Based on Simon's theory of complexity architecture, we investigate the near decomposability of complex networks, including network near decomposable hierarchy (NDH) and near decomposability in behavior and properties (NDBP): the related conceptions and approaches are given and illustrated with an example. Different from existing researches, we emphasize the network NDBP; and follow Simon's generation mechanism of complex things to find the network NDH; In addition, based on Simon's theory that most things in the real world are complex systems with the NDH; and the situation that the approach for analyzing the NDH of a simple room system in Simon's example requires that the system adjacency matrix be in the near block diagonal form, which is often not the case for the adjacency matrices of complex systems in the real world, we propose an approach for analyzing the NDH of complex networks (complex systems) by splitting network adjacency matrices, which is suitable for solving real-world problems faced rather than for scientific discovery. The near decomposability of complex networks, due to the advantages of network language, can provide a more convenient and effective way than the near decomposability of complex systems for understanding and dealing with the problems of complex things.

H9N2亚型禽流感病毒(AIV)抗原变异迅速,相继于2009年及2013年分别出现新的抗原亚群病毒,对该病毒的防控带来了极大的挑战.目前,关于H9N2禽流感病毒形成不同抗原亚群机制,尚不清楚.流感病毒血凝素蛋白(HA)的单克隆抗体是研究该机制的基础工具.为此,本研究通过血凝抑制(HI)实验筛选到1株新型的抗原亚群H9N2病毒H514.通过杂交瘤细胞融合技术以及间接免疫荧光的方法,筛选到5株抗H514病毒HA蛋白的单抗,均属于IgG亚型.Western blot结果表明5株单克隆抗体中只有5E9作用的是线性表位,其余4株皆是针对构象表位.进一步地研究发现,这5株单克隆抗体对H514株皆有HI活性,最高达到28.这些结果为H9N2亚型禽流感病毒的抗原性分子研究提供了重要材料.

鸭坦布苏病毒是一种黄病毒科黄病毒属单股正链RNA病毒,可造成蛋鸭、种鸭产蛋下降,给养鸭业造成巨大经济损失的重要疾病.目前,坦布苏弱毒活疫苗是临床上有效预防该病的重要手段之一.CpG ODN作为疫苗佐剂被证实能够有效提高疫苗的免疫效果,但其对弱毒活疫苗的是否具有免疫增强的效果尚不清楚.本研究选择鸭坦布苏弱毒活疫苗FX2010-180P,以CpG ODN为免疫佐剂,在鸭体内进行免疫以及攻毒保护实验.通过检测鸭体内抗体水平、免疫保护率、组织切片来进行免疫效果的评估.结果显示,FX2010-180P免疫组鸭子在免疫后3～4 d即可产生特异性抗体,而添加CpG ODN疫苗佐剂组,免疫鸭的抗体产生时间滞后于未添加组.攻毒保护实验结果显示,两种方式免疫方式都可以保护鸭子不受坦布苏病毒野毒的感染.本研究结果显示添加CpG ODN对鸭坦布苏FX2010-180P弱毒活疫苗并不具有免疫增强的效果.

Since 2016, frequent outbreaks of egg-reducing syndromes caused by an unknown virus in duck farms have resulted in huge economic losses in China. The causative virus was isolated and identified as a novel species in Avihepatovirus of the picornavirus family according to the current guidelines of the International Committee on Taxonomy of Viruses (ICVT), and was named the duck egg-reducing syndrome virus (DERSV). The DERSV was most closely related to wild duck avihepatovirus-like virus (WDALV) with 64.0%, 76.8%, 77.5%, and 70.7% of amino acid identities of P1, 2C, 3C, and 3D proteins, respectively. The DERSV had a typical picornavirus-like genomic structure, but with the longest 2A region in the reported picornaviruses so far. Importantly, the clinical symptoms were successfully observed by artificially infecting ducks with DERSV, even in the contact exposed ducks, which suggested that DERSV transmitted among ducks by direct contact. The antibody levels of DERSV were correlated with the emergence of the egg-reducing syndromes in ducks in field. These results indicate that DERSV is a novel emerging picornavirus causing egg-reducing syndrome in ducks.

