Junfeng Sun

Comparative proteome analysis of tracheal tissues in response to infectious bronchitis coronavirus, Newcastle disease virus, and avian influenza virus H9 subtype virus infection.

Infectious bronchitis coronavirus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) H9 subtype are major pathogens of chickens causing serious respiratory tract disease and heavy economic losses. To better understand the replication features of these viruses in their target organs and molecular pathogenesis of these different viruses, comparative proteomic analysis was performed to investigate the proteome changes of primary target organ during IBV, NDV, and AIV H9 infections, using 2D‐DIGE followed MALDI‐TOF/TOF‐MS. In total, 44, 39, 41, 48, and 38 proteins were identified in the tracheal tissues of the chickens inoculated with IBV (ck/CH/LDL/97I, H120), NDV (La Sota), and AIV H9, and between ck/CH/LDL/97I and H120, respectively. Bioinformatics analysis showed that IBV, NDV, and AIV H9 induced similar core host responses involved in biosynthetic, catabolic, metabolic, signal transduction, transport, cytoskeleton organization, macromolecular complex assembly, cell death, response to stress, and immune system process. Comparative analysis of host response induced by different viruses indicated differences in protein expression changes induced by IBV, NDV, and AIV H9 may be responsible for the specific pathogenesis of these different viruses. Our result reveals specific host response to IBV, NDV, and AIVH9 infections and provides insights into the distinct pathogenic mechanisms of these avian respiratory viruses.

Recombinant Newcastle disease virus expressing the infectious bronchitis virus S1 gene protects chickens against Newcastle disease virus and infectious bronchitis virus challenge

The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV.

Genetic, antigenic, and pathogenic characteristics of avian infectious bronchitis viruses genotypically related to 793/B in China

Abstract In this study, 20 infectious bronchitis virus (IBV) strains, which were genotypically related to 793/B, as assessed by an S1 gene comparison and a complete genomic sequence analysis, were isolated and identified from 2009 to 2014 in China. Phylogenetic analysis, network tree, similarity plot analysis, Recombination Detection Program 4(RDP4) and sequence comparison revealed that 12 of the 20 isolates were likely the reisolated vaccine virus. One isolate, ck/CH/LSD/110857, was shown to have originated from recombination events between H120- and 4/91-like vaccine strains that did not result in changes of antigenicity and pathogenicity. The remaining seven IBV isolates were shown to have originated from recombination events between a 4/91-like vaccine strain and a GX-LY9-like virus, which were responsible for the emergence of a novel serotype. A vaccination-challenge test found that vaccination with the 4/91 vaccine strain did not provide protection against challenge with the recombinant viruses. In addition, the results showed that the recombination events between the vaccine and field strains resulted in altered genetics, serotype, antigenicity, and pathogenicity compared with those of their deduced parental viruses. The results are important not only because this virus is of economic importance to poultry industry, but also because it is important for elucidating the origin and evolution of other coronaviruses.

Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin–neuraminidase (HN) glycoprotein

Galectin-1 is an important immunoregulatory factor and can mediate the host–pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro. We confirmed that CG-1B interacted with NDV hemagglutinin–neuraminidase (HN) glycoprotein, in which the specific G4 N-glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N-glycans on HN glycoprotein.

Genome characterization, antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south China

Abstract In 2014, three infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0111/14, γCoV/ck/China/I0114/14 and γCoV/ck/China/I0118/14, were isolated and identified from chickens suspected to be infected with IBV in Guangxi province, China. Based upon data arising from S1 sequence and phylogenetic analyses, the three IBV isolates were genetically different from other known IBV types, which represented a novel genotype (GI-29). Virus cross-neutralization tests, using γCoV/ck/China/I0111/14 as a representative, showed that genotype GI-29 was antigenically different from all other known IBV types, thus representing a novel serotype. Complete genomic analysis showed that GI-29 type viruses were closely related to and might originate from a GX-YL5-like virus by accumulation of substitutions in multiple genes. These GI-29 viral genomes are still evolving and diverging, particularly in the 3′ region, although we cannot rule out the possibility of recombination events occurring. For isolate γCoV/ck/China/I0114/14, we found that recombination events had occurred between nsps 2 and 3 in gene 1 which led to the introduction of a 4/91 gene fragment into the γCoV/ck/China/I0114/14 viral genome. In addition, we found that the GI-29 type γCoV/ck/China/I0111/14 isolate was a nephropathogenic strain and high pathogenic to 1-day-old specific pathogen-free (SPF) chickens although cystic oviducts were not observed in the surviving layer chickens challenged with γCoV/ck/China/I0111/14 isolate.

