流感病毒感染诱导MDCK细胞调亡的研究

用荧光染色、ＤＮＡ凝胶电泳等方法检测了Ａ型流感病毒株Ａ１／京防８６－１和Ｂ型流感病毒株Ｂ／沪防９３－１诱导狗肾传代细胞（ＭＤＣＫｃｅｌｌｓ）的凋亡情况,并采用ＭＴＴ法和流式细胞仪比较了这２株病毒对ＭＤＣＫ细胞毒力和调亡诱导能力水平。结果显示：病毒感染６ｈ后,细胞ＤＮＡ发生断裂,病毒感染１２ｈ后,可见明显的染色质凝聚;在一定范围内,细胞调亡强度表现出明显的时间和剂量依赖关系;并且,Ａ型流感病毒株的毒     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

黑色素抑制流感病毒诱导宿主细胞凋亡

报道了流感病毒体外诱导狗肾细胞系（ＭＤＣＫ）细胞凋亡的检测结果,对黑色素选择性抑制流感病毒诱导细胞凋亡的可能性进行了探讨,同时与临床上常用的抗病毒药物病毒唑的效果进行比较。结果显示：病毒感染６ｈ后,即可观测到宿主细胞核固缩现象、ＤＮＡ凝胶电泳出现特征性的梯状图谱,感染１２ｈ后,细胞核可见明显的裂解;并且流感病毒株Ａ１／京防８６１诱导细胞凋亡能力强于Ｂ沪防／９３－１;在２０～１２５μｇ／ｍＬ浓度范围内,黑色素可有效抑制６４个血凝单位（ＨＵ）的流感病毒感染诱导的细胞凋亡而无细胞毒性作用,其抑制效率类似病毒唑。初步研究结果表明：黑色素抗流感病毒诱导细胞凋亡机理与其阻断病毒吸附侵入宿主细胞有关     

H1N1亚型流感病毒诱导外周血单个核细胞凋亡研究

A型流感病毒能诱导淋巴细胞、单核巨噬细胞的凋亡,为进一步探讨淋巴细胞和单核巨噬细胞在凋亡中可能存在的相互作用,用H1N1亚型流感病毒诱导人外周血淋巴细胞和单核巨噬细胞的凋亡．结果显示,前48 h,H1N1流感病毒能诱导淋巴细胞和单核巨噬细胞的凋亡,但在培养48 h后,流感病毒对单核巨噬细胞表现为凋亡抑制作用,同时流感病毒对淋巴细胞吸附不同时间后,荧光染色和流式细胞术检测凋亡未见明显差异,说明细胞凋亡与病毒吸附时间长短并无相关性．检测p53抑制剂Pifithrin-α(PFT-α)加入前后淋巴细胞和单核巨噬细胞的凋亡情况,结果显示,淋巴细胞和单核巨噬细胞的凋亡均被抑制, 提示通过p53诱导的凋亡可能是流感病毒诱导细胞凋亡的一条重要途径．     

神经酰胺诱导药物敏感型和耐药型细胞凋亡机制的探讨

以药物敏感型细胞株Ｋ５６２／Ｓ和耐药型细胞株Ｋ５６２／Ａ０２为对象．观察原癌基因Ｂｃｌ－２的表达量在两种细胞中的差异,以及神经酰胺作为一个新的脂质第二信使诱导细胞凋亡的能力,并利用酪氨酸激酶抑制剂ｇｅｎｉｓｔｅｉｎ,酪氨酸磷酸酯酶抑制剂ｖａｎａｄａｔｅ,观察酪氨酸可逆磷酸化与细胞凋亡间的关系．结果显示：在Ｋ５６２／Ａ０２中Ｂｃｌ－２的表达量明显高于Ｋ５６２／Ｓ;外源性神经酰胺能成功地诱导Ｋ５６２／Ｓ,Ｋ５６２／Ａ０２细胞凋亡,凋亡细胞具有典型的形态学改变和ＤＮＡ“Ｌａｄｄｅｒ”形成,ＦＣＭ检测出现凋亡细胞峰,但在同样的诱导条件下,Ｋ５６２／Ｓ细胞凋亡明显高于Ｋ５６２／Ａ０２细胞．ＦＣＭ检测ｇｅｎｉｓｔｅｉｎ能显著改变这两种细胞生长周期,但细胞阻滞于Ｇ２／Ｍ期,便对神经酰胺诱导的细胞凋亡无明显作用,ｖａｎａｄａｔｅ单独对细胞地明显作用,但与神经酰胺共同作用能明显提高细胞凋亡率．以上结果表明在药物诱导的细胞调亡中Ｂｃｌ－２基因起重要作用,神经酰胺能诱导Ｋ５６２／Ｓ和Ｋ５６２／Ａ０２细胞调亡．     