近几年,禽流感病毒(AIV)跨越禽-犬屏障产生新型H3N2犬流感病毒,后者在亚洲多国的犬中广泛流行,造成潜在的公共卫生安全威胁.尽管如此,H3N2AIV如何跨宿主传播至犬的分子机制尚不清楚.为此,本研究利用早期筛选到的一株不感染犬和鼠,但感染禽的H3N2禽流感病毒A/Duck/Shanghai/01/2009(H3N2)(简称为SH01),通过RT-PCR技术扩增了病毒的8个全基因片段,并分别克隆至PLLB双向表达载体上.将构建成功的8个质粒纯化后共转染293T细胞,转染48 h后加入TPCK胰酶作用2h,将上清液和细胞一同接种9～11日龄SPF鸡胚,检测其血凝效价.经测序分析,确定获救病毒的8个基因片段序列与亲本毒株的序列完全一致,表明该病毒拯救成功.H3N2禽流感病毒反向遗传操作平台的成功建立为探索该病毒跨宿主传播犬的分子机制提供了工具.

A live attenuated duck Tembusu virus (TMUV) vaccine FX2010-180P (180P) was successfully utilized to prevent TMUV infections in ducks in China. Compared with wild-type TMUV, 180P was highly attenuated and lost transmissibility in ducks. However, the mechanism of the attenuation of 180P remains poorly understood. To explore the key molecular basis of attenuation, chimeric and site mutant viruses in the background of the wild-type TMUV-FX2010 (FX) strain were rescued, and the replication, tissue tropism, and transmissibility were characterized in ducks. The results show that the envelope (E) protein was responsible for attenuation and loss of transmission in ducks. Further studies showed that a D120N amino acid mutation located in domain II of the E protein was responsible for the attenuation and transmissibility loss of 180P in ducks. The D120N substitution resulted in an extra high-mannose type N-linked glycosylation (NLG) in the E protein of 180P compared with the wild-type TMUV, which might restrict the tissue tropism and transmissibility of TMUV in ducks. Our findings elucidate that N120 in the E protein is a key molecular basis of TMUV attenuation in ducks and provide new insight into the role of NLG in TMUV tissue tropism and transmissibility.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in B.194 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 x 10(-3) substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 x 10(-3 )substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 ATVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

本研究选取了一株H9N2亚型禽流感病毒A/Chicken/Shanghai/2093/2009毒株(简称A2093),分别构建了共表达PA-X和PA蛋白的真核表达质粒、单表达PA蛋白和PA-X蛋白的真核表达质粒,研究PA-X对宿主蛋白表达及流感病毒聚合酶活性的影响.结果显示:同时表达PA-X和PA蛋白可以强烈抑制宿主蛋白表达,单独表达A2093的PA-X同样具有较强抑制宿主蛋白表达的功能,而仅表达PA蛋白不影响宿主蛋白表达;在A2093的复制子系统中PA-X缺失后聚合酶活性明显提高;利用反向遗传操作技术拯救出缺失PA-X的A2093ΔPA-X毒株,在MDCK和LMH细胞上比较其与亲本A2093毒株复制能力的差异,发现在MDCK和LMH细胞上,从接种后12～36 h的不同时间点A2093ΔPA-X比A2093病毒低度明显降低.

人类历史上数次流感大流行,都与不同流感病毒之间的基因重组有关,为了研究新发现的蝙蝠流感病毒与禽流感病毒的重组可能性,本研究利用反向遗传操作技术对嵌合蝙蝠流感病毒(Bat09:mH9mN2)与禽流感病毒(A2093/H9N2)进行替换重组,研究发现仅H9病毒的M基因可单向替换Bat病毒的M基因获得重组嵌合蝙蝠流感病毒,命名为Bat09:mH9mN2-A2093(M),其他基因均不能互换拯救.该重组病毒可以在鸡胚上、MDCK细胞上复制良好,但在禽源LMH细胞上几乎不复制.本研究发现蝙蝠流感病毒与H9N2亚型禽流感病毒之间的基因兼容性并不强,仅有禽源病毒M基因可以单向替换重组,这与之前的研究报道一致,关于M基因的重组兼容性及重组病毒生物学特性还有待进一步研究.

BACKGROUND:Reassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes. METHODS:The polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS). RESULTS:We discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice. CONCLUSIONS:The higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

微管相关蛋白1轻链3(LC3)是一种在流感病毒感染过程中被病毒M2蛋白招募至细胞膜处聚集的宿主细胞自噬相关蛋白.为了解其在流感病毒感染过程中行使的功能,利用CRISPR/Cas9技术构建LC3蛋白缺失细胞系,用以观察病毒生长复制的差异.设计sgRNA并插入至核心载体LentiCRISPRv2中,与辅助质粒共同转染293T细胞行慢病毒的包装.包装完成后用以感染A549细胞并进行嘌呤霉素压力筛选,对存活细胞的LC3蛋白含量进行检测,结果显示具有嘌吟抗性的细胞中未检测到LC3蛋白条带,LC3敲除细胞系构建成功.运用核染色法对比敲除细胞和正常细胞的细胞活性,发现缺失LC3蛋白细胞活性小幅度下降.流感病毒感染两种细胞绘制生长曲线,缺少LC3蛋白的条件下病毒复制水平下降.本研究构建的LC3蛋白缺失细胞系能够为将来研究LC3蛋白在细胞自噬与病毒感染过程中发挥功能,以及分子机制的研究提供了良好的细胞模型.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

