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.

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.

为了解猪圆环病毒型(porcine circovirus3,PCV3)在河北省的流行情况及遗传变异特点,本研究在2017年2月至2018年7月间,从河北省不同地区60个猪场采集了310份不同临床症状表现的发病猪组织样本,应用PCR方法对其进行检测.结果 显示该地区PCV3阳性率为16.5％(51/310),猪场阳性率为45.0％(27/60),对不同临床症状发病率分析显示PCV3在表现为繁殖障碍及腹泻猪群中具有较高的阳性率,且易与PCV2发生共同感染.对获得的8株PCV3的cap基因进行测序及同源性分析显示,所得8条序列之间的核苷酸相似性为98.0％～99.7％,与24条国内外参考株之间的核苷酸相似性为98.0％～100.0％.对cap基因所推导的氨基酸序列进行分析发现cap基因的主要突变位点发生在24,27,77及150位,其中24和27位主要发生了A24V和R27K变异.对32条序列进行进化树的构建发现,进化树可分为3a、3b和3c 3个基因群,河北分离株主要分布于3a基因群和3c基因群,表明了河北省PCV3流行株相对稳定.我们的研究结果为PCV3在河北省的流行及进化特点提供了理论参考,这些数据可为该地区PCV3的诊断及综合防控提供科学依据.

Porcine circovirus 3 (PCV3) is a recently identified virus that is associated with reproductive failure, porcine dermatitis and nephropathy syndrome, and multi-systemic inflammation. To investigate the molecular epidemic characteristics and genetic evolution of PCV3 in northern China, a commercial TaqMan-based real-time quantitative PCR kit was used to detect PCV3 in 435 tissue specimens collected from pigs with various clinical signs from 105 different swine farms in northern China. The results showed that 48 out of 105 (45.7%) farms and 97 out of 435 (22.3%) samples tested positive for PCV3. Of the 97 PCV3-positive samples, 80 (82.5%) tested positive for other pathogens. PCV3 was found more frequently in pigs with reproductive failure than in those with other clinical signs. This study is the first to detect PCV3 in Tianjin. The complete genome sequences of six PCV3 isolates and the capsid (Cap) protein gene sequences of 11 isolates were determined. Based on the predicted amino acids at positions 24 and 27 of the Cap protein and their evolutionary relationships, the 17 PCV3 strains obtained from northern China and 49 reference strains downloaded from the GenBank database were divided into four major groups (3a-3d). An analysis of selection pressure and polymorphism indicated that the PCV3 Cap protein seems to be evolving under balancing selection, that the population is in dynamic equilibrium, and that no population expansion occurred during the study period. Our results provide new information about the molecular epidemiology and evolution of PCV3.

This study was conducted to understand porcine deltacoronavirus (PDCoV) epidemic situation, and provide a tool for PDCoV serum antibody detection and the epidemiological investigation. Prokaryotic expression of recombinant PDCoV partial spike (S) protein was used as detection antigen, and an indirect ELISA antibody detection method based on PDCoV S1 protein was established. The method was used to detect 570 serum samples collected from some pig farms in Hebei from January to December 2017. The results showed that the established ELISA method of PDCoV IgG antibody in this study could specifically detect PDCoV antibody; no cross-reaction with antisera against porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever (CSF) and pseudorabies virus (PRV) was found. The coefficients of variation (CV) of repeated in-batch test was 1.9%-5.4%, CV of batch-to-batch test was 2.3%-5.1%. In 570 blood samples, 105 were positive, and the positive rate was 18.4% (105/570), which was higher than the positive rate of PDCoV IgG antibody (11%) in Hebei from January 2016 to October 2016. It is suggested that the infection rate of PDCoV in Hebei is on the rise. The ELISA method established in this study had the advantages of high sensitivity, strong specificity and good reproducibility, and can be used for the detection of clinical PDCoV serum antibodies.Copyright © 2019, Chinese Journal of Animal Science and Veterinary Medicine Co., Ltd. All right reserved.

为建立同时快速定量检测猪博卡病毒(PBoV)G1基因群和猪流行性腹泻病毒(PEDV)的方法,本研究参照GenBank登录的PBoV的NP1基因和PEDV的M基因保守序列,设计了引物和探针,经优化反应条件后,建立了能够同时检测PBoV G1基因群和PEDV的双重TaqMan荧光定量PCR方法.特异性试验结果显示该方法与PCV2、PRV、PDCoV、PRoV和TGEV无交叉反应,敏感性试验结果显示该方法对PBoV、PEDV的质粒标准品检测下限分别为21.8拷贝/μL和31.7拷贝/μL;且组内、组间变异系数均小于4％,重复性好.应用本实验建立的方法对2017年5月～2018年8月,河北省部分地区采集的142份仔猪腹泻样品检测结果显示,PBoV G1阳性率为18.3％(26/142),PEDV阳性率为62.7％(89/142),其中PBoV G1与PEDV共感染率为10.6％(15/142),该方法优于常规PCR方法.本研究建立的双重TaqMan荧光定量PCR方法对PBoV和PEDV的准确检测、 病原监测、 流行病学调查等均具有重要意义.

Porcine bocavirus (PBoV), which belongs the genus Bocaparvovirus, has been identified throughout the world. However, serological methods for detecting anti-PBoV antibodies are presently limited. In the present study, an indirect enzyme-linked immunosorbent assay (PBoV-rNP1 ELISA) based on a recombinant form of nucleoprotein 1 (NP1) of PBoV was established for investigating the seroprevalence of PBoV in 2025 serum specimens collected in north-central China from 2016 to 2018, and 42.3% of the samples tested positive for anti-PBoV IgG antibodies, indicating that the seroprevalence of PBoV is high in pig populations in China.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea,vomiting,dehydration and mortality in neonatal piglets.In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV,two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank.The genes of the conservative region were amplified and cloned into pMD19-T vector.Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template,a RT-qPCR to PDCoV and PEDV was established.The sensitivity,specificity and repeatability were tested.Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies · μL 1 for PDCoV and PEDV,respectively.No cross-reaction was detected to PBoV,TGEV,PRV and PCV2.The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017.PDCoV positive rate was 16.9％,PEDV positive rate was 66.2％,and co-infection was 2.3 ％.The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR.These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.

猪流行性腹泻(porcine epidemic diarrhea,PED)是由PEDV引起 仔猪腹泻,呕吐的一种高致死性的肠道 染病.本病在英国首先爆发,后逐渐扩展到欧洲,亚洲乃至全球,在2010年,我国多省份大面积发病,对我国的养猪业造成了严重的经济损失. PEDV属于冠状病毒科冠状病毒属,与猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV),猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV)同科同属,且发病症状和病理变化极为相似,极难通过常规手段进行区分,因此,建立一种快速且准确的诊断PEDV的方式对PED的防治具有极其重要的意义.