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Ducks are an economically important waterfowl but a natural reservoir for some zoonotic pathogens, such as influenza virus and flaviviruses. Our understanding of the duck immune system and its interaction with viruses remains incomplete. In this study, we constructed the transcriptomic landscape of duck circulating immune cells, the first line of defense in the arthropod-borne transmission of arboviruses, using high-throughput single-cell transcriptome sequencing, which defined 14 populations of peripheral blood leukocytes (PBLks) based on distinct molecular signatures and revealed differences in the clustering of PBLks between ducks and humans. Taking advantage of in vivo sex differences in the susceptibility of duck PBLks to avian tembusu virus (TMUV) infection, a mosquito-borne flavivirus newly emerged from ducks with a broad host range from mosquitos to mammals, a comprehensive comparison of the in vivo dynamics of duck PBLks upon TMUV infection between sexes was performed at the single-cell level. Using this in vivo model, we discovered that TMUV infection reprogrammed duck PBLks differently between sexes, driving the expansion of granulocytes and priming granulocytes and monocytes for antiviral immune activation in males but decreasing the antiviral immune activity of granulocytes and monocytes by restricting their dynamic transitions from steady states to antiviral states with a decrease in the abundance of circulating monocytes in females. This study provides insights into the initial immune responses of ducks to arthropod-borne flaviviral infection and provides a framework for studying duck antiviral immunity.

Fowlpox virus (FPV) is used as a vaccine vector to prevent diseases in poultry and mammals. The insertion site is considered as one of the main factors influencing foreign gene expression. Therefore, the identification of insertion sites that can stably and efficiently express foreign genes is crucial for the construction of recombinant vaccines. In this study, we found that the insertion of foreign genes into ORF054 and the ORF161/ORF162 intergenic region of the FPV genome did not affect replication, and that the foreign genes inserted into the intergenic region were more efficiently expressed than when they were inserted into a gene. Based on these results, the recombinant virus rFPVNX10-NDV F–E was constructed and immune protection against virulent FPV and Newcastle disease virus (NDV) was evaluated. Tests for anti-FPV antibodies in the vaccinated chickens were positive within 14 days post-vaccination. After challenge with FPV102, no clinical signs of FP were observed in vaccinated chickens, as compared to that in the control group (unvaccinated), which showed 100% morbidity. Low levels of NDV-specific neutralizing antibodies were detected in vaccinated chickens before challenge. After challenge with NDV ck/CH/LHLJ/01/06, all control chickens died within 4 days post-challenge, whereas 5/15 vaccinated chickens died between 4 and 12 days post-challenge. Vaccination provided an immune protection rate of 66.7%, whereas the control group showed 100% mortality. These results indicate that the ORF161/ORF162 intergenic region of FPVNX10 can be used as a recombination site for foreign gene expression in vivo and in vitro.

Highlights • A novel genotype, designated as GVII-1, has been identified in south China over a 5-year period. • GVII-1 was derived from two recombination events that replaced the spike gene in a GI-18-like virus with an as-yet-unidentified sequence. • GVII-1 represented a novel serotype. • GVII-1 showed a low affinity to the respiratory tract in chickens., Recombination events are known to contribute to the emergence of novel infectious bronchitis virus (IBV) genotypes. In this study, we carried out detailed phylogenetic analysis and sequence comparisons based on 74 complete nucleotide sequences of the IBV S1 gene, including strain I0636/16 and 73 representative sequences from each genotype and lineage. The results showed that strain I0636/16 represented a novel genotype, designated as lineage 1 within genotype VII (GVII-1). Further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage 18 within genotype I (GI-18)-like virus with an as-yet-unidentified sequence, likely derived from another IBV strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. To the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/CH/LGX/111119. These results emphasize the importance of limiting exposure to novel IBVs that may serve as a source of genetic material for emerging viruses, as well as the importance of IBV surveillance in chicken flocks.

The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1), which is located in the early endosomes, could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and phorbol 12-myristate 13-acetate treatment, overexpression of dominant-negative (DN) caveolin-1, or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. In addition, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. Moreover, NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and that Rab5 is involved in the traffic and location of NDV. IMPORTANCE Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other members of the Avulavirus genus in the Paramyxoviridae family.