Previous studies have shown that chimeric bat influenza viruses can be generated by reverse genetic system. However, the roles of the surface or internal genes of chimeric bat influenza viruses in viral replication and virulence in different host species were still not completely understood. In this study, we generated a chimeric H9N2 bat virus with both HA and NA surface genes from the avian A2093/H9N2 virus and compared its replication and virulence with the chimeric H1N1 bat virus with both HA and NA from the PR8/H1N1 virus in vitro and in mice. The chimeric H1N1 virus showed significantly higher replication in mammalian and avian cells and significantly higher virulence in mice than the chimeric H9N2 virus. Moreover, the chimeric H9N2 virus with the bat influenza internal M gene showed a higher replication in mammalian cells than in avian cells. While the chimeric H9N2 virus with the avian-origin viral M gene displayed a higher replication than that with the bat influenza M gene in avian cells, which likely resulted from increased receptor binding ability to α 2,3 sialic acid linked glycans of the former virus. Our study indicates that bat influenza internal genes are permissive in both mammalian and avian cells, and the bat influenza internal M gene shows more compatibility in mammals than in the avian host. Although the surface genes play more critical roles for viral replication in different host substrates, influenza M gene also potentially impacts on replication, virulence and host tropism.

细胞培养流感病毒生产疫苗过程中细胞系的选择至关重要,本研究建立了可控表达H9N2亚型禽流感病毒(AIV)HA蛋白的MDCK细胞系,能有效支持H9N2流感病毒的扩增.本研究应用插入四环素调控元件和H9N2亚型禽流感病毒HA基因的重组质粒V2-H9,与辅助质粒pMDSV、psPAX2共转染293T细胞包装慢病毒,将包装出的慢病毒感染MDCK细胞,通过添加嘌呤霉素筛选出阳性的单克隆细胞.再用PR8 SH441病毒感染筛选出来的MDCK单克隆细胞.结果显示,流感病毒在其中4株MDCK单克隆细胞的HA效价接近于8;生长曲线结果显示,流感病毒在第34株MDCK单克隆细胞中复制效果最好;间接免疫荧光(IFA)和Western blot检验结果显示,第34株MDCK单克隆细胞H9 HA的表达量最高;将第34株MDCK单克隆细胞驯化后进行悬浮培养,每隔24 h测定细胞密度,结果表明,第34株MDCK单克隆细胞可以悬浮培养,且流感病毒在第34株MDCK悬浮细胞中HA效价可达9log2.以上结果表明,本研究获得了一株可高效扩增H9亚型禽流感病毒的MDCK细胞株,为流感弱毒疫苗的研发提供了新的研究方法和实验基础.

本研究分离得到两株猪源H1N1流感病毒,通过进化树分析发现分别是欧亚类禽H1N1猪流感病毒和类鸭源H1N1猪流感病毒,为了研究两株毒株的NS1蛋白对Ⅰ型IFN产生的抑制能力,分别构建了2个毒株的NS1基因的真核表达载体,将NS1真核表达质粒、Ⅰ型IFN报告质粒与干扰素刺激质粒RIG-I共转染293T细胞,利用双荧光素酶报告基因检测Ⅰ型IFN激活水平.结果显示:欧亚类禽H1N1猪流感病毒NS1蛋白对人源细胞干扰素的拮抗作用明显强于类鸭源H1N1猪流感病毒;2种NS1蛋白对RIG-I与TBK-1激活的IFN-I抑制能力存在显著的差别,但是对IRF-3激活的IFN-I抑制能力没有显著差别.本研究结果初步揭示了欧亚类禽与类鸭源H1N1猪流感病毒抑制IFN-I能力的差异,及H1N1禽流感适应猪群的潜在分子机制.

