禽流感H5N1亚型病毒感染ICR小鼠的动物模型

目的 建立H5N1禽流感病毒感染ICR小鼠的疾病动物模型.方法 将100 μL H5N1 禽流感病毒原液(EID50为105.37/0.2 mL) 鼻腔接种ICR小鼠,设生理盐水组、正常尿囊液组对照,接毒后14 d内每隔12 h观察一次,主要观测指标有临床体征、体重和体温变化、死亡率、病理变化、病毒分离和血清抗体检测 (ELISA方法).结果 被感染的ICR小鼠的病程可以划分为潜伏期 (第0～1天)、急性感染期 (第2～7天)、恢复期 (第8～14天),急性感染期表现出活动明显减少,弓背,反应性差,扎堆;接毒后第1天开始体温和体重下降,第6天体温和体重停止下降;接毒组ICR小鼠累计的死亡率为60%;急性感染期ICR小鼠的肺部病变最严重,表现为间质性肺炎,肺间质充血、水肿和淋巴细胞浸润,毛细血管扩张,上皮细胞变性、坏死、脱落,并有充血和单核细胞浸润;接毒后第1天至第8天可在小鼠的肺、脑、气管和心、肝、脾、肾分离到病毒;接毒后第6天从ICR小鼠血清中检测到抗体.结论 本实验室建立的H5N1禽流感病毒感染ICR小鼠的模型在临床表现、体重变化、死亡率、病理变化、病毒复制指标能达到禽流感病毒疾病模型的造模要求,符合人类禽流感感染疾病的基本特征.     

Oxidative Stress in Lungs of Mice Infected with Influenza a Virus

As oxidative stress has been implicated in the pathogenesis of certain viral diseases we determined antioxidant and prooxidant parameters in lungs and bronchoalveolar lavage fluid (BALF) of mice infected with a lethal dose of influenza A/PR8/34 virus. Viral infection was characterized by massive infiltration of leukocytes, mainly polymorphonuclear leukocytes, into the alveolar space. The total number of BALF cells increased up to 8-fold (day 3 post-infection) and these cells appeared activated as judged by their increased rates of superoxide anion radical (O₂^-) generation upon stimulation. Maximal rates of radical generation by BALF cells during the early stages of infection were 15- or 70-fold higher than those of cells from control animals when expressed per cell or total BALF cells, respectively. At the terminal stages of infection the total capacity of BALF cells to release of declined to ≈ 35-fold the control values. Infection also resulted in increased in vivo formation of hydrogen peroxide (H²O²) within the lungs at a time that coincided with the maximal capacity of BALF cells to release O_2-.

Whereas pulmonary activities of glutathione peroxidase and reductase remained unaltered, levels of ascorbate in the cell-free BALF decreased significantly during the early stages of the infection and then returned to normal levels and above, late in infection. The oxidation state of the dehydroascorbic acid/ ascorbate couple increased concomitantly with the decrease in ascorbate concentrations early in infection and remained elevated throughout the infection. As assessed by the prevention of peroxyl radical-induced loss of phycoerythrin fluorescence, the total antioxidant capacity present in lung tissue homogenate from terminally ill animals was not diminished when compared to that prepared from lungs of control mice. We conclude that although early stages of influenza infection are associated with the presence of oxidative stress in the lung tissue and alveolar fluid lining the epithelial cells, this stress does not appear to overwhelm local antioxidant defenses. The results therefore do not support a direct causative role of oxidative tissue damage in the pathogenesis of influenza virus infection.     

葡萄NIP基因家族的鉴定与表达分析

类根瘤菌26膜内在蛋白(nodulin 26 like intrinsic proteins,NIPs)是水通道蛋白的亚类,在植物营养获取和胁迫应答过程中发挥着重要作用。该研究利用多种生物信息学软件,对葡萄NIP家族基因进行分析,并采用RT PCR方法克隆得到4个NIP家族基因,利用qRT PCR方法分析非生物胁迫下NIP基因的表达特征。结果显示：(1)在葡萄基因组中,共鉴定到8个NIP基因,分布于葡萄4条染色体上,主要定位在质膜中;结构上含有6个跨膜结构域和两个典型的保守结构域NPA;氨基酸序列中存在很多个可能的磷酸化位点。(2)进化分析表明葡萄和拟南芥NIP基因具有较高的同源性,基因结构包含外显子数4~6个,保守基序种类和数量相似;基因启动子上游2 kb包含多种应答逆境和激素的顺式调控元件,其数量差异可能与基因本身功能相关。(3)NIP家族基因在不同组织中表达水平差异较大,多数成员在叶中表达水平较高,在茎中较低;成功克隆得到4个葡萄VvNIP基因,其长度分别为789 bp、606 bp、897 bp、789 bp,分别编码262、201、298、293个氨基酸。(4)qRT PCR结果显示,不同胁迫处理下NIP基因在葡萄叶片中的表达水平不同：低温处理下葡萄NIP基因大多呈显著下调表达;盐胁迫下,除VvNIP2 1、VvNIP4 2外其余家族基因均呈下调表达;干旱胁迫下VvNIP4 2显著上调。研究表明,VvNIP基因对多种胁迫均有响应,为葡萄逆境胁迫机制研究提供了参考。     

Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.     