Highlight • The GI-19 strain was shown to be the dominant IBV lineage worldwide. • Isolate I0305/19 belongs to GI-19 lineage. • Isolate I0305/19 emerged through recombination events. • Isolate I0305/19 is a highly nephropathogenic strain. • Isolate I0305/19 showed broad tissue tropism in infected chickens., In the present study, an IBV strain I0305/19 was isolated from a diseased commercial broiler flock in 2019 in China with high morbidity and mortality. The isolate I0305/19 was clustered together with viruses in sublineage D of GI-19 lineage on the basis of the complete S1 sequence analysis. Isolate I0305/19 and other GI-19 viruses isolated in China have the amino acid sequence MIA at positions 110 -112 in the S protein. Further analysis based on the complete genomic sequence showed that the isolate emerged through at least four recombination events between GI-19 ck/CH/LJS/120848- and GI-13 4/91-like strains, in which the S gene was found to be similar to that of the GI-19 ck/CH/LJS/120848-like strain. Pathological assessment showed the isolate was a nephropathogenic IBV strain that caused high morbidity of 100% and mortality of 80% in 1-day-old specific-pathogen-free (SPF) chicks. The isolate I0305/19 exhibited broader tropisms in different tissues, including tracheas, lungs, bursa of Fabricius, spleen, liver, kidneys, proventriculus, small intestines, large intestines, cecum, and cecal tonsils. Furthermore, subpopulations of the challenge virus were found in tissues of infected chickens; this finding is important in understanding how the virulent IBV strains can potentially replicate and evolve to cause disease. This information is also valuable for understanding the mechanisms of replication and evolution of other coronaviruses such as the newly emerged SARS-CoV-2.

In this study, we isolated and identified 2 infectious bronchitis virus (IBV) strains from layer chickens soon after vaccination with the Massachusetts–Connecticut bivalent vaccine (Conn) and H120 and 4/91 booster vaccines in China in 2011. The results of cross-virus-neutralization tests and phylogenetic analysis of the S1 subunit of spike gene of these vaccine strains and other reference strains showed that strain LJL/110302 was of GI-19 lineage, whereas LLN/111169 was of the GI-1 lineage of the Conn serotype. Further comparative genomic analysis revealed that LLN/111169, an IBV strain with novel traits, originated from multiple recombination events (at least 3 recombination sites) between GI-19 and the Conn and 4/91 vaccine strains. LLN/111169 was pathogenic to specific pathogen-free (SPF) chickens. This is of prime importance because while IBV prevention measures worldwide are mainly dependent on modified live vaccine strains, our results showed that recombination between field and vaccine strains has produced a novel pathogenic IBV strain. In addition, LLN/111169 showed relatively broad tissue tropism (trachea, lungs, kidneys, and cecal tonsils) in infected SPF chickens. These results emphasize the importance of IBV surveillance in chicken flocks.

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.

Highlights • For the first time using reverse genetics to reveal the roles of HVRs in coronavirus. • The HVRs exchange from IBV S1 subunit weakened the adsorption during IBV infection in vitro. • The HVRs exchange in IBV S1 reduced ARV with Beaudette, but not sufficiently change serotypes. • The recombinant IBVs provided insights into reverse genetic vaccines., To study the roles of hypervariable regions (HVRs) in receptor-binding subunit S1 of the spike protein, we manipulated the genome of the IBV Beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three HVRs of the Beaudette strain with the corresponding fragments from a QX-like nephropathogenic isolate ck/CH/LDL/091022 from China. We characterized the growth properties of these recombinant IBVs in Vero cells and embryonated eggs, and their pathogenicity, tropism, and serotypes in specific pathogen-free (SPF) chickens. All seven recombinant IBVs proliferated in Vero cells, but the heterogenous HVRs could reduce their capacity for adsorption during in vitro infection. The recombinant IBVs did not significantly increase the pathogenicity compared with the Beaudette strain in SPF chickens, and they still shared the same serotype as the Beaudette strain, but the antigenic relatedness values between the recombinant strain and Beaudette strain generally decreased with the increase in the number of the HVRs exchanged. The results of this study demonstrate the functions of HVRs and they may help to develop a vaccine candidate, as well as providing insights into the prevention and control of IBV.

Highlights • IBV causes a highly contagious disease in chickens worldwide. • Recombination among IBVs is thought to contribute to the emergence of new IBV variants. • Strain I1101/16 is originated from recombination events between ck/CH/LJL/140901- and 4/91-like strains. • Strain I1101/16 is different from its putative parental viruses in terms of genome, antigenicity, pathogenicity, tissue tropism and viral shedding., 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.

