Huixin Li

Molecular characterization and pathogenicity of infectious bronchitis coronaviruses: complicated evolution and epidemiology in china caused by cocirculation of multiple types of infectious bronchitis coronaviruses.

Objective: To monitor and study the molecular epidemiology, evolution and pathogenicity of infectious bronchitis viruses (IBVs) in China in recent years and further our knowledge of the evolution of IBVs. Methods: Thirty-seven IBV isolates were isolated from commercial chickens in China. The isolates were characterized by RT-PCR, sequencing, typing and analyzing the entire S1 gene. In addition, 4 selected IBV isolates were used to experimentally infect the specific pathogen-free chickens to study their pathogenicity. Results: Three types of IBV have been cocirculating in chicken flocks in China in recent years. Unique insertions and deletions in S1 protein regions were identified among different types of IBV. Moreover, a new IBV strain was isolated and identified in a layer hen. S1 gene analysis showed that a recombination event had occurred in the virus’s evolutionary process. In addition, experimental infection has shown that IBV isolates have been nephropathogenic in China in recent years. Conclusion: Mutations, insertions, deletions and recombination of the S1 protein gene contribute to the genetic diversity of IBV in China. Cocirculation of multiple types of IBV in field conditions in China renders its epidemiology and evolution very complicated, indicating the necessity for development of new vaccines or vaccine strategies.

Altered pathogenicity, immunogenicity, tissue tropism and 3'-7 kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos

Abstract In this study, we attenuated a Chinese LX4-type nephropathogenic infectious bronchitis virus (IBV) strain, CK/CH/LHLJ/04V, by serial passage in embryonated chicken eggs. Based on sequence analysis of the 3′-7kb region, the CK/CH/LHLJ/04V virus population contained subpopulations with a mixture of genetic mutants. The titers of the virus increased gradually during serial passage, but the replication capacity decreased in chickens. The virus was partially attenuated at passage 40 (P40) and P70, and was fully attenuated at P110. It lost immunogenicity and kidney tropism at P110 and P70, respectively. Amino acid substitutions were found in the 3′-7kb region, primarily in the spike (S) protein. Substitutions in the S1 subunit occurred between P3 and P40 and all subpopulations in a virus passage showed the same substitutions. Other substitutions that occurred between P70 and P110, however, were found only in some subpopulations of the virus passages. A 109-bp deletion in the 3′-UTR was observed in most subpopulations of P70 and P110, and might be related to virus replication, transcription and pathogenicity. The changes described in the 3′-7kb region of the virus are possibly responsible for virus attenuation, immunogenicity decrease and tissue tropism changes; however, we cannot exclude the possibility that other parts of the genome may also be involved in those changes.

Serotype shift of a 793/B genotype infectious bronchitis coronavirus by natural recombination.

Abstract An infectious bronchitis coronavirus, designated as ck/CH/LHLJ/140906, was isolated from an infectious bronchitis virus (IBV) strain H120-vaccinated chicken flock, which presented with a suspected infectious bronchitis virus (IBV) infection. A phylogenetic analysis based on the S1 gene clustered ck/CH/LHLJ/140906 with the 793/B group; however, a pairwise comparison showed that the 5′ terminal of the S1 gene (containing hypervariable regions I and II) had high sequence identity with the H120 strain, while the 3′ terminal sequence was very similar to that of IBV 4/91 strain. A SimPlot analysis of the complete genomic sequence, which was confirmed by a phylogenetic analysis and nucleotide similarities using the corresponding gene fragments, suggested that isolate ck/CH/LHLJ/140906 emerged from multiple recombination events between parental IBV strains 4/91 and H120. Although the isolate ck/CH/LHLJ/140906 had slightly higher S1 amino acid sequence identity to strain 4/91 (88.2%) than to strain H120 (86%), the serotype of the virus was more closely related to that of the H120 strain (32% antigenic relatedness) than to the 4/91 strain (15% antigenic relatedness). Whereas, vaccination of specific pathogen-free chickens with the 4/91 vaccine provided better protection against challenge with ck/CH/LHLJ/140906 than did vaccination with the H120 strain according to the result of virus re-isolation. As the spike protein, especially in the hypervariable regions of the S1 domain, of IBVs contains viral neutralizing epitopes, the results of this study showed that recombination of the S1 domain resulted in the emergence of a new serotype.

Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China.

Abstract In this study, 418 IBVs were isolated in samples from 1717 chicken flocks. Twenty-nine of the isolates were classified as the Massachusetts genotype. These 29 isolates, as well as two previously isolated Massachusetts genotype IBV strains, were studied further. Of the 31 strains, 24 were H120-like and two were M41-like isolates as determined by complete genomic sequence analysis, indicating that most of the IBV isolates were likely the reisolated vaccine virus. The remaining five IBV isolates, ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and ck/CH/LHB/130573, were shown to have originated from natural recombination events between an H120-like vaccine strain and other types of viruses. The virus cross-neutralization test found that the antigenicity of ck/CH/LHB/111172, ck/CH/LSD/111219, and ck/CH/LHB/130598 was similar to that of H120. Vaccination with the H120 vaccine offered complete protection against challenge with these isolates. However, isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 were serotypically different from their parental viruses and from other serotypes in this study. Furthermore, vaccination with the H120 vaccine did not provide protection against challenge with these two isolates. The results of this study demonstrated that recombination is the mechanism that is responsible for the emergence of new serotype strains, and it has the ability to alter virus serotypes. Therefore, IBV surveillance of chicken flocks vaccinated with IBV live vaccines, as well as the consideration of new strategies to effectively control IBV infection using inactivated or/and genetically engineered vaccines, is of great importance.

Origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus isolate ck/CH/LJL/111054.

Abstract Recombination among infectious bronchitis viruses (IBVs), coupled with point mutations, insertions, and deletions that occur in the genome, is thought to contribute to the emergence of new IBV variants. In this study an IBV, ck/CH/LJL/111054, was isolated from a H120-vaccinated chicken, which presented with a suspected IBV infection. Phylogenetic analysis of the S1 subunit sequence confirmed that strain ck/CH/LJL/111054 is of the Connecticut-type; however, further extensive full-length genomic analysis identified the occurrence of recombination events. Therefore, strain ck/CH/LJL/111054 may have originated from recombination events between Conn- and Mass-like strains at three recombination breakpoints: two located within the nsp3 gene sequence and one in the nsp12 gene sequence. Further, the uptake of the 5′ untranslated regions, nsp2, parts of nsp3, nsp4–11, and parts of nsp 12 from Mass-like virus by ck/CH/LJL/111054 might have resulted in changes in viral replication efficiency rather than antigenic changes, via cross-neutralization analysis with the H120 strain. Recombination events coupled with the accumulation of mutations in the ck/CH/LJL/111054 genome may account for its increased virulence in specific-pathogen free chickens.

A novel B-cell epitope of avian infectious bronchitis virus N protein.

This report describes the identification of a novel linear B-cell epitope at the N-terminus of the avian infectious bronchitis virus (IBV) nucleocapsid (N) protein. A monoclonal antibody designated 2D2 was prepared against the IBV N protein, and a series of 23 partially overlapping, tagged fragments of the IBV N gene were expressed. These peptides and five heterogeneous IBV strains were used to identify the epitope recognized by 2D2 using enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. The linear motif (11)DSPAPIIKLGG(21), located at the N-terminus of the N protein, was identified as the epitope bound by 2D2. ELISA and Western blotting also showed that this epitope was recognized by IBV-positive chicken serum. We propose that this motif is a linear B-cell epitope of N protein, and is the core sequence for reactivity, as demonstrated by binding of 2D2 to the truncated peptides and different IBV strains. Alignment and comparison of the 2D2-defined epitope with other coronavirus sequences showed that the epitope is conserved among avian coronaviruses. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the IBV N protein.

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

ABSTRACT 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.