Cytopathogenesis and Inhibition of Host Gene Expression by RNA Viruses

Many viruses interfere with host cell function in ways that are harmful or pathological. This often results in changes in cell morphology referred to as cytopathic effects. However, pathogenesis of virus infections also involves inhibition of host cell gene expression. Thus the term “cytopathogenesis,” or pathogenesis at the cellular level, is meant to be broader than the term “cytopathic effects” and includes other cellular changes that contribute to viral pathogenesis in addition to those changes that are visible at the microscopic level. The goal of this review is to place recent work on the inhibition of host gene expression by RNA viruses in the context of the pathogenesis of virus infections. Three different RNA virus families, picornaviruses, influenza viruses, and rhabdoviruses, are used to illustrate common principles involved in cytopathogenesis. These examples were chosen because viral gene products responsible for inhibiting host gene expression have been identified, as have some of the molecular targets of the host. The argument is made that the role of the virus-induced inhibition of host gene expression is to inhibit the host antiviral response, such as the response to double-stranded RNA. Viral cytopathogenesis is presented as a balance between the host antiviral response and the ability of viruses to inhibit that response through the overall inhibition of host gene expression. This balance is a major determinant of viral tissue tropism in infections of intact animals.     

异型流感病毒感染小鼠肺细胞因子水平变化

为了制备能够抵御不同型别流感病毒感染的疫苗,揭示机体对异型流感病毒感染交叉免疫保护作用的主要机制,用流感病毒疫苗免疫小鼠后分别感染同型、异型流感病毒,另设使用免疫增强剂IL-2后感染异型流感病毒组,观察小鼠的一般状况和肺指数,并用ELISA方法测定肺匀浆中细胞因子IFN-γ、IL-2、IL-4及IL-10的含量。结果显示,异型免疫组和异型免疫加强组病毒感染后细胞因子IFN-γ含量明显高于感染前（P〈0.05）。研究表明,异型病毒感染后IFN-γ水平明显增高,此细胞因子可能在流感病毒异型间交叉保护免疫反应中起重要作用,其机制有待进一步研究确定。     

禽流感H5N1病毒感染BALB/c小鼠的细胞免疫动态变化

[目的]测定H5N1病毒感染BALB/c的小鼠模型的细胞免疫动态变化,探讨病毒对机体免疫系统的影响。[方法]通过流式细胞仪测定CD3+T、CD4+T、CD8+T等细胞免疫变化。[结果]感染H5N1病毒的小鼠血液中CD3+T、CD4+T、CD8+T细胞数量下降(P<0.05),脾脏中T细胞数量下降的趋势与血液相同,CD4+T/CD8+T的比例上升,只是两者的时间有所差别。[结论]说明病毒对细胞免疫T细胞数量影响较大,而且CD8+T受到的影响更为明显,反应了机体特异性细胞免疫功能受抑制,并且彼此之间的平衡受到破坏。     

Ultrastructure of the Surfaces of Cells Infected with Avian Leukosis-Sarcoma Viruses

When stained with ruthenium red (RR), chick embryo cells infected with various strains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 and RAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) at their surfaces as compared with uninfected cells. This increase was most prominent in cells infected with the Fujinami strain of RSV. The layer was resistant to digestion with neuraminidase or trypsin but was readily removed by exposure to hyaluronidase. The thickness of this AMPS layer was not correlated with the varying degree of loss of contact inhibition exhibited by cells infected with the different strains of virus. The staining of the cell envelope with a solution of phosphotungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins. The outer surface of the virions showed the same staining as the cell surface with RR and PTA-CR, and the budding virus particle was seen to incorporate the RR layer of the cell into its structure. The RR layers of cells and virions appeared to fuse, as did those between virus particles, suggesting that these layers play a role in the aggregation of virus particles and in their adherence to the surface of the cell.     

Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

禽流感病毒HA基因真核表达质粒的构建与表达

血凝素蛋白（HA）基因是禽流感病毒（AIV）重要的保护性抗原基因.为了研究 HA基因疫苗,用PCR扩增H5亚型AIV HA基因,将其克隆到质粒pcDNA4/HisMax和pRc/CMV上得到真核表达质粒pC4H5和pCMVH5.采用Tfx^TM-20、Superfect转染试剂和电转染法转染HeLa细胞,转染后的HeLa细胞经蛋白质印迹和血凝试验检测HA蛋白及其活性.结果表明,Superfect转染和电转染均能正确表达HA蛋白并具有生物学活性,蛋白质印迹检测到HA和HA裂解的HA1和HA2,与AIV 的HA、HA1、HA2蛋白的分子质量一致.从血凝试验结果看,Superfect和电转染表达的HA均具有血凝活性,而经Superfect转染的pC4H5的表达量是pCMVH5的8倍,表明pC4H5是一高效的真核表达质粒.     