鱼呼肠孤病毒诱导草鱼肾细胞凋亡

采用荧光显微镜、电子显微镜、琼脂糖凝胶电泳、流式细胞仪分析等技术研究鱼呼肠孤病毒诱导草鱼细胞（ＣＩＫ）调亡。结果显示,鱼呼肠孤病毒感染ＣＩＫ细胞后,光镜下可见空斑形成;荧光染色观察到细胞调亡碎裂核;且电镜下呈现细胞核裂解,核周裂隙增大,细胞膜内陷并出泡形成调亡小体现象;琼脂糖凝胶电泳出现１８０－２００ｂｐ整数倍的ＤＮＡ梯形带;流式细胞仪检测到典型的细胞调亡峰,在病毒感染４８ｈ,细胞调亡百分率达１５     

新生隐球菌对角质形成细胞活力的作用研究

目的研究新生隐球菌体外对角质形成细胞活力的影响。方法将新生隐球菌父代标准株与子代荚膜缺陷株于体外分别与角质形成细胞分别共培养,同时设立热灭活的菌体、空白对照,再分别设立菌体与细胞直接接触与不接触共培养相互对照,分别作用0.5 h、1 h和2 h后,采用流式细胞仪检测隐球菌角质形成细胞的调亡率。结果随着时间延长,与空白对照组及热灭活组比较,实验组角质形成细胞的凋亡率逐渐增加。无荚膜株与父代有荚膜株比较,无荚膜株对细胞活力的影响在作用后1 h、2 h明显低于有荚膜株。2种菌株不直接接触培养使细胞的凋亡率明显下降;不直接接触的有荚膜株与热灭活的菌体之间比较差异不显著。结论虽然有荚膜株与无荚膜株隐球菌均可以使角质形成细胞活性明显降低,但荚膜可以显著增强菌体对细胞活力的影响;角质形成细胞活力的降低主要是通过与菌体接触培养后产生的,诱导细胞调亡需要菌体与细胞的直接接触。     

细胞色素c(Cyt c)诱导烟草悬浮细胞(BY-2)凋亡

用不同浓度细胞色素c(Cyt c)诱导继代时间不同的烟草悬浮细胞48 h后观察形态学特征的结果表明,继代培养10和13 d的细胞均在10 mmol·L-1Cyt c时出现最高的细胞凋亡率,而继代5 d的细胞在Cyt c浓度为12.5 mmol·L-1时细胞凋亡的诱导率仍表现上升趋势;DNA电泳检测结果显示凋亡处理的细胞中DNA呈现较明显的DNA梯度.     

蛋氨酸脑啡肽（MEK）抗B型流感病毒感染作用的研究

研究MEK抗B型流感病毒感染的作用。采用MDCK细胞和9～10日龄鸡胚,按不同的顺序加入不同剂量MEK和B型流感病毒,共培养72 h后做血凝实验。所有加入B型流感病毒的MDCK细胞均培养出病毒,HA滴度为1：64。在鸡胚尿囊腔中,先注入MEK孵育24 h后,再注入B型流感病毒的鸡胚也培养出病毒,HA滴度为1：6.8,与病毒对照组比较P〈0.01,有统计学意义。实验结果未见MEK直接抗B型流感病毒感染MDCK细胞株的作用,但可见MEK抗B型流感病毒感染鸡胚的作用。     

Sindbis病毒的繁殖与宿主细胞BHK—21的凋亡

详细报道了Sindbis病毒诱导BHK-21细胞凋亡的过程,病毒感染6h后即可观测到核染色质的断裂,病毒感染12h后染色质可见明显的凝集,感染后24h DNA电泳出现明显的DNA“阶梯”(DNA ladder)。电镜观察更清楚地显示了凋亡小体形成的某些细节:在染色质凝集处核外膜突起,最后与细胞核分离形成凋亡小体。在此基础上将一段病毒非结构蛋白nsP2基因克隆到真核表达载体pMAMneo中,并得到瞬间表达,在其中一些细胞中出现DNA断裂这一细胞凋亡的基本特征,通过对nsP2氨基酸序列的分析,结合以前的实验结果推测nsP2可能与诱导细胞凋亡直接相关。     

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.     