Genetic diversity of avian infectious bronchitis virus in China in recent years

Abstract In this study, 213 infectious bronchitis viruses (IBVs) were isolated from samples collected from 801 flocks suspected to be infected with IBV from January 2016 to December 2017 in China. By using complete nucleotide sequences of S1 gene we determined the phylogeny of these IBV isolates, which in turn allowed us to define six lineages/genotypes, a number of recombinants and a novel variant. The GI-19 lineage was the most frequently isolated type in China in recent years. Although scattered mutations in the S1 gene among the GI-19 lineage viruses were observed, we also noted different sublineages in the GI-19 lineage with unique mutations, suggesting a high degree of S1 gene variation since they were first isolated in the mid-1990s. We also isolated a number of vaccine-like viruses from vaccinated diseased chickens, although more work is needed to differentiate the reisolation of vaccine strains from field strains of the same serotype. One of the important findings in this study is that the prevalence of the TW I type viruses in GI-7 lineage has been increasing in recent years in China. Another important finding is that recombination events occurred between the predominant GI-19 lineage and the commonly used 4/91 vaccine, which gave rise to distinct IBV isolates. In addition, a novel IBV isolate, together with a reference strain in GenBank database, were found to form a novel lineage/genotype that was remarkably distinct from established lineages. The characteristics of the antigenicity, tissue tropism, pathogenicity and complete genome were required for further investigation for the recombinants and the viruses in different sublineages and novel lineages. Meanwhile, permanent monitoring of circulating strains was needed to monitor the emerging viruses and rationally modify vaccination strategies in the field situation.

Genetic, antigenic, and pathogenic characteristics of Newcastle disease viruses isolated from geese in China

Four Newcastle disease virus (NDV) strains were isolated from domestic, commercial geese that showed clinical signs that were believed to be the result of NDV infections. The genetic, antigenic, and pathogenic characteristics of the 4 NDVs were compared with those of NDV strains that were isolated from chickens. The complete genomes of 2 of the NDV strains contained 15,186 nucleotides (nt); the other 2 contained 15,192 nt, and exhibited the typical genomic organization of genotype II NDV and molecular characteristics of VIId NDVs. Phylogenetic analysis confirmed that the genotype II and VIId NDVs that were isolated from geese belonged to the same clusters as the corresponding genotypes of the chicken isolates. A serologic assay demonstrated that the antigenic relatedness among the NDVs was associated with their genotypes, rather than their hosts, and that amino acid substitutions in the F and/or HN proteins may contribute to the antigenic differences among these NDV genotypes. Geese infected with genotype VIId NDVs that were isolated from geese and chickens showed similar pathologic characteristics. NDVs that were isolated from geese did not differ in genetic, serologic, and pathogenic characteristics from those isolated from chickens, indicating that these NDVs were derived from chicken NDVs. Given the significance of geese in NDV epidemiology, effective biosecurity measures should be adopted to prevent the interspecies transmission of NDVs.

Genetics, antigenicity and virulence properties of three infectious bronchitis viruses isolated from a single tracheal sample in a chicken with respiratory problems