Highlights • 793/B IBV was among the most important serotype to be recognized worldwide. • Twelve out of 20 IBVs genetically related to 793/B are reisolates of 4/91 vaccine strain. • ck/CH/LSD/110857 was originated from recombination events between H120- and 4/91-like strains. • Seven isolates were from recombination events between a 4/91-like strain and a GX-LY9-like virus., 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.

Highlights • The naturally recombinant TW I genotype represents a novel serotype. • Neither the commercial vaccines nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the naturally recombinant TW I serotype. • The naturally recombinant TW I serotype is very pathogenic, and is able to cause cystic oviducts in a high percentage of birds., Since 2009, strains of the naturally recombinant TW I genotype of infectious bronchitis virus (IBV) have caused considerable damage to the Chinese poultry industry. To better understand the antigenicity and pathogenesis of this genotype, the characteristics of the ck/CH/LDL/140520 strain were compared to those of four commercial IB vaccine strains that are used commonly in China, as well as four attenuated viruses that represent two types of IBV strains, which are believed to have originated in China and are the predominant IBV types circulating in chicken flocks in China and many other parts of the world. The results showed that all eight strains were genetically and serotypically different from the strain ck/CH/LDL/140520. Furthermore, neither the vaccine strains nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the ck/CH/LDL/140520 strain, indicating that it is necessary to develop new live vaccines or to evaluate the use of established vaccines in combination to control naturally recombinant TW I-type IBV strains in the future. Our results showed that strain ck/CH/LDL/140520 is very pathogenic, and that it is able to cause cystic oviducts in a high percentage of birds, as well as mortality due to nephritis and respiratory distress with complete tracheal ciliostasis, especially in chickens infected at 1 day of age.

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.

The study was conducted to evaluate whether avian β-defensins (AvBDs) could be induced by Newcastle disease virus (NDV) infection, and to investigate the potential signaling pathway of AvBD2 induction in response to NDV infection as well. First, mRNA expression of AvBDs (1–14) was evaluated in the chicken embryo fibroblasts (CEFs) infected with NDV strain F48E9 at 6, 12, 24, 36, and 48 h post-inoculation (hpi), respectively. The results demonstrated a significant induction of AvBD2 in CEFs elicited by the NDV strain. Then, we expressed and purified the AvBD2 proteins in both eukaryotic cells and prokaryotic cells. Of the two recombinant AvBD2 proteins, only the protein expressed in eukaryotic cells showed directly antiviral activity against NDV strain F48E9 in vitro. Ligands of toll-like receptors (TLRs) were chosen as alternatives to NDV to further study signaling pathway of AvBD2 induction here, due to insufficient upregulation of AvBD2 expression elicited by NDV. We found that the mRNA expression of AvBD2 was highly upregulated by Pam3CSK4, FLA-ST, and ODN-M362. Then, four inhibitors of signaling pathway, including inhibitors of JNK, ERK1/2, p38 MAPK, and NF-κB, were used in this study. Of the four inhibitors, only inhibition of the p38 MAPK signaling pathway significantly reduced AvBD2 expression after stimulation with Pam3CSK4, FLA-ST and ODN-M362, respectively. Taken together, these results revealed that AvBD2 play a pivotal role in host innate immunity response to NDV infection. The mRNA expression of AvBD2 might be regulated in a p38 MAPK-dependent manner.

Highlights • Recombination accounts for the emergence of IBV variants. • Strain ck/CH/LGX/130530 is originated from recombination events between a pathogenic tl/CH/LDT3/03- and H120-like strain. • Recombination event occurred in the 5′ end of the Gene 1 region did not result in changes in the genotype, serotype, and protectotype of the IBV. • The replicase gene of IBV ck/CH/LGX/130530 isolate is associated with viral pathogenicity., 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.

Highlights • Infectious bronchitis virus causes a respiratory disease in domestic chickens worldwide. • Recombination is thought to contribute to the emergence of IBV variants. • Strain ck/CH/LHLJ/140906 is originated from recombination events between 4/91- and H120-like strains. • Recombination of the S1 domain resulted in the emergence of a novel serotype of IBV., 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.

Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the "rule of six" that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don't form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs.

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.

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.

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.

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.

Highlights • Infectious bronchitis virus causes a respiratory disease in domestic chickens worldwide. • Recombination is thought to contribute to the emergence of IBV variants. • Strain ck/CH/LJL/111054 is originated from recombination events. • Concerns were raised regarding the recombination between different IBV types., 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.

