In the present study, we report the detection of a novel infectious bronchitis virus lineage within genotypes GVII (GVII-2) in commercial flocks in China since 2008. Virus strains within this lineage have been isolated from different geographic regions in China. S1 genes of these viruses have an intralineage divergence of 0.2%-4.4% nucleotides and 0.1%-7.5% amino acids. The pairwise distances of nucleotide and amino acid sequences based on the S1 subunit between GVII-2 and the closest GVII-1 were 28.3% -28.8% and 28.1%-28.9%, respectively. Complete genomic sequence analysis of the first detected strain, ck/CH/LAH/08I (LAH/08I), suggested that it is derived from recombination events with a genomic backbone of the GI-19 ck/CH/LGS/08I-like strain and distinct S, 3a, and 5a genes. The GVII-2 strain represents a unique serotype that differs from the GVII-1 I0636/16 strain in the same genotype and its deduced parental virus GI-19 I0305/19. Strain LAH/08I not only showed strong tissue tropism in the kidneys of SPF chickens but also exhibited high replication capacities in the trachea, kidney, digestive tract, and immune system of infected chickens, attributed to its pathogenicity in SPF chickens. Strain LAH/08I also showed tropism in the oviducts and the ability to induce cystic oviduct formation in adult chickens after infection in baby chicks.

Due to the scarcity of cell-adapted strains and limitations of current reverse genetics techniques, the factors determining cell tropism of infectious bronchitis virus (IBV), a prototype gammacoronavirus, remain unclear. Here, we demonstrated that the expanded cell tropism of CEA, a chicken embryo fibroblast-adapted strain derived from tl/CH/LDT3/03, was related to its enhanced attachment capacity. Then, a reverse genetics platform for IBV based on circular polymerase extension reaction (CPER) was established, enabling the successful recovery of rCEA and rLDT, which retained the biological properties of their respective parental viruses. Using this platform, we constructed a series of chimeric viruses based on the CEA strain by incorporating the entire S gene, the S1 and S2 subunits, and substitutions at differential sites derived from the tl/CH/LDT3/03 strain. Our findings indicated that subunit S1, rather than S2, was associated with viral adaptation in DF-1 cells, with the amino acid residue at position 413 in the C-terminal domain of the S1 protein as a key determinant. Evaluation with recombinant S1 proteins and chimeric viruses revealed that the histidine residue at position 413 (His413) promoted enhanced attachment of both S1 proteins and viruses to cells, while exerting no direct effect on viral entry. Mechanistically, His413 facilitated efficient viral binding to α-2,3-linked sialic acids presented on gangliosides rather than glycoproteins. This interaction activated Src kinase and triggered caveolae-mediated endocytosis, initiating viral entry and subsequent replication. Collectively, the CPER-based reverse genetics platform established in this study represents a significant technical advancement for IBV, and our findings provide novel insights into the cell adaptation and entry mechanisms of IBV.IMPORTANCEAlthough infectious bronchitis virus (IBV) was the first discovered coronavirus, the cellular receptors and cofactors that mediate successful infection, as well as the specific pathways and mechanisms of viral entry into host cells, remain to be elucidated. In this study, we established a reverse genetics platform for IBV using circular polymerase extension reaction for the first time. A series of chimeric viruses were subsequently recovered, and a specific amino acid substitution at position 413 in the C-terminal domain of the S1 subunit was identified as a key determinant for the expanded DF-1 cell tropism of IBV. The histidine residue at this site facilitated viral binding to α-2,3-linked sialic acids on gangliosides, thereby activating the caveola-mediated endocytosis and enabling viral entry into DF-1 cells. This study provides novel insights into the receptor-binding function of the IBV S protein and the strategies employed by the virus for cell adaptation. Additionally, our findings offer new perspectives for developing cell-culture-based vaccines.

