Analysis of a QX-like avian infectious bronchitis virus genome identified recombination in the region containing the ORF 5a, ORF 5b, and nucleocapsid protein gene sequences

The complete genome of a QX-like infectious bronchitis virus (IBV) strain Sczy3 isolated recently in Sichuan was sequenced. The genome contains 27,695 nucleotides (nt), and possesses a genomic structure similar to other IBV strains. Sequence comparisons demonstrated that the Sczy3 genome had the highest nt sequence identity with QX-like IBVs and was most dissimilar to the Massachusetts type IBV. Differences in the sequences of genes present in the Sczy3 genome and other IBVs gene sequences were also identified. Phylogenic analysis showed that the entire genome and most of the Sczy3 genes were located in the same cluster as LX4. Recombination analysis showed that Sczy3 is a chimeric strain derived from LX4 (major parental sequence) and H120 (minor parental sequence) suggesting that recombination occurred in a region containing the 3′ terminal 5a sequence (83 nt), the 5′ terminal 5b sequence (222 nt), and the 5′ terminal nucleocapsid protein gene sequence (132 nt). Mutations and intergenic recombination may have played an important role in the evolution of IBVs.     

Sequencing, phylogenetic analysis, and potential recombination events of infectious bronchitis viruses isolated in Korea

The S2 glycoprotein and membrane (M) protein genes and S1 glycoprotein and nucleocapsid (N) genes of 11 Korean infectious bronchitis virus (IBV) isolates were amplified by RT-PCR, cloned, and sequenced. The resultant nucleotide sequences were compared with the published sequences for non-Korean IBV strains. Korean IBV isolates formed two independent subclusters within the phylogenetic tree based on S2 glycoprotein gene sequences. However, four and two different clusters were formed in the phylogenetic tree based on S1 glycoprotein and M gene sequences, respectively. In particular, Korean IBV K446-01 and K203-02 strains appeared to be the result of recombination between an indigenous Korean IBV strains and a vaccine strain (Massachusetts serotype) currently used in Korea. The recent IBV isolate, K026-10, formed a new subgroup that was closely related to traditional Korean IBV group in a phylogenetic tree based on the S1 and S2 genes, but it was grouped into the traditional Korean IBV cluster in a phylogenetic tree based on the M and N genes. Our data show that field IBVs in Korea are continuing to evolve and that vaccine strains might actually play a critical role in the appearance of new IBV strains via recombination in the field.     

Complete genome sequence analysis of a predominant infectious bronchitis virus (IBV) strain in China

Infectious bronchitis (IB) is one of the major diseases in poultry flocks all over the world caused by infectious bronchitis virus (IBV). In the study, the complete genome sequence of strain A2 was sequenced and analyzed, which was a predominant IBV strain in China. The results indicated that there were mutations, insertions, and deletions distributed in the whole genome. The A2 virus had the highest identity to S14 and BJ in terms of full genome, whereas had a further distance to Massachusetts strains. Phylogenetic analysis showed that A2 isolate clustered together with most Chinese strains. The results of this study suggest that strain A2 may play an important role in IBV’s evolution and A2-like IBVs are predominant strains in China.     

Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model

Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. ≥95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3′UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3′UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3′UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.     

Naturally occurring recombination between distant strains of infectious bronchitis virus

New variants of infectious bronchitis virus (IBV) have emerged in Australia despite its geographical isolation and intensive vaccination programs. In the present study, the 3′ terminal 7.2 kb of the genome of a recently isolated variant of IBV (N1/03) was sequenced and compared with the sequences of classical and novel strains of IBV, the two main groups of these viruses in Australia. The comparison revealed that recombination between classical and novel IBVs was responsible for the emergence of the new variant. It was concluded that novel IBVs, which have not been detected since 1993, and which are phylogenically more distant from classical IBVs than turkey coronaviruses, might still be circulating and contributing to the evolution of IBV in Australia.     

Emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in Chinese chicken flocks

The emergence of novel infectious bronchitis viruses (IBVs) has been reported worldwide. Between 2011 and 2014, eight IBV isolates were identified from disease outbreaks in northeast China. In the current study we analysed the S1 gene of these eight IBV isolates in addition to the complete genome of five of them. We confirmed that these isolates emerged through the recombination of LX4 and Taiwan group 1 (TW1) viruses at two switch sites, one was in the Nsp 16 region and the other in the spike protein gene. The S1 gene in these viruses exhibited high nucleotide similarity with TW1-like viruses; the TW1 genotype was found to be present in southern China from 2009. Pathogenicity experiments in chickens using three of the eight virus isolates revealed that they were nephropathogenic and had similar pathogenicity to the parental viruses. The results of our study demonstrate that recombination, coupled with mutations, is responsible for the emergence of novel IBVs.     