鸭病毒性肝炎(DVH)是危害雏鸭的一种急性高度致死性传染病,引发DVH的主要病原体为鸭甲肝病毒(DHAV),其中1型鸭肝炎病毒在中国各省流行最广.本研究于2018年从山东某养鸭场的病料中分离得到两株DHAV,经分子生物学鉴定、序列比对和进化树分析,鉴定引起雏鸭患病的病原体为DHAV-1.经过纯化后,对SPF雏鸭进行致病性试验,用鸭胚滴定和荧光定量的方法测定脏器病毒含量,致病性分析结果显示,两株DHAV-1对雏鸭的致病性不同,SD37毒株引起雏鸭致死率为100％,而SD63引起雏鸭致死率为12.5％,这可能与VP1氨基酸差异有关.本研究通过对山东地区两株DHAV-1的鉴定和致病性研究,为我国DHAV的分子流行病学、防治以及疫苗研制提供了材料和理论指导.

Based on our previous studies, we show that the M gene is critical for the replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing the bat M gene with those from human and swine influenza A viruses. However, the key amino acids of the M1 and/or M2 proteins that are responsible for virus replication and pathogenicity remain unknown. In this study, replacement of the PR8 M gene with the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of the chimeric H17 bat influenza virus. Further studies revealed that M1 was more crucial for viral growth and pathogenicity than M2 and that the amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins identified in this study might be important for influenza virus surveillance and could be used to produce live attenuated vaccines in the future. IMPORTANCE The M1 and M2 proteins influence the morphology, replication, virulence, and transmissibility of influenza viruses. Although a few key residues in the M1 and M2 proteins have been identified, whether other residues of the M1 and M2 proteins are involved in viral replication and pathogenicity remains to be discovered. In the background of the chimeric H17 bat influenza virus, the Eurasian avian-like M gene from the A/California/04/2009 virus significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 in viral growth and pathogenicity in vitro and in vivo and that the key amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine applications in the future. These findings provide important contributions to knowledge of the genetic basis of the virulence of influenza viruses.

2015年暴发了一种由新型鹅细小病毒(NGPV)引起的鸭喙萎缩-侏儒症(BADS)的疾病,造成了严重的经济损失.本研究根据GenBank中NGPV相对保守的VP3基因序列,设计1对特异性引物和1条TaqMan探针,并对其反应条件进行优化,建立了一种检测NGPV的TaqMan荧光定量PCR方法.敏感性试验结果表明,该方法检测敏感性达到37.2拷贝/μL,比常规PCR灵敏度高100倍,而且该方法对鸭其他主要病原核酸检测均为阴性,具有良好的特异性.批内和批间的检测变异系数均小于等于2.2％.对收集的50份病料进行检测,本研究建立的荧光定量PCR方法NGPV检出率为78％,普通PCR方法为50％,荧光定量PCR方法的敏感性明显高于普通常规PCR方法.研究结果表明该方法具有操作简便、敏感性高和特异性强的特点,可用于NGPV的快速诊断.

H6 subtype avian influenza viruses spread widely in birds and pose potential threats to poultry and mammals, even to human beings. In this study, the evolution and pathogenicity of H6 AIVs isolated in live poultry markets from 2011 to 2017 were investigated. These H6 isolates were reassortant with other subtypes of influenza virus with increasing genomic diversity. However, no predominant genotype was found during this period. All of the H6N2 and most of the H6N6 isolates replicated efficiently in lungs of inoculated mice without prior adaptation. All of the H6N2 and two H6N6 isolates replicated efficiently in nasal turbinates of inoculated mice, which suggested the H6N2 viruses were more adaptive to the upper respiratory tract of mice than the H6N6 viruses. One of H6N2 virus caused systemic infection in one out of three inoculated mice, which indicated that H6 avian influenza virus, especially the H6N2 viruses posed a potential threat to mammals. Five H6 strains selected from different genotypes caused no clinical signs to inoculated chickens, and their replication were limited in chickens since the viruses have been detected only from a few tissues or swabs at low titers. Our study strongly suggests that the H6 avian influenza virus isolated from live poultry markets pose potential threat to mammals.

H9N2亚型禽流感病毒(AIV)在自然界中广泛存在和传播,给养禽业造成了巨大损失.为了进一步揭示该病毒的致病机制,本研究采用RT-PCR技术扩增禽流感病毒A/Chicken/Shanghai/1/2006(简称SH1)的PB2、PB1、PA、HA、NP、NA、M、NS 8个基因片段,并分别克隆至PLLB双向表达载体上.采用8质粒系统共转染293T细胞,转染48 h后加入TPCK胰酶作用2 h,将上清液和细胞一同接种9～11日龄SPF鸡胚,并检测其血凝效价.经序列比对,拯救获得的病毒rSH1的8个基因片段序列均与亲本病毒SH1的序列相同.实验结果表明,本研究成功建立了H9N2亚型禽流感病毒反向遗传操作系统,为该病毒的致病机理和传播机制研究等奠定了技术平台.