As a unique antigenic variant of Newcastle disease virus (NDV) in pigeons, genotype VI NDVs cause serious disease in pigeons and pose a potential threat to domestic poultry. In this study, analyses of genetic variation and evolution of global genotype VI NDVs revealed the worldwide distribution and continuous evolution of genotype VI NDVs. Of importance, VI.2.1.1.2.1 and VI.2.1.1.2.2 are the two prevalent sub-genotypes worldwide. The virulence, replication capacity, and pathogenesis in chickens of representative strains from VI.2.1.1.2.1 and different clusters in VI.2.1.1.2.2 were further evaluated. Compared with the VI.2.1.1.2.1 strain, increased replication capacity and virulence in chickens were observed in strains of VI.2.1.1.2.2. VI.2.1.1.2.2 strains showed preferential binding for α-2,3-linked sialic acids and superior performance in viral entry, cell-cell fusion, and release of progeny virions. Evaluation with recombinant viruses demonstrated that residues at positions 365 and 497 in the HN protein contributed to the differences in biological characteristics of VI.2.1.1.2.2 strains, and residue 365 was a key determinant. Moreover, our results showed that the activity of the viral replication complex contributed to the differences in replication capacity among these viruses, with the P protein being the major individual contributor. The optimal effect was achieved when the NP and L proteins were homologous. Moreover, the 5' terminal trailer region was also found to be involved in the replication capacity of genotype VI NDVs; however, the viral V protein was not related to the replication and virulence of these viruses. Our findings highlight the potential risk of VI.2.1.1.2.2 NDVs due to their persistent circulation and evolution. IMPORTANCE:Genotype VI is the most diverse group of Newcastle disease viruses (NDVs). In addition to infectious disease in pigeons, the potential threat to chicken flocks and the public health implications associated with genotype VI NDVs also need to be addressed. Herein, comprehensive genetic evolution analysis revealed a global distribution pattern and continuous evolution of genotype VI NDVs worldwide. The biological characteristics of genotype VI NDVs belonging to different sub-genotypes were also evaluated. In particular, the widespread transmission, circulation, and constant evolution of currently prevalent sub-genotype VI.2.1.1.2.2 have led to the alteration of receptor binding preference, an increase in replication capacity, and a resultant increase in virulence in chickens. These findings expand our current understanding of the evolution and pathogenesis of genotype VI NDVs.

Entry of Newcastle disease virus (NDV) into target cells via caveolae-mediated endocytosis has been presented. However, the viral factors that mediate the endocytic entry and fusion in intracellular endosomes remain poorly understood. Herein, the potential contributions of HN stalk region and second receptor biding site (site II) located at the globular head region on the endocytosis of NDV and the viral-cell membrane fusion in endosomes were investigated. HN protein mutants with substitutions at positions 89 and 94 in the stalk region and at position 516 in site II were firstly constructed. We demonstrated that mutations in HN stalk region restricted its interaction with F protein and fusion promotion activity. While mutation in HN site II resulted in slightly decreased fusion promotion activity without affected the HN-F interactions. Further evaluation of receptor binding and entry capacities with HN mutant NDVs obtained by reverse genetics demonstrated that both mutations at stalk region and site II reduced the binding abilities of NDV to receptors. Interestingly, site II in HN was not responsible for the endocytic entry of NDV and subsequent viral-cell membrane fusion in endosomes. While the residues at 89 and 94 in HN stalk region were involved in the fusion of NDV in endosomes. Of particular note, the acidic condition was indispensable for efficiently fusion triggering of NDV within endosomes. Besides, residues at 89 and 94 in HN were conserved in different genotypes of NDVs and the endocytic entry of NDV into cells was not determined by viral virulence and genotype.

Newcastle disease virus (NDV) genotype VI from pigeon origin is an important causative agent for serious disease in pigeons. Although the biological characteristics of genotype VI NDV have been extensively studied, the understanding of the thermostability of this genotype is still incomplete. In this study, an NDV strain, designated P0506, was isolated from a diseased pigeon in China and classified as genotype VI. Phylogenetic analysis on the basis of the Fusion gene coding sequence indicated that P0506 belonged to sub-genotype VI.2.1.1.2.2 of class II. The thermostability may be a universal characteristic of genotype VI NDV. Thus, the thermostability of two strains, including P0506 identified in this study and P0713 identified previously, belonging to VI.2.1.1.2.2, and another previously isolated strain, P0813, in VI.2.1.1.2.1, was investigated. It was indicated that all three viruses presented resistance to heat treatment, but P0713 was more robust than P0813 and P0506. By constructing a series of HN protein mutants, amino acid residues at both residues 365 and 497 in HN protein were found to be involved in the heat resistance. Furthermore, the effects of residues 365 and 497 in HN protein on the thermostability of the virus were further evaluated by using recombinant viruses generated by the reverse genetic system. Our results showed that residue at position 365 in HN protein was the key thermostable determinant of sub-genotype VI.2.1.1.2.2 NDV. These findings will help us better understand the thermostable mechanism of NDV and serve as a foundation for the further development of novel thermostable vaccines.

