi 7,14. Further examination of the tissue tropism of Kr/D64/05 in chickens will be needed.
With respect to vaccine efficacy, Ladman et al. (2006) claimed that the IBV S1 gene sequence identity value is a better predictor of protective immunity in chickens than is serotyping by virus neutralization 5. Our results revealed that IBV field isolates (except for K-II isolates to KM91 strain) had amino acid identities of less than 72.5% compared to the current IBV vaccine strains H120 and KM91, indicating that the IBV vaccine strains H120 and KM91 may offer partial or no protection against different genetic type strains. Therefore, further study should be considered to determine whether currently available IBV vaccines confer cross-protection against field IBV strains. If currently available IBV vaccines cannot provide protection against virus strains occurring in the field, alternative local vaccines should be considered for effective control of IB in Korea.
This research was supported by the National Veterinary Research and Quarantine Service (NVRQS), Ministry of Agriculture and Forestry, South Korea (Project B-AD15-2003-07-02).