Background: We previously attenuated the infectious bronchitis virus (IBV) strain CK/CH/LDL/97I and found that it can convey protection against the homologous pathogenic virus. Objective: To compare the full-length genome sequences of the Chinese IBV strain CK/CH/LDL/97I and its embryo-passaged, attenuated level to identify sequence substitutions responsible for the attenuation and define markers of attenuation. Methods: The full-length genomes of CK/CH/LDL/97I P5 and P115 were amplified and sequenced. The sequences were assembled and compared using the MEGALIGN program (DNAStar) and a phylogenetic tree was constructed using MEGA4 software. Results: The CK/CH/LDL/97I virus population contained subpopulations with a mixture of genetic mutants. Changes were observed in nsp4, nsp9, nsp11/12, nsp14, nsp15, nsp16, and ORF3a, but these did not result in amino acid substitutions or did not show functional variations. Amino acid substitutions occurred in the remaining genes between P5 and P115; most were found in the S region, and some of the nucleotide mutations resulted in amino acid substitutions. Among the 9 nsps in the ORF1 region, nsp3 contained the most nucleotide substitutions. Conclusions: Sequence variations in different genes, especially the S gene and nsp3, in the genomes of CK/CH/LDL/97I viruses might contribute to differences in viral replication, pathogenicity, antigenicity, immunogenicity, and tissue tropism.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/μL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Highlights • Two pheasant coronaviruses (PhCoVs) were isolated in 2017 in China. • The two PhCoVs were genetically similar to IBV. • Pathogenicity, replication, and shedding of PhCoV were obvious different when infected chickens and pheasants. • PhCoVs isolated from different outbreaks may have evolved independently from IBVs by adaption in pheasants., Two viruses were isolated in 2017 from commercial pheasants with severe clinical signs and mortality in Shandong and Anhui provinces, China, respectively. We examined the pathogenic effects of the viruses in chicken embryos and the size and morphology of the virus particles, performed phylogenetic analysis based on the S1 gene and complete genomic sequences, and examined the antibody responses against infectious bronchitis virus (IBV). The results suggested that the viruses I0623/17 and I0710/17 were avian coronaviruses and were identified as pheasant coronaviruses (PhCoV), with greatest similarity to IBV. Further investigations of the antigenicity, complete genome organization, substitutions in multiple genes, and viral pathogenicity, replication, and shedding in chickens and pheasants showed obvious differences between PhCoV and IBV in terms of antigenicity, and viral pathogenicity, replication, and shedding in chickens and pheasants. The close genetic relationship, but obvious differences between PhCoVs and IBVs suggested the IBVs could be the ancestors of PhCoVs, and that PhCoVs isolated from different outbreaks may have evolved independently from IBVs circulating in the specific region by adaption in pheasants. This hypothesis was supported by analysis of the S1 gene fragments of the two PhCoVs isolated in the current study, as well as PhCoVs isolated in the UK and selected IBV strains. Such analyses indicated different evolution patterns and different tissue tropisms between PhCoVs isolated in different outbreaks. Further studies are needed to confirm this hypothesis by studying the complete genomic sequences of PhCoVs from different outbreaks and the pathogenicity of IBVs in pheasants to compare and clarify the relationships between PhCoVs and IBVs.

Highlights ► Infectious bronchitis virus is one of the most important coronavirus causing respiratory disease in chicken. ► Analysis of protein epitope in virus could provide important insights that could facilitate the development of specific diagnostics and further understanding of the antigenic structure of protein. ► Two linear B-cell epitopes that were recognized by the mAbs 6D10 and 4F10, which corresponded to the amino acid sequences 242FGPRTK247 and 195DLIARAAKI230, respectively, in the IBV N protein. ► The two epitopes are very conserved among IBV serotypes., The nucleocapsid (N) protein of the infectious bronchitis virus (IBV) may play an essential role in the replication and translation of viral RNA. The N protein can also induce high titers of cross-reactive antibodies and cell-mediated immunity, which protects chickens from acute infection. In this study, we generated two monoclonal antibodies (mAbs), designated as 6D10 and 4F10, which were directed against the N protein of IBV using the whole viral particles as immunogens. Both of the mAbs do not cross react with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV) and subtype H9 avian influenza virus (AIV). After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 6D10 and 4F10, which corresponded to the amino acid sequences 242FGPRTK247 and 195DLIARAAKI203, respectively, in the IBV N protein. Alignments of amino acid sequences from a large number of IBV isolates indicated that the two epitopes, especially 242FGPRTK247, were well conserved among IBV strains. This conclusion was further confirmed by the relationships of 18 heterologous sequences to the 2 mAbs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections.

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

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.

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.

Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2010, and they were genotyped by comparison with 19 reference strains in the present study. The results showed that LX4-type isolates are still the predominant IBVs circulating in chicken flocks in China, and these isolates could be grouped further into two clusters. Viruses in each cluster had favored amino acid residues at different positions in the S1 subunit of the spike protein. In addition, a recombination event was observed to have occurred between LX4- and tl/CH/LDT3/03I-type strains, which contributed to the emergence of a new strain. The most important finding of the study is the isolation and identification of Taiwan II-type (TW II-type) strains of IBV in mainland China in recent years. The genome of TW II-type IBV strains isolated in mainland China has experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. Pathogenicity testing and sequence analysis of the 3' terminal untranslated region revealed that TW II-type IBV strains isolated in mainland China have a close relationship with the embryo-passaged, attenuated TW2296/95.

A comprehensive study of the epidemiology and pathogenicity of infectious bronchitis virus (IBV) in China was carried out by molecular characterization of the S1 gene from 46 isolates obtained for this study and 174 reference strains isolated over a 15-year period. Nine types were found according to sequence analysis and phylogenetic study of the S1 gene. The co-circulation of multiple IBV types and the ongoing emergence of IBV variants are the epidemiological challenges in China. Factors contributing to the continual emergence include mutations, insertions and deletions in the S1 protein genes; recombination between local IBV strains circulating in chicken flocks in China; and recombination between local strains and vaccine strains. Vaccination-challenge analysis between circulating field strains and Mass-type H120 vaccine indicated the need to develop new vaccines from local IBV strains. These results also emphasize the importance of continued IBV surveillance in China.

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′-7 kb 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′-7 kb 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′-7 kb 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.

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.

Twelve infectious bronchitis virus (IBV) isolates obtained from commercial chickens in China between 2005 and 2006 were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and the sequencing of the entire S1 gene. CK/CH/LSD/05I--an IBV variant, which was unlike the nephropathogenic IBV isolates found in China--exhibited an affinity for the respiratory tract. The variant was identified by phylogenic analysis and basic local alignment search tool (BLAST) searches of the entire S1 gene and by the vaccination-challenge test that was performed using heterologous strains. Further, it was demonstrated that the commercially used H120 vaccine did not provide sufficient protection against this variant; however, the attenuated heterologous IBV tl/CH/LDT3/03 P120, whose parent virus was isolated in China, showed a better efficacy of protection against CK/CH/LSD/05I. This study thus may demonstrate that the use of a combination of commercially available vaccines or of attenuated heterologous strains would provide satisfactory protection against the variant CK/CH/LSD/05I. In addition, the study also revealed that IBV strains exhibiting different pathogenicities were found cocirculating in the chicken flock in China.

Objective: To understand the evolutionary relationship of duck enteritis virus (DEV) in the family Herpesviridae and the genome organization of DEV. Methods: A modified ‘targeted gene walking PCR’ method was used to amplify the unknown gene of DEV Clone-03 strain adjacent to a short stretch of known sequence. The assembled unknown fragment was confirmed to be a homologue of herpesviruses by BLAST analysis and comparison with other herpesviruses homologues. Open reading frames (ORFs) were determined and analyzed by Gene Runner software and an online transcriptional element search engine. Two evolutionary analysis approaches were used to conduct phylogenetic trees of DEV in relation to herpesviruses based on the deduced amino acid sequences. Results: A genomic fragment encompassing 25,625 nt and containing 16 ORFs was amplified, determined, and compared with homologues in other herpesviruses. Phylogenetic trees indicated that DEV and other herpesviruses generated from one ancestor and DEV were grouped into the subfamily Alphaherpesvirinae. In most cases, DEV showed a close relationship with the genus Mardivirus, but formed a single branch. Conclusion: Partial genomic organization and phylogenetic analysis in the present study provides evidence that DEV was a member of the subfamily Alphaherpesvirinae and should be assigned as a single group.

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 genotypic relationship between CK/CH/LDL/97I-type strains and reference IBVs based on S1 gene comparisons and the protection provided by vaccination with commercial vaccines and attenuated homologous and heterologous strains. Phylogenetic analysis and the comparison of S1 showed that CK/CH/LDL/97I-type virus might be a new serotype compared to vaccine strains and other types of IBV isolates in China. Protection efficacy was evaluated by morbidity, mortality, and virus re-isolation from the challenged chicks. Complete protection by IBV vaccination was provided by the homologous strain but sufficient respiratory protection was not provided by the commercial vaccines. Heterologous strains against CK/CH/LDL/97I challenge and the development of a vaccine against CK/CH/LDL/97I-type IBV will be necessary to control infectious bronchitis disease in poultry. Further development of the attenuated CK/CH/LDL/97I strain may provide a valuable contribution towards this goal.

