Membrane (M) protein genes of 20 infectious bronchitis virus (IBV) strains isolated in China between 1995 and 2004 were sequenced and analyzed. The M genes of twenty isolates were composed of 672 to 681 nucleotides, encoding polypeptides of 223 to 226 amino acid residues. Variations of the deduced amino acids of M gene mainly occurred at positions 2 to 17 and 221 to 233, comparing with that of the IBV strain LX4. There were deletions or insertions in the M gene of Chinese isolates at amino acid position 2 to 6, leading to the loss or gain of a glycosylation site. Phylogenetic tree based on amino acid sequences of M genes from 20 Chinese isolates and 34 reference strains showed that they were classified into five distinct clusters. Most of the Chinese IBV strains were included in clusters II and IV, forming distinct groups. The isolates in cluster II showed a close evolutionary relationship with Taiwan isolates. Furthermore, recombination especially the recombination between field isolates and vaccine strains had been observed while comparing the phylogeny of M genes with those of S1 and N genes.
Amino Acid Sequence ; China ; Genetic Variation ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Sequence Homology, Amino Acid ; Viral Matrix Proteins ; genetics

Monovalent antisera of 3 vaccine strains and 7 representative field isolates were prepared based on the comparison of genetic diversity of the hypervariable region I of S1 gene (HVR I from 3 infectious bronchitis (IB) vaccine strains (H120, Ma5 and 4/91) ,one reference strain M41 and 26 IB field isolates. These 30 strains were classified in 7 different genotypes, respectively. Virus-neutralizing test on tracheal organ cultures (TOC) with chicken embryo were used to evaluate relatedness values of the antigenicity based on the antibody titer, to analyze the antigenic relationships between the isolates and vaccine strains, as well as to determine the serotypes of 26 IB viruses isolated from the field in Guangxi between 1985 and 2008. The results showed 30 strains were classified into 7 distinct serotypes and there were two predominant serotypes within the 26 isolates, serotypes 1 (totally 13 isolates) and serotype 2 (totally 5 isolates), respectively. In addition, there were some differences observed between the results of serotyping and the genotyping (including the S1, N, M and 3'UTR). The results of the study demonstrated that there were different predominant serotypes and multiple serotypes of IBV circulated in Guangxi in recent years, antigenic variation existed between Guangxi field isolates and vaccine strains.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Chick Embryo ; Chickens ; China ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genetic Variation ; Genotype ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Phylogeny ; Poultry Diseases ; immunology ; virology ; Viral Envelope Proteins ; genetics ; immunology

The entire S1 protein gene of five infectious bronchitis (IB) vaccine strains (JAAS, IBN, Jilin, J9, H120) used in China were compared with that of the IB field isolate CK/CH/LDL/97 I present in China. The nucleotide and deduced amino acid similarities between the five IB vaccine strains and the field strain, CK/CH/LDL/97 I, were not more than 76.4% and 78.7%, respectively. Phylogenetic analysis based on the S1 gene showed that the vaccine strains and the field strain belonged to different clusters and had larger evolutionary distances, indicating that they were of different genotypes. The five vaccine strains were used for protection test against challenge of the field isolate CK/CH/LDL/97 I. The chickens inoculated with five vaccine strains showed morbidity as high as 30%-100% after challenged with the CK/CH/ LDL/97 I strain. The organ samples at 5 days post challenge showed that the viral detection rates were 50%-90% and 10%-30% for trachea and kidney, respectively. The live attenuated vaccines only provided partial protection to the vaccinated chickens against heterologous IBV infection, CK/CH/LDL/97 I.
Animals ; Antibodies, Viral ; blood ; Chickens ; virology ; Coronavirus Infections ; prevention & control ; veterinary ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Membrane Glycoproteins ; genetics ; Phylogeny ; Poultry Diseases ; prevention & control ; Spike Glycoprotein, Coronavirus ; Vaccines, Attenuated ; immunology ; Viral Envelope Proteins ; genetics ; Viral Vaccines ; immunology

The S1 gene hypervariable region I (HVR I) of 22 infectious bronchitis virus (IBV) strains isolated in Guangxi during the period of 1985-2007 were sequenced and compared to that of the other IBV reference strains and the pigeon coronavirus isolates. A phylogenetic tree based on nucleotide sequences of HVR I of all the IBV showed that they were classified into 5 distinct Clusters. 16 out of 22 IBV isolates were grouped into Cluster I, and had higher homology with pigeon coronavirus isolates but lower homology with the Massachusetts (Mass) type vaccine strains. There were 4 and 3 amino-acid residues inserted at the sites of 33-34 and 34-35 respectively within HVR I in 15 isolates, except in isolate GX-NN6 there had 4 amino-acid residues inserted at the both sites; isolates GX-YL1 and GX-NN2 had close relationship with Mass type vaccine strains, and they shared Cluster II; isolates GX-G and GX-XD of Cluster III had close relationship with the Japanese strain JP Miyazaki 89 which was isolated at the same period; isolates GX-YL6 and GX-NN7 of Cluster V had close relationship with the European strain 4/91. The results showed that there were high phylogenetic diversity among the IBVs prevailed in the field in Guangxi resulting from the commonly occurred mutation or insertion within the S1 gene HVR I of the viruses, and majority of the isolates had lower homology with the commonly used Mass type vaccine strains. There was much higher homology among viruses isolated in the same period of time, but without distinct difference in geographical origins.
Amino Acid Sequence ; Animals ; Chickens ; virology ; Genetic Variation ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Membrane Glycoproteins ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; chemistry ; genetics