ABSTRACT As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors. IMPORTANCE In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

对2020-2021年期间分离自10个省(自治区)活禽市场不同宿主与部分环境中的新城疫病毒(NDV)进行鉴定,获得了 31株class Ⅰ类NDV.F基因分子特征分析显示,class Ⅰ类NDVF基因中起始密码子存在突变,导致产生不同长度的编码序列.氨基酸序列分析结果显示,大部分毒株的F蛋白具有典型的弱毒株裂解位点基序,但个别毒株的裂解位点存在特征性的突变.F蛋白中融合肽、N-连接的糖基化位点、中和表位以及七肽重复区域在这31个毒株之间高度保守,而信号肽区域中存在众多的氨基酸替换.HN蛋白中仅N-连接的糖基化位点在31个毒株之间保守,中和表位和七肽重复区域均存在氨基酸替换.此外,首次发现了 HN编码区长度为1 728 nt的突变毒株.遗传进化分析显示,与其他国家和地区的毒株不同,我国的class Ⅰ类NDV形成了独特的进化分支,即基因亚型1.1.2.耐热性测定结果显示,class Ⅰ类NDV分离株表现出不同程度的耐热特性.本研究结果为我国classⅠ类NDV的遗传变异和生物学特性研究奠定了基础.

本研究利用荧光染料标记的新城疫病毒(NDV)检测病毒的吸附、融合和进入,旨在为研究NDV入侵细胞的机制提供一种工具.首先通过DiOC标记NDV(DiOC-NDV)检测病毒的吸附和进入,随后利用R18标记NDV(R18-NDV)检测病毒的融合,最后利用R18和DiOC同时标记NDV(DiOC/R18-NDV)进一步检测NDV在内体中的融合.病毒吸附的检测结果显示,代表NDV的绿色荧光定位于细胞表面.病毒进入的检测结果显示,60～120 min不能被台盼蓝淬灭以及被台盼蓝淬灭的绿色荧光强度均无明显变化(P＞0.05);病毒融合的检测结果显示,30～120 min时均能在细胞表面和细胞质中观察到红色荧光,且60～120 min时细胞的总荧光强度无显著差异(P＞0.05).以上结果表明,NDV吸附细胞后,在细胞表面与内体中发生膜融合及进入,且该过程在60 min内完成.综上所述,荧光标记的NDV能直观、可靠地展现病毒的吸附、进入和融合过程,荧光标记NDV可作为一种研究NDV入侵细胞机制的工具.

In this study, 232 class I Newcastle disease viruses (NDVs) were identified from multiple bird species at nationwide live bird markets (LBMs) from 2017 to 2019 in China. Phylogenetic analysis indicated that all 232 isolates were clustered into genotype 1.1.2 of class I on the basis of the fusion (F) gene sequences, which were distinct from the genotypes identified in other countries. Most of the isolates (212/232) were shown to have the typical F gene molecular characteristics of class I NDVs, while a few (20/232) contained mutations at the site of the conventional start codon of the F gene, which resulted in open reading frames (ORFs) altered in length. The isolates with ACG, CTA, and ATA mutations showed different levels of increased virulence and replication capacity, suggesting that these viruses may be transitional types during the evolution of class I NDVs from avirulent to virulent. Further evaluation of biological characteristics with recombinant viruses obtained by reverse genetics demonstrated that the ATG located at genomic positions 4523 to 4525 was the authentic start codon in the F gene of class I NDV, and the specific ATA mutations which contributed to the expression of F protein on the surface of infected cells were the key determinants of increased replication capacity and virulence. Interestingly, the mutation at the corresponding site of genotype II LaSota of class II had no effects on the virulence and replication capacity in chickens. Our results suggest that the alteration of virulence and replication capacity caused by specific mutations in the F gene could be a specific characteristic of class I NDVs and indicate the possibility of the emergence of virulent NDVs due to the persistent circulation of class I NDVs. IMPORTANCE The available information on the distribution, genetic diversity, evolution, and biological characteristics of class I Newcastle disease viruses (NDVs) in domestic poultry is currently very limited. Here, identification of class I NDVs at nationwide live bird markets (LBMs) in China was performed and representative isolates were characterized. A widespread distribution of genotype 1.1.2 of class I NDVs was found in multiple bird species at LBMs in China. Though most isolates demonstrated typical molecular characteristics of class I NDVs, a few that contained specific mutations at the site of the conventional start codon of the fusion gene with increased virulence and replication capacity were identified for the first time. Our findings indicate that the virulence of class I NDVs could have evolved, and the widespread transmission and circulation of class I NDVs may represent a potential threat for disease outbreaks in poultry.