The study was conducted to evaluate effects of dietary supplementation with Ligustrum lucidum (LL, 10 g/kg), Schisandra chinensis (SC, 10 g/kg), LL (10 g/kg) + mannan oligosaccharides (MOS, 50 mg/kg), or SC (10 g/kg) + MOS (50 mg/kg) on growth performance and parameters of antioxidative and immunological status of broilers. The results showed that feeding LL, SC, LL + MOS, or SC + MOS had no significant effect on growth performance of broilers relative to the control. However, compared to the control, LL, SC, LL + MOS, or SC + MOS significantly decreased malondialdehyde concentration in serum, thigh, and heart of broilers. In addition, glutathione reductase activity of heart and sera of the birds were significantly elevated by supplementation LL, SC, LK + MOS, or SC + MOS. Furthermore, LL, SC, LL + MOS, or SC + MOS significantly improved antibody titres against Newcastle disease virus and lymphocyte proliferation of broilers (p < 0.05). Whereas, no cooperating effect between LL (or SC) and MOS on antioxidant status and immunity of broilers were found.

Duck enteritis virus (DEV) is classified to the family Herpesviridae, but has not been grouped into any genus so far. Four overlapped fragments were amplified from the DEV genome with polymerase chain reaction (PCR). The assembled length of the four fragments was 6,202 bp, which contained the genes encoding unique long (UL) 24, thymidine kinase (TK) and glycoprotein H (gH) proteins. The UL24 overlapped with TK by 64 nucleotides (nt), in a head-to-head transcription orientation, and the TK and gH had the same transcription orientation. The comparison of amino acid sequences of these 3 deduced DEV proteins with other 12 alphaherpesviruses displayed 5 highly conserved sites in the UL24, as well as another 5 consensus regions in the TK and 4 consensus regions in the gH. The RNA polymerase II transcriptional control elements were identified in all the UL24, TK and gH of DEV. These elements included core promoters, TATA motifs and polyadenylation sites. Phylogenetic analysis for the genetic classification of DEV in the Alphaherpesvirinae subfamily with other 12 alphaherpesviruses was computed. The result showed that DEV was more closely related to avian herpesviruses, except infectious laryngotracheitis virus (ILTV), than to other alphaherpesviruses. Conclusively, according to the phylogenesis-based analysis and the homology comparison of functional domains of UL24, TK and gH, DEV should be classified to a separate genus of the Alphaherpesvirinae subfamily in the family Herpesviridae.

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.

Coronavirus-like viruses, designated peafowl/China/LKQ3/2003 (pf/CH/LKQ3/03) and teal/China/LDT3/2003 (tl/CH/LDT3/03), were isolated from a peafowl and a teal during virological surveillance in Guangdong province, China. Partial genomic sequence analysis showed that these isolates had the S–3–M–5–N gene order that is typical of avian coronaviruses. The spike, membrane and nucleocapsid protein genes of pf/CH/LKQ3/03 had >99 % identity to those of the avian infectious bronchitis coronavirus H120 vaccine strain (Massachusetts serotype) and other Massachusetts serotype isolates. Furthermore, when pf/CH/LKQ3/03 was inoculated experimentally into chickens (specific-pathogen-free), no disease signs were apparent. tl/CH/LDT3/03 had a spike protein gene with 95 % identity to that of a Chinese infectious bronchitis virus (IBV) isolate, although more extensive sequencing revealed the possibility that this strain may have undergone recombination. When inoculated into chickens, tl/CH/LDT3/03 resulted in the death of birds from nephritis. Taken together, this information suggests that pf/CH/LKQ3/03 might be a revertant, attenuated vaccine IBV strain, whereas tl/CH/LDT3/03 is a nephropathogenic field IBV strain, generated through recombination. The replication and non-pathogenic nature of IBV in domestic peafowl and teal under field conditions raises questions as to the role of these hosts as carriers of IBV and the potential that they may have to transmit virus to susceptible chicken populations.