Altered pathogenicity of a tl/CH/LDT3/03 genotype infectious bronchitis coronavirus due to natural recombination in the 5′- 17 kb region of the genome

Abstract An infectious bronchitis coronavirus, designated as ck/CH/LGX/130530, was isolated from an IBV strain H120-vaccinated chicken in this study. Analysis of the S1 gene showed that isolate ck/CH/LGX/130530 was a tl/CH/LDT3/03-like virus, with a nucleotide sequence similarity of 99%. However, a complete genomic sequence analysis showed that ck/CH/LGX/130530 was more closely related to a Massachusetts type strain (95% similarity to strain H120) than to the tl/CH/LDT3/03 strain (86%), suggesting that recombination might have occurred during the origin of the virus. A SimPlot analysis of the complete genomic sequence confirmed this hypothesis, and it showed that isolate ck/CH/LGX/130530 emerged from a recombination event between parental IBV H120 strain and pathogenic tl/CH/LDT3/03-like virus. The results obtained from the pairwise comparison and nucleotide similarity showed that the recombination breakpoint was located in the nsp14 gene at nucleotides 17055–17083. In line with the high S1 gene sequence similarity, the ck/CH/LGX/130530 isolate was serotypically close to that of the tl/CH/LDT3/03 strain (73% antigenic relatedness). Furthermore, vaccination with the LDT3-A vaccine, which was derived from the tl/CH/LDT3/03 strain by serial passaging in chicken eggs, provided good protection against challenge with the tl/CH/LDT3/03 strain, in contrast to the poor protection offered with the H120 vaccine. Interestingly, isolate ck/CH/LGX/130530 exhibited low pathogenicity toward specific-pathogen-free chickens compared with the nephropathogenic tl/CH/LDT3/03 strain, which was likely due to natural recombination in the 5′ 17-kb region of the genome. Our results also indicate that the replicase gene of IBV isolate ck/CH/LGX/130530 is associated with viral pathogenicity.

Identification of a novel linear B-cell epitope in the M protein of avian infectious bronchitis coronaviruses

This report describes the identification of a novel linear B-cell epitope at the C-terminus of the membrane (M) protein of avian infectious bronchitis virus (IBV). A monoclonal antibody (MAb) (designated as 15E2) against the IBV M protein was prepared and a series of 14 partially-overlapping fragments of the IBV M gene were expressed with a GST tag. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using MAb 15E2 to identify the epitope. A linear motif, 199FATFVYAK206, which was located at the C-terminus of the M protein, was identified by MAb 15E2. ELISA and western blotting also showed that this epitope could be recognized by IBV-positive serum from chicken. Given that 15E2 showed reactivity with the 199FATFVYAK206 motif, expressed as a GST fusion protein, in both western blotting and in an ELISA, we proposed that this motif represented a linear B-cell epitope of the M protein. The 199FATFVYAK206 motif was the minimal requirement for reactivity as demonstrated by analysis of the reactivity of 15E2 with several truncated peptides that were derived from the motif. Alignment and comparison of the 15E2-defined epitope sequence with the sequences of other corona-viruses indicated that the epitope is well conserved among chicken and turkey coronaviruses. The identified epitope should be useful in clinical applications and as a tool for the further study of the structure and function of the M protein of IBV.

Unique sequence characteristics of genes in the leftmost region of unique long region in duck enteritis virus.

Objective: Our objective was to further understand the evolutionary position of duck enteritis virus (DEV) in the family Herpesviridae and to determine the genomic structure in the leftmost region of the unique long region (UL) of DEV. Methods: ‘Targeted gene walking PCR’ was used to amplify the unknown gene of DEV clone-03 strain adjacent to known sequences in the UL region. Open reading frames (ORFs) were determined by Gene Runner and an online transcriptional element search engine. The phylogenetic tree was constructed by DNAStar based on the deduced amino acid sequences. Results: A fragment of 13,947 bp, containing 5 ORFs (designated ICP4, ORF1, ORF2, LORF2 and LORF3, respectively), was amplified from the DEV clone-03 strain genome. In addition, a tandem repeat region of α-type-like sequences was detected between ICP4 and LORF2. Phylogenetic analysis of ICP4 indicated that DEV ICP4 should be clustered into Varicellovirus. DEV ORF1 and ORF2 were unique among herpesviruses. Additionally, the existence and characteristics of LORF2 and LORF3 indicated a close relationship with Mardivirus members. Conclusion: The characteristics and gene content in the leftmost end of the UL region of the DEV genome showed that the DEV genome is different from those of other herpesviruses.