目前,新城疫病毒(NDV)融合蛋白(F)的融合前构象是未知的,为获得具有融合前构象的NDV F蛋白,本研究对基因Ⅶ型NDV CK/CH/LHLJ/1/06株的F基因胞外区编码序列进行了修饰和密码子优化,将其克隆至真核表达载体中构建获得了重组质粒pCAG-optiF.通过SWISS-MODEL网站对蛋白结构进行预测,结果显示,质粒pCAG-optiF编码的蛋白能够形成与人呼吸道合胞体病毒F蛋白融合前构象相似的结构.将质粒pCAG-optiF转染至鸡源细胞LMH中,通过间接免疫荧光试验证实构建的蛋白在鸡源细胞中成功表达.将质粒pCAG-optiF转染悬浮培养的Expi293F细胞,表达并纯化获得重组蛋白roptiF.SDS-PAGE电泳结果显示获得了与预期大小相符的蛋白.Western-blotting结果显示,表达的roptiF蛋白能够与NDVCK/CH/LHLJ/1/06株的抗血清反应.进一步通过负染色电子显微镜观察roptiF蛋白结构,结果显示,roptiF蛋白具有典型的F蛋白融合前构象的结构特征.本研究成功获得了具有融合前构象的基因Ⅶ型NDV F蛋白,并且该蛋白能够与抗NDV的血清反应,本研究结果为研制新型NDV疫苗奠定了基础.

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.

为探索新城疫病毒(NDV)F基因及其蛋白诱导细胞免疫的能力,本研究对基因Ⅶ型NDV CK/CH/LHLJ/1/06株F基因进行了密码子优化和修饰,将其克隆至真核表达载体,获得了重组质粒pCAF.将质粒pCAF转染至悬浮培养的细胞中,上清中表达的蛋白经纯化获得了重组蛋白proF.以构建的质粒pCAF及其重组蛋白proF作为DNA和亚单位疫苗,采用pCAF单独免疫、proF单独免疫、pCAF和proF联合免疫的方式分别免疫SPF鸡,免疫后分离外周血淋巴细胞,通过淋巴细胞增殖试验和细胞因子检测试验评估pCAF和proF诱导的细胞免疫反应.结果显示,proF可刺激分离自pCAF单独免疫组或proF单独免疫组的外周血淋巴细胞增殖和细胞因子IL-4和IFN-γ的分泌,并且proF刺激后,pCAF和proF联合免疫组的外周血淋巴细胞的增殖水平、细胞因子IL-4和IFN-γ的分泌水平显著高于pCAF单独免疫组或proF单独免疫组.上述结果表明,pCAF和proF均可诱导细胞免疫应答,且与pCAF或proF单独免疫相比,pCAF和proF联合免疫组诱导的免疫效果更好.本研究结果为研制新型NDV疫苗提供了思路和理论基础.

本研究旨在明确新城疫病毒(NDV)溶瘤作用是否依赖其复制水平,并改进NDV溶瘤机制研究模型.以NDV疫苗株LaSota感染人肿瘤细胞系HCT116、A549和人非肿瘤细胞系HEK293T为研究模型;RT-qPCR检测NDV在各细胞系的基因组复制水平;Western blot检测NDV在各细胞系的蛋白表达水平;利用流式细胞术检测NDV感染对各细胞系的细胞周期和细胞凋亡的影响.结果 表明,NDV在三个细胞系内的基因组复制水平和蛋白表达水平没有显著差异,但对三个细胞系的溶瘤作用存在明显差异;进一步流式细胞术检测发现,NDV感染能诱发HEK293T和HCT116的细胞周期发生G1期停滞,但对A549的细胞周期没有明显影响;此外,NDV感染24 h后,A549细胞以早期凋亡为主,而HCT116细胞以坏死/晚期细胞凋亡为主.NDV的溶瘤作用并不依赖于其复制水平,而且NDV对不同类型肿瘤细胞的溶瘤作用存在不同机制.