We wished to ascertain the prevalence as well as the genetic and antigenic variation of infectious bronchitis viruses (IBVs) circulating in the Guangxi Province of China in recent years. The S1 gene of 15 IBV field isolates during 2012-2013 underwent analyses in terms of the similarity of amino-acid sequences, creation of phylogenetic trees, recombination, and serologic identification. Similarities in amino-acid sequences among the 15 isolates of the S1 gene were 54.3%-99.6%, and 43.3%-99.3% among 15 isolates and reference strains. Compared with the vaccine strain H120, except for GX-YL130025, the other 14 isolates showed a lower similarity of amino-acid sequences of the S1 gene (65.1-81.4%). Phylogenetic analyses of the S1 gene suggested that 15 IBV isolates were classified into eight genotypes, with the predominant genotype being new-type II. Recombination analyses demonstrated that the S1 gene of the GX-NN130048 isolate originated from recombination events between vaccine strain 4/91 and a LX4-like isolate. Serotyping results suggested that seven serotypes prevailed during 2012-2013 in Guangxi Province, and that only one isolate was consistent with the vaccine strain H120 in serotype (which has been used widely in recent years). The serotype of recombinant isolate GX-NN130048 was different from those of its parent strains. These results suggested that not only the genotype, but also the serotype of IBV field isolates in Guangxi Province had distinct variations, and that increasing numbers of genotypes and serotypes are in circulation. We showed that recombination events can lead to the emergence of new serotypes. Our study provides new evidence for understanding of the molecular mechanisms of IBV variations, and the development of new vaccines against IBVs.
Animals ; Antibodies, Viral ; blood ; Chickens ; China ; Coronavirus Infections ; blood ; veterinary ; virology ; Genetic Variation ; Genotype ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; blood ; virology ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; immunology

The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005, while insertions and deletions were located within the S1 gene. These results indicated that CK/CH/SD09/005 is a novel variant that may be derived from the QX-like strains that are prevalent in China. Multiple genetic mechanisms, including recombinations, mutations, insertions and deletions, are likely to have contributed to the emergence of this IBV strain.
Animals ; Chickens ; China ; Coronavirus Infections ; veterinary ; virology ; Genome, Viral ; Genomics ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand.
Animals ; Chickens ; Coronavirus Infections/*veterinary/virology ; Infectious bronchitis virus/*genetics/*isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases/*virology ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid ; Thailand ; Viral Proteins/chemistry

In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.
Amino Acid Sequence ; Animals ; Chick Embryo ; Coronavirus Infections ; veterinary ; virology ; Ducks ; virology ; Female ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment

In order to investigate the prevalence and track genetic and antigenic evolutions of infectious bronchitis virus (IBV) and their prevalence in Guangxi, China since 1985, gene amplification and sequencing and virus neutralization (VN) test on chicken embryo tracheal organ cultures were used in genotyping and serotyping of 28 IBV isolates during 2009-2011 in Guangxi. The results of N gene sequencing and comparison showed that the 28 isolates and reference strains were classified into three groups, and most isolates belonged to group Ill, while the isolates in 1985-2008 belonged to groups IV and II. The data of VN test indicated that the 28 isolates belonged to 6 serotypes; among them, 71. 4% belonged to serotypes 1, 2, and 3, and 11 (39.3%) shared the same serotype with the current vaccine strains. Given the data of our previous study, it is found that prevalent serotypes and their proportions varied in different areas of Guangxi and during different periods. These data lay a good foundation for developing an oil-emulsified inactivated polyvalent vaccine containing local dominant serotypes for the effective prevention and control of infectious bronchitis.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Chickens ; China ; epidemiology ; Coronavirus Infections ; epidemiology ; immunology ; veterinary ; virology ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; epidemiology ; immunology ; virology

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.
Amino Acid Sequence ; Animals ; Coronavirus Infections/*veterinary/virology ; Glycoproteins/chemistry/*genetics ; Infectious bronchitis virus/*classification/*genetics/isolation & purification ; Molecular Sequence Data ; *Phylogeny ; Polymorphism, Restriction Fragment Length ; Poultry ; Poultry Diseases/*virology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Alignment/veterinary ; Sequence Analysis ; Sequence Homology, Amino Acid ; Viral Proteins/chemistry/genetics