Five strains of infectious bronchitis virus (IBV) were isolated from five layer flocks that had nephropathogenic infection in four provinces in China. Among them, three of the five flocks had been vaccinated against infectious bronchitis. Virulence studies indicated that the five Chinese IBV isolates caused 10 to 30% mortality in 15-day-old specific pathogen free chickens and gross lesions were mainly confined to the kidneys in all of the dead chickens. Two oligonucleotide pairs, S1Uni2 and S1Oligo3' or S1Oligo5' and S1Oligo3', were used after propagation of the isolates in embryonated eggs to amplify the S1 protein genes of the spike protein. The cDNA derived by reverse transcriptase-polymerase chain reaction was cloned and sequenced. The nucleotide and amino acid sequence of S1 protein gene had a similar degree of identity (or =92%) among the five Chinese IBV isolates. The nucleotide and amino acid identity of the S1 protein gene between the five Chinese IBV isolates and 16 strains of other IBVs varied from 60 to 81%. This clearly showed that the five Chinese IBV isolates comprised a separate genotype. These results demonstrated, for the first time, that there is a new genotype of nephropathogenic IBV circulating in vaccinated and non-vaccinated flocks in China.

Background Infectious bronchitis virus (IBV), a prototype of the Coronaviridae family, is an economically important causative agent of infectious bronchitis in chickens and causes an acute and highly contagious upper respiratory tract infections that may lead to nephritis. However, the molecular antiviral mechanisms of chickens to IBV infection remain poorly understood. In this study, we conducted global gene expression profiling of chicken kidney tissue after nephropathogenic IBV infection to better understand the interactions between host and virus. Results IBV infection contributed to differential expression of 1777 genes, of which 876 were up-regulated and 901 down-regulated in the kidney compared to those of control chickens and 103 associated with immune and inflammatory responses may play important roles in the host defense response during IBV infection. Twelve of the altered immune-related genes were confirmed by real-time RT-PCR. Gene ontology category, KEGG pathway, and gene interaction networks (STRING analysis) were analyzed to identify relationships among differentially expressed genes involved in signal transduction, cell adhesion, immune responses, apoptosis regulation, positive regulation of the I-kappaB kinase/NF-kappaB cascade and response to cytokine stimulus. Most of these genes were related and formed a large network, in which IL6, STAT1, MYD88, IRF1 and NFKB2 were key genes. Conclusions Our results provided comprehensive knowledge regarding the host transcriptional response to IBV infection in chicken kidney tissues, thereby providing insight into IBV pathogenesis, particularly the involvement of innate immune pathway genes associated with IBV infection.

Highlights • Massachusetts-type is the most prevalent serotype of infectious bronchitis virus worldwide. • Analysis of genomic sequences provides more information about occurrence, origin and evolution. • Pathogenic, non-pathogenic and recombinant Mass-type IBVs are co-existing in chicken flocks in China. • Data of Mass-type from isolates in China can help to explain the emergence of Mass-type IBV., Four Massachusetts-type (Mass-type) strains of infectious bronchitis coronavirus (IBV) were compared genetically with the pathogenic M41 and H120 vaccine strains using the complete genomic sequences. The results revealed that strains ck/CH/LNM/091017 and ck/CH/LDL/101212 were closely related to the H120 vaccine, which suggests that they might represent re-isolations of vaccine strains or variants of vaccine strains that have resulted from the accumulated point mutations after several passages in chickens. In contrast, strains ck/CH/LHLJ/07VII and ck/CH/LHLJ/100902 had a close genetic relationship with the pathogenic M41 strain. In addition, molecular markers have been identified that distinguish between field and vaccine (or vaccine-like) Mass-type viruses, which may be able to differentiate between field and vaccine strains for diagnostic purposes. Phylogenetic analysis, and pairwise comparison of full-length genomes and the nine genes, identified the occurrence of recombination events in the genome of strain CK/VH/LHLJ/07VII, which suggests that this virus originated from recombination events between M41- and H120-like strains at the switch site located at the 3′ end of the nucleocapsid (N) genes. To our knowledge, this is the first time that evidence for the evolution and natural recombination under field conditions between Mass-type pathogenic and vaccinal IBV strains has been documented. These findings provide insights into the emergence and evolution of the Mass-type IB coronaviruses and may help to explain the emergence of Mass-type IBV in chicken flocks all over the world.