To develop an alternative vectored vaccine against both Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV), the glycoprotein C (gC) gene was first deleted from an avirulent ILTV. Based on this gC-deleted ILTV mutant, a recombinant ILTV expressing the fusion protein (F) of a genotype VII NDV (designated ILTV-Delta gC-F) was then constructed. Expression of the NDV F protein in ILTV-Delta gC-F-infected LMH cells was examined with an immunofluorescence assay and western blotting. The F gene was stably maintained in the genome of ILTV-Delta gC-F and the F protein was stably expressed. Compared with the parental virus, ILTV-Delta gC-F demonstrated an increased penetration capacity in vitro, and an increased replication rate in vitro and in vivo. Both the parental virus and ILTV-Delta gC-F were avirulent in chickens. Vaccination of specific-pathogen-free chickens with ILTV-Delta gC-F induced ILTV-specific antibodies, detected with an enzyme-linked immunosorbent assay (ELISA), and provided complete clinical protection against virulent ILTV, although viral shedding and replication were detected in the respiratory tract in the early stage of infection in a very small number of birds. Vaccination with ILTV-Delta gC-F also provided significant protection against challenge with a virulent genotype VII NDV, although the level of NDV-specific antibodies detected with an ELISA was low. Notably, the numbers of birds that were positive for the virulent genotype VII NDV and the replication of the challenge virus NDV in selected target tissues were significantly lower in the ILTV-Delta gC-F-vaccinated chickens than in the control birds. Our results indicate that ILTV-Delta gC-F has potential utility as a bivalent candidate vaccine against both infectious laryngotracheitis and Newcastle disease.

Nine infectious bronchitis virus (IBV) strains belonging to the GI-7 lineage were isolated between 2009 and 2017 in China. Phylogenetic analysis and comparisons of full-length sequences of the S1 gene suggested that the GI-7 lineage should be further classified as Taiwan (TW)-I and TW-II sublineages, which correspond to the previous TW-I and TW-II genotypes. The nine IBV strains were clustered in the TW-II sublineage. Further investigation revealed that viruses in the TW-I and TW-II were not only genetically but also antigenically different. Moreover, the TW-II sublineage contained various clades and recombinants. A recombinant was found to originate from recombination events between field strains (TW-II ck/CH/LJL/090608- and GI-19 ck/ CH/LDL/091022-like viruses) in which the recombination in the S1 subunit coding sequences had led to changes in antigenicity of the viruses. A more in-depth investigation demonstrated that TW-II viruses appear to have undergone a significant evolution following introduction in mainland China, which resulted in the viruses diverging into different clades. The viruses between the different clades in TW-II sublineage exhibited a significant change in genetic and antigenic characteristics. In addition, the five TW-II viruses selected on the basis of the results of S1 nucleotide sequence phylogenetic trees showed different pathogenicity to specific-pathogen-free chickens, although they could induce nephritis in the infected chickens and thus were identified as nephropathogenic strains.

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.

Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

In this study, four codon optimized plasmids (designated as pCAG-optiF-1, 2, -3, and -4) containing modified F genes from the epidemic and virulent NDV genotype VII strain isolated in China that is expected to express the pre-fusion conformation of the F protein were constructed. The expression of these F variants in chicken-derived cells was detected by an indirect immunofluorescence assay and western blot analysis. Two soluble F variants (roptiF-1 and 2) potentially with the pre-fusion conformation were expressed and purified from suspended cells. Vaccination with each of the plasmids as a DNA vaccine conferred partial clinical protection to chicks against NDV. Comparatively, the plasmid pCAG-optiF-2 encoded a soluble protein with a mutant cleavage site and the potential pre-fusion conformation provided better protection than the other plasmids. Further investigation of the combined vaccinations with the plasmid DNA pCAG-optiF-2 prime + protein roptiF-2 boost vaccination strategy elicited more robust immunity, as confirmed by the detection of antibodies against NDV using enzyme-linked immunosorbent assay and virus neutralization assay, as compared to those vaccinated with only the plasmid pCAG-optiF-2 or protein roptiF-2. More importantly, the DNA prime + protein boost vaccination provided more efficacious protection against virulent NDV challenge, as evidenced by the complete clinical protection, reduced viral shedding, and limited virus replication in tissues of the challenge chicks. These results indicated that the pre-fusion conformation of the F protein could be considered as the target immunogen for the development of novel NDV vaccines.

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 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.