A type-specific blocking ELISA for the detection of infectious bronchitis virus antibody

Infectious bronchitis virus (IBV) infection has been a major threat to the poultry industry worldwide. Current commercially available ELISA kits detect group-specific antibodies; however, to understand the status of field infection, a monoclonal antibody (mAb) blocking ELISA (b-ELISA) against local IBVs was developed. The selected mAb showed specificity to Taiwan IBV strains but had no cross-reactivity with the vaccine strain H120. Using the hemagglutination inhibition (HI) test as a standard, the cut-off value, sensitivity, and specificity of a b-ELISA using this mAb were evaluated in 390 field samples. The type-specificity of detection was validated using a panel of chicken hyperimmune sera. The results showed that the b-ELISA demonstrated high sensitivity (98.0%) and specificity (97.2%) of detection. The agreement between the results of the b-ELISA and the HI test was statistically significant (Kappa = 0.95), and there was no significant difference between these two methods (McNemar p = 0.72). The b-ELISA specifically detected Taiwan IBV serotypes but not three non-Taiwan IBV serotypes nor sera against other avian pathogens. This b-ELISA provides type-specific antibody detection of local IBV strains. It has the potential to serve as a rapid and reliable diagnostic method for IBV clinical infections in the field in Taiwan.     

Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan

Summary In order to differentiate recent isolates of avian infectious bronchitis virus (IBV) in Taiwan, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and direct sequencing methods were used to type 25 IBV Taiwan isolates. Two conserved sequences that flank the hypervariable region I (HVR I) in the N-terminus of S1 protein gene were chosen as primers. Sequences of 228–231 base pairs (bp) were amplified by PCR from 25 Taiwan isolates and 4 reference strains (H120, Conn, JMK, Holte). PCR products were digested with 5 restriction endonucleases,BsoFI,DdeI,MboII,AluI,RsaI, and different IBV isolates were grouped according to their RFLP patterns. The RFLP patterns of the 4 reference strains in this study matched the published sequences in GenBank. Except 1 vaccine strain, the other 24 Taiwan isolates were different from these 4 and 18 other IBV strains whose sequences were published. The data from PCR-RFLP and sequencing of IBV genomes showed that the 24 Taiwan isolates can be divided into 2 distinct groups, I and II. Seven RFLP patterns are identified in group I and only 1 in group II.     

Genetic analysis revealed LX4 genotype strains of avian infectious bronchitis virus became predominant in recent years in Sichuan area,China

Between 2008 and 2010, 19 strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in Sichuan province, China. The S1 genes of the isolates were amplified and sequenced. Phylogenetic analysis revealed that the 19 isolates and 37 reference IBV strains can be grouped into eight genotypes. Although IBVs of Taiwan-I type, massachusetts type, and proventriculitis type were isolated, but most isolates were LX4 genotype. Homology analysis of the sequences of S1 genes of the 19 isolates and 37 reference IBV strains revealed that the identity of the nucleotides and amino acid sequences of the S1 genes between the 15 LX4-type isolates and other IBV strains were 71.9–99.3% and 72.1–99.1%, respectively, while those of the analyzed IBV of LX4 type were 96.0–99.9% and 94.3–99.8%, respectively. The results from this study and other published results in the GenBank database showed that isolates circulating in Sichuan province in recent years were mainly LX4 genotype, which is the predominant genotype circulated in China in recent years.     

Evidence of genetic diversity generated by recombination among avian coronavirus IBV

Summary.  Previously, we demonstrated that the DE072 strain of IBV is a recombinant which has an IBV strain D1466-like sequence in the S gene. Herein, we analyzed the remaining 3.8 kb 3′ end of the genome, which includes Gene 3, Gene 4, Gene 5, Gene 6, and the 3′ non-coding region of the DE072 and D1466 strains. Those two viruses had high nucleotide similarity in Gene 4. However, the other individual genes had a much different level of sequence similarity with the same gene of the other IBV strains. The genome of five IBV strains, of which the complete sequence of the 3′ end of the genome has been determined, were divided at an intergenic (IG) consensus sequence (CTGAACAA or CTTAACAA) and compared phylogenetically. Phylogenetic trees of different topology indicated that the consensus IG sequences and the highly conserved sequence around this regions may serve as recombination ‘hot spots’. Phylogenetic analysis of selected regions of the genome of the DE072 serotype field isolates further support those results and indicate that isolates within the same serotype may have different amounts of nucleotide sequence similarity with each other in individual genes other than the S gene. Presumably this occurs because the consensus IG sequence serves as the template switching site for the viral encoded polymerase. Received December 1, 1999 Accepted March 24, 2000     

Comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains

Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5' end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PL(pro)), main protease (M(pro)), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase gene are important for virus replication. We have sequenced four regions of the replicase genes corresponding to the 5'-terminal sequence, PL(pro), M(pro), and RdRp domains from 20 heterologous IBV strains, and compared them with previously published coronavirus sequences. All the coronavirus 5'-termini and PL(pro) domains were divergent, unlike the M(pro) and the RdRp domains that were highly conserved with 28% and 48% conserved residues, respectively. Among IBV strains, the 5' untranslated region including the leader sequence was highly conserved (>94% identical); whereas, the N-terminal coding region and the PL(pro) domains were highly variable ranging from 84.6% to 100%, and 77.6% to 100% identity, respectively. The IBV M(pro) and RdRp domains were highly conserved with 82.7% and 92.7% conserved residues, respectively. The BJ strain was the most different from other IBVs in all four regions of the replicase. Phylogeny-based clustering based on replicase genes was identical to the antigen-based classification of coronaviruses into three groups. However, the IBV strain classification based on replicase gene domains did not correlate with that of the type-specific antigenic groups. The replicase gene sequences of many IBVs recovered from infected chickens were identical to those of vaccine viruses irrespective of serotype, suggesting that either there has been an exchange of genetic material among vaccine and field isolates or that there is a convergent evolution to a specific replicase genotype. There was no correlation between the genotype of any region of the replicase gene and pathotype, suggesting that the replicase is not the sole determinant of IBV pathogenicity.     

Genetic diversity of avian infectious bronchitis coronavirus strains isolated in China between 1995 and 2004

Summary. Twenty-six avian infectious bronchitis (IB) viruses (IBV) were isolated from outbreaks in chickens in China between 1995 and 2004. They were characterized by comparison with twenty-six Chinese reference strains and five other IBV strains. Chinese IBVs, which were mainly nephropathogenic, were placed into seven genotypes. Fourteen Chinese IBV isolates were placed in genotype I, having small evolutionary distances from each other. Genotype II included 6 strains that were isolated in the 1990s in China. Genotype III consisted of eight Chinese isolates that showed close relationship with Korean IBV isolates. Another eight IBV isolates clustered in genotype IV and showed larger evolutionary distances. The Massachusetts serotype was present in China in 1990s and was in a separate genotype. Two isolates, HN99 and CK/CH/LHN/00I, which might be a reisolation of vaccine strains, clustered into genotype VI. Four Chinese IBV isolates formed another genotype and showed larger evolutionary distances from other Chinese IBV genotypes (genotype VII). IBVs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%. The results showed that IBVs in China came from genetic changes both in IBV populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. IBVs showing various genetic differences are cocirculating in China.     

Evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains

The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3′ end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.     

Comparisons of envelope through 5B sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes

A 1.78 kb sequence, including the E, M, 5a and 5b genes, and the intergenic region between the M and 5a genes, of six US strains of infectious bronchitis (corona)virus (IBV) were sequenced and compared to the published sequences for two additional strains. The overall identities as determined through pairwise analyses of nucleotide sequences of the entire 1.78 kb region ranged from 90 to 99%, with the 5b open reading frame (ORF) having the greatest identity (94–99%) while the identities of the E, 5a and M ORFs ranged from 87 to 100%. Nucleotide sequencing of recent field isolates from Alabama (Ala1) and California (Cal3) revealed distinct shifts in homology in the M gene, indicating two apparent recombination events between the Holland 52/Mass41-like strain and an Ark-like strain, both origins of commonly used vaccine strains. Putative sites of recombination could also be identified in both the E and M ORFs of laboratory strains of IBV.     

Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene

Infectious bronchitis virus (IBV) isolates recovered in Russia, Ukraine, and Kazakhstan between 2007 and 2010 were subjected to molecular characterization and compared with those isolated a decade ago. The IBV genome was detected in 202 out of 605 field samples from chickens with various clinical signs. Partial sequencing of the S1 gene revealed 153 vaccine strains and 49 field isolates of several genetic groups. Massachusetts, 793/B and D274 remained the predominant IBV genotypes along with QX, whereas B1648, Italy-02, Arkansas and variants accounted for about 12% of the total number. Three IBVs contained recombinant S1 gene sequences comprising genome fragments of QX-type field isolates and vaccine strains H120 (UKR/02/2009) or 4/91 (RF/03/2010), and vaccine strains H120 and D274 (RF/01/2010). The results of the present study showed a significant decline in prevalence of variant IBVs and a further spread of QX-type isolates in commercial chicken flocks in Russia as compared with the 1998 to 2002 data.     

Protective effect of vaccination in chicks with local infectious bronchitis viruses against field virus challenge.

Avian infectious bronchitis virus (IBV) causes tremendous economic losses to the poultry industry worldwide. Different serotypes of this virus show little cross-protection. The present study investigated the relationship between differences in the genotype based on the N-terminus of the spike protein and the protection provided by vaccination with IBVs. Cross-immunization tests were performed using both killed and live viruses in specific-pathogen-free chicks. One-day-old chicks were immunized with killed or high [10(5) 50% embryo infectious dose (EID50)] or low (10(3) EID50) doses of live IBV viruses and challenged with homologous or heterologous IBV strains. The immunization efficacy was evaluated by virus isolation from the challenged chicks. In the killed-virus and high-dose live-virus test groups, IBV vaccination protected against challenge by homologous but not heterologous IBV strains. However, a low dose of live IBV showed no protection against virulent IBV challenge. These results indicate that both the genotype based on the N-terminus of the spike protein and the virus dose are essential to IBV protection from immunization. Thus, development of vaccines from different local strains is necessary to control infectious bronchitis in poultry.