Primary study on the lesions and specific proteins in BEAS-2B cells induced with the 2009 A (H1N1) influenza virus

In order to investigate the lesions and proteins with differential expression in cells infected with the 2009 A (H1N1) virus and to determine the specific proteins involved in cell damage, the present study has been performed. BEAS-2B cells were infected with the 2009 A (H1N1) influenza virus or the seasonal H1N1 influenza virus for 12, 24, 48, and 72 h, and cell cycle and apoptosis were analyzed with flow cytometry. Total cellular proteins were extracted and underwent two-dimensional gel electrophoresis. The differentially expressed proteins underwent mass spectrometry for identification. The results showed that after 12 h, cells infected with the virus strain sourced from severe cases had the highest apoptosis rate (P?P?P?Galectin-1 was specifically observed in BEAS-2B infected with 2009 A (H1N1) influenza viruses, and cofilin-1 was specifically observed in BEAS-2B cells in the late stage of 2009 A (H1N1) influenza virus infection. In conclusion, differential effects of the 2009 A (H1N1) influenza virus and seasonal H1N1 influenza virus were identified on the cell cycle and apoptosis, and galectin-1 may play a role in cell apoptosis induced by 2009 A (H1N1) influenza virus.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

Potential Role of HPA Axis and Sympathetic Nervous Responses in Depletion of B Cells Induced by H9N2 Avian Influenza Virus Infection

Except severe pulmonary disease caused by influenza virus infection, an impaired immune system is also a clinic characteristic. However, the mechanism(s) of influenza virus infection-induced depletion of B cells was unknown. Here, we compared the effect of two variant virulence H9N2 virus infections on mouse B cells. Our study found that the infection with highly pathogenic virus (V) of led to depletion of spleen B cells and bone marrow (BM) early B cells, compared to lowly pathogenic virus (Ts). Moreover, high apoptosis and cell cycle arrest in spleen and BM were detected, suggesting important factors for the reduction of B cells in both organs. Further, this effect was not caused by virus replication in spleen and BM. Compared to Ts virus infection, V virus resulted in higher glucocorticoids (GCs) and lower leptin level in plasma. Intraperitoneal GCs receptor antagonist RU486 injection was sufficient to prevent the loss of spleen B cell and BM pro- and immature B cells, but similar result was not observed in leptin-treated mice. Depletion of spleen B cells and BM pro-B cells was also reversed by chemical sympathectomy mediated by the norepinephrine (NE) analog 6-hydroxydopamine (6-OHDA), but the treatment didn''t affect the GCs level. This study demonstrated that depletion of B cells induced by H9N2 AIV was dependent on HPA axis and sympathetic response.     

Implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic 2009 H1N1 influenza A virus after host adaptation

While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.     

Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.     

Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells

The nucleocapsid protein (NP) (56 kDa) of human influenza A viruses is cleaved in infected cells into a 53-kDa form. Likewise, influenza B virus NP (64 kDa) is cleaved into a 55-kDa protein with a 62-kDa intermediate (O. P. Zhirnov and A. G. Bukrinskaya, Virology 109:174-179, 1981). We show now that an antibody specific for the N terminus of influenza A virus NP reacted with the uncleaved 56-kDa form but not with the truncated NP53 form, indicating the removal of a 3-kDa peptide from the N terminus. Amino acid sequencing revealed the cleavage sites ETD16*G for A/Aichi/68 NP and sites DID7*G and EAD61*V for B/Hong Kong/72 NP. With D at position -1, acidic amino acids at position -3, and aliphatic ones at positions -2 and +1, the NP cleavage sites show a recognition motif typical for caspases, key enzymes of apoptosis. These caspase cleavage sites demonstrated evolutionary stability and were retained in NPs of all human influenza A and B viruses. NP of avian influenza viruses, which is not cleaved in infected cells, contains G instead of D at position 16. Oligopeptide DEVD derivatives, specific caspase inhibitors, were shown to prevent the intracellular cleavage of NP. All three events, the NP cleavage, the increase of caspase activity, and the development of apoptosis, coincide in cells infected with human influenza A and B viruses. The data suggest that intracellular cleavage of NP is exerted by host caspases and is associated with the development of apoptosis at the late stages of infection.