Host Restrictions of Avian Influenza Viruses: In Silico Analysis of H13 and H16 Specific Signatures in the Internal Proteins

Gulls are the primary hosts of H13 and H16 avian influenza viruses (AIVs). The molecular basis for this host restriction is only partially understood. In this study, amino acid sequences from Eurasian gull H13 and H16 AIVs and Eurasian AIVs (non H13 and H16) were compared to determine if specific signatures are present only in the internal proteins of H13 and H16 AIVs, using a bioinformatics approach. Amino acids identified in an initial analysis performed on 15 selected sequences were checked against a comprehensive set of AIV sequences retrieved from Genbank to verify them as H13 and H16 specific signatures. Analysis of protein similarities and prediction of subcellular localization signals were performed to search for possible functions associated with the confirmed signatures. H13 and H16 AIV specific signatures were found in all the internal proteins examined, but most were found in the non-structural protein 1 (NS1) and in the nucleoprotein. A putative functional signature was predicted to be present in the nuclear export protein. Moreover, it was predicted that the NS1 of H13 and H16 AIVs lack one of the nuclear localization signals present in NS1 of other AIV subtypes. These findings suggest that the signatures found in the internal proteins of H13 and H16 viruses are possibly related to host restriction.     

Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×10² vs. 2.3×10⁴ vs. 8.7×10⁴; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.     

H9N2亚型禽流感病毒NS1基因的进化分析

利用RT-PCR方法,扩增了1998～2005年间分离的9株H9N2亚型禽流感病毒的NS1基因,对其进行了序列测定和进化分析.序列分析表明,9株AIV NS1基因完整的阅读框均为654bp,编码217个氨基酸,其核苷酸和推导的氨基酸同源性分别为95.4%～99.8%和93.6%～100%;9株病毒的NS1蛋白的C端均有13个氨基酸的缺失;进化分析表明,9株AIV属于A群,且形成一个独立分支,在该分支中,只有Ck/HN/A3/98株属于Ck/HK/Y280/97-like亚类,且与Ck/BJ/8/98的进化关系最近,其余8株属于Ck/SH/F/98-like亚类,说明Ck/SH/F/98-like亚类的H9N2亚型AIV在中国大陆的鸡群中广泛存在.NS1基因的进化及其编码产物的特性分析,为AIV的毒力变异、致病机制、药物靶位点的设计及鉴别诊断的研究奠定了基础.     

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

Virologica Sinica - The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between...     

Neuraminidase Subtyping of Avian Influenza Viruses with PrimerHunter-Designed Primers and Quadruplicate Primer Pools

We have previously developed a software package called PrimerHunter to design primers for PCR-based virus subtyping. In this study, 9 pairs of primers were designed with PrimerHunter and successfully used to differentiate the 9 neuraminidase (NA) genes of avian influenza viruses (AIVs) in multiple PCR-based assays. Furthermore, primer pools were designed and successfully used to decrease the number of reactions needed for NA subtyping from 9 to 4. The quadruplicate primer-pool method is cost-saving, and was shown to be suitable for the NA subtyping of both cultured AIVs and uncultured AIV swab samples. The primers selected for this study showed excellent sensitivity and specificity in NA subtyping by RT-PCR, SYBR green-based Real-time PCR and Real-time RT-PCR methods. AIV RNA of 2 to 200 copies (varied by NA subtypes) could be detected by these reactions. No unspecific amplification was displayed when detecting RNAs of other avian infectious viruses such as Infectious bronchitis virus, Infectious bursal disease virus and Newcastle disease virus. In summary, this study introduced several sensitive and specific PCR-based assays for NA subtyping of AIVs and also validated again the effectiveness of the PrimerHunter tool for the design of subtyping primers.     

2004年初广东省高致病性禽流感(H5N1)疫点野生鸟类禽流感研究简报

2004年初禽流感疫情期间,针对广东省疫点地区开展了野生鸟类禽流感病毒（H5、H7、H9）调查取样工作,共获得血样76份、拭子125份、器官样21份。血清检测采用琼脂扩散（AGP）和血凝抑制试验（H1）,病毒检测用鸡胚法鉴定。分析结果：（1）只在非疫点的汕头南澳的八哥上检测到H9病毒;（2）雷州、罗定、海丰及汕头4个地区的血清检测结果中H5和H9阳性率分别为31．6％和23．7％;沿海地区阳性率极显著高于非沿海地区;（3）候鸟的阳性率极显著高于留鸟。因此,该次疫情中候鸟中存在传播禽流感病毒的可能性。