Background Duck enteritis virus (DEV) is an unassigned member in the family Herpesviridae. To demonstrate further the evolutionary position of DEV in the family Herpesviridae, we have described a 42,897-bp fragment. We demonstrated novel genomic organization at one end of the long (L) region and in the entire short (S) region in the Clone-03 strain of DEV. Results A 42,897-bp fragment located downstream of the LOFR11 gene was amplified from the Clone-03 strain of DEV by using 'targeted gene walking PCR'. Twenty-two open reading frames (ORFs) were predicted and determined in the following order: 5'-LORF11-RLORF1-ORF1-ICP4-S1-S2-US1-US10-SORF3-US2-MDV091.5-like-US3-US4-US5-US6-US7-US8-ORFx-US1-S2-S1-ICP4 -3'. This was different from that of the published VAC strain, both in the linkage of the L region and S region, and in the length of the US10 and US7 proteins. The MDV091.5-like gene, ORFx gene, S1 gene and S2 gene were first observed in the DEV genome. The lengths of DEV US10 and US7 were determined to be 311 and 371 amino acids, respectively, in the Clone-03 strain of DEV, and these were different from those of other strains. The comparison of genomic organization in the fragment studied herein with those of other herpesviruses showed that DEV possesses some unique characteristics, such as the duplicated US1 at each end of the US region, and the US5, which showed no homology with those of other herpesviruses. In addition, the results of phylogenetic analysis of ORFs in the represented fragment indicated that DEV is closest to its counterparts VZV (Varicellovirus) and other avian herpesviruses. Conclusion The molecular characteristics of the 42,897-bp fragment of Clone-03 have been found to be different from those of the VAC strain. The phylogenetic analysis of genes in this region showed that DEV should be a separate member of the subfamily Alphaherpesvirinae.

Background Avian infectious bronchitis (IB) is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV). Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). Results 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection. Conclusions To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.

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.

In this study, a newly identified avian beta-defensin (AvBD) orthologue was isolated from Chinese painted quail (Coturnix chinensis) lung and bone marrow tissues. The complete nucleotide sequence of the gene contained a 204-bp open-reading frame encoding 67 amino acids. Homology, characterization, and comparison of the gene with AvBD from other avian species confirmed that it was quail AvBD9. To analyze and compare the expression pattern of AvBD9 in tissues from young and adult quails, layer hens, and broilers, reverse transcription polymerase chain reaction was performed using mRNA isolated from 21 different tissues. The AvBD9 expression pattern distribution was differed among quails of different ages, layer hens, and broilers. It was widely expressed in all the tissues except the trachea, liver, and kidney and was highly expressed in the lung and heart of young quails. Similarly, it was widely expressed in all the tissues of adult quails except for the liver, kidney, spleen, thymus, and Harderian gland. In layer hens, AvBD9 was widely expressed in all the tissues except the trachea, glandular stomach, and cecum. Similarly, it was widely expressed in all the tissues of broilers except for the trachea, glandular stomach, rectum, cecum, bone marrow, and cecal tonsil. Recombinant AvBD9 (rAvBD9) was produced and purified by expressing the gene in Escherichia coli. Additionally, peptide according to quail AvBD9 sequence was synthesized, named sAvBD9. As expected, both rAvBD9 and sAvBD9 exhibited strong bactericidal properties against 11 strains of bacteria, including Gram-positive and Gram-negative forms.

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, (199)FATFVYAK(206), 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 (199)FATFVYAK(206) 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 (199)FATFVYAK(206) 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.

In this study, a novel avian beta-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, -20 degrees C to 100 degrees C) and pH values (range, 3 to 12).

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.

Background Infectious bronchitis virus (IBV) is first to be discovered coronavirus which is probably endemic in all regions with intensive impact on poultry production. In this study, we used two-dimensional gel electrophoresis (2-DE) and two-dimensional fluorescence difference gel electrophoresis (2-DIGE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), to explore the global proteome profiles of trachea and kidney tissues from chicken at different stages infected in vivo with the highly virulent ck/CH/LDL/97I P5 strain of infectious bronchitis virus (IBV) and the embryo-passaged, attenuated ck/CH/LDL/97I P115 strain. Results Fifty-eight differentially expressed proteins were identified. Results demonstrated that some proteins which had functions in cytoskeleton organization, anti-oxidative stress, and stress response, showed different change patterns in abundance from chicken infected with the highly virulent ck/CH/LDL/97I P5 strain and those given the embryo-passaged, attenuated P115 stain. In addition, the dynamic transcriptional alterations of 12 selected proteins were analyzed by the real-time RT-PCR, and western blot analysis confirmed the change in abundance of heat shock proteins (HSP) beta-1, annexin A2, and annexin A5. Conclusions The proteomic alterations described here may suggest that these changes to protein expression correlate with IBV virus' virulence in chicken, hence provides valuable insights into the interactions of IBV with its host and may also assist with investigations of the pathogenesis of IBV and other coronavirus infections.