Abstract Three different IBV genotypes/serotypes, designated ck/CH/LDL/150434–I (LDL/150434–I), ck/CH/LDL/150434–II (LDL/150434–II) and ck/CH/LDL/150434–III (LDL/150434–III), were detected in a single tracheal sample from a chicken showing signs of respiratory disease. The viruses were isolated using a cross-neutralization test and limiting dilution in embryonated specific-pathogen-free (SPF) eggs. Isolate LDL/150434–I was a re-isolation of H120 vaccine strain that was introduced into the chicken flock by vaccination, transmitted between chickens, and later accumulated several genomic mutations. Isolate LDL/150434–II was a novel variant that originated from recombination events between H120 and ck/CH/LDT3/03-like viruses. The widespread use of H120 vaccine, which offered incomplete protection against heterotypic IBVs in the fields, may play important roles in the emergence of such a novel genetic variant. Based on the analysis of S1 and complete genomic sequence, isolate LDL/150434–III was related genetically but distinct from the established strains of nrTW I type viruses of GI-7 lineage circulating in Mainland China since 2009. The three IBV isolates were avirulent when they infected SPF chickens. Furthermore, synergistic effects on pathogenicity were not observed when the different types co-infected the SPF chickens. However, the isolates persisted in the respiratory tracts longer in combined infected birds than those in individual infected birds. The results provide insights into the evolution of the viruses and co-infection of chickens with different virus serotypes.

Characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/CH/LJL/140901-like backbone and an S2 fragment from a 4/91-like virus

Abstract In this study, we isolated an infectious bronchitis virus, designated I1101/16, from broiler breeders in China. Analysis of the S1 gene showed that isolate I1101/16 was genetically close to strain ck/CH/LJL/140901, which belongs to the TW I genotype (also known as lineage GI-7 based on the recent IBV classification), however the S2 gene showed genetic diversity comparing to that of S1 gene. Comparison of the genomic sequences showed that the genome of isolate I1101/16 was similar to that of strain ck/CH/LJL/140901 from the 5′ end of the genome to the 5′ end of the S2 gene and from the 5′ end of the 3a gene to the end of the genome, whereas the remaining parts of the genome sequences were more closely related to those of strain 4/91 than those of ck/CH/LJL/140901, thereby suggesting that recombination might have occurred during the origin of the virus. SimPlot and Bootscan analysis of the complete genomic sequence confirmed this hypothesis, where it showed that isolate I1101/16 arose through recombination events between ck/CH/LJL/140901- and 4/91-like viruses. Isolate I1101/16 and strain ck/CH/LJL/140901 shared identical amino acids in almost all five of their B cell epitopes, but the two viruses had a serotype relatedness value of 65, which is well below 80, i.e., the lower cutoff value for viruses of the same serotype. In addition, pathogenicity tests demonstrated that isolate I1101/16 was more pathogenic to 1-day-old specific-pathogen-free chickens than strain ck/CH/LJL/140901, according to analysis of the clinical signs, whereas strain ck/CH/LJL/140901 exhibited prolonged replication and shedding after challenge compared with isolate I1101/16. This study did not provide evidence that recombination can directly alter the antigenicity, virulence, replication, shedding, and tissue tropism of a virus, but it did show that recombination events are likely to be major determinants of viral evolution.

Isolation and Characteristics of the Arkansas-Type Infectious Bronchitis Virus in China

SUMMARY Two infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0712/11 (I0712/11) and γCoV/ck/China/I0108/17 (I0108/17), were isolated from diseased chicken flocks in different provinces in China and genotyped as Arkansas (Ark)-type viruses with three other Chinese Ark field strains, the Jilin vaccine strain, and the American Ark- and Ark DPI-like viruses. Complete genomic sequence analysis and pairwise comparison of nucleotide sequences encoding the S1 subunit of the spike protein and other structural and accessory proteins revealed that Chinese Ark field isolates were genetically closely related to the Jilin vaccine and American ArkDPI11 strains, although extensive nucleotide changes were found across the genomes of Chinese Ark field isolates. This suggests that Chinese Ark-type isolates are derived from the Jilin vaccine, and have diverged and evolved independently by point mutations since introduction into China. It is also possible that the Chinese Ark viruses have arisen as a result of different introductions of American ArkDPI11-like strains from the United States; this hypothesis requires further investigation. Pathogenicity testing showed that Chinese Ark viruses had comparable virulence to that of the Massachusetts-type M41 strain, although they had lower affinity for the kidneys of chickens than the M41 strain had. Although Ark-type viruses are not widespread in China, surveillance and updating the currently applied vaccination strategy for sound protection against IBV disease are important because this type of virus has caused heavy economic losses in the United States.