流感病毒感染诱导MDCK细胞凋亡的研究

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诱导狗肾体代细胞(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MMCK细胞的毒力和凋亡诱导能力水平。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,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。     

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     

miR26a和miR939调控H1N1型流感病毒在MDCK细胞中的复制

摘要：【目的】研究发现microRNAs（miRNAs）可以参与调控病毒在宿主细胞内感染和复制的过程。作者研究了两条miRNAs对H1N1型流感病毒在宿主细胞内复制的影响。【方法】构建miR26a和miR939的高效表达载体,并将这两种表达载体转入MDCK细胞中,24 h后用H1N1型流感病毒感染转染后的MDCK (Madin dardy canine kidney) 细胞,接种72 h后,检测流感病毒的复制情况,研究miR26a和miR939对H1N1型流感病毒在MDCK细胞内复制的影响。【结果】实验结果表明,miRNAs的表达载体可以在细胞内高效表达miRNAs,不同的miRNAs对流感病毒在MDCK细胞中复制的调控作用不同, miR26a可以有效抑制流感病毒在MDCK细胞中的复制,而miR939则促进流感病毒在MDCK细胞中的复制的作用。【结论】细胞内miRNAs可以调控H1N1型流感病毒在宿主细胞中的复制过程,本文首次报导miR26a和miR939在流感病毒复制过程中的调控作用。     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

柯萨奇病毒A组10型感染RD细胞的转录组学分析

探讨柯萨奇病毒A组10型（coxsackievirus A10, CV-A10）感染人横纹肌肉瘤（rhabdomyosarcoma,RD）细胞的基因表达谱变化,为深入理解CV-A10致病机制提供重要基础数据。本研究将CV-A10以感染复数（MOI）为0.1的剂量感染RD细胞12 h后提取总RNA,通过RNA-Seq技术获取RD细胞中的全部转录本信息,基于生物信息学的方法对差异表达基因进行GO(gene ontology, GO)和KEGG(Kyoto encyclopedia of genes and genomes, KEGG)分析。结果显示,共筛选到2 610个差异表达基因,其中表达上调的基因有1 582个,表达下调的基因有1 028个。随机选取8个差异表达基因,经RT-qPCR验证后发现,其变化趋势与转录组数据一致。GO和KEGG富集分析结果显示,差异表达基因主要聚焦于细胞内信号转导、mRNA加工、抗病毒免疫反应等过程。结果表明,CV-A10感染可能会引起RD细胞发生自噬。     

人H7N9禽流感病毒、高致病H5N1禽流感病毒及H1N1流感病毒感染小鼠特征分析

目的对比分析人高致病H5N1禽流感病毒、H7N9禽流感病毒及H1N1流感病毒分别感染BALB/c小鼠后的机体反应特征。方法分别以H7N9病毒、H5N1病毒和H1N1病毒滴鼻感染BALB/c小鼠,观察小鼠存活率、体征变化及感染后肺组织病理损伤差异,检测小鼠感染流感病毒后肺组织增殖细胞核抗原(PCNA)表达并观察小鼠感染后修复状况。结果 H7N9病毒、H5N1病毒和H1N1病毒均感染BALB/c小鼠,小鼠存活率依次为H7N9H1N1H5N1,肺组织病理损伤严重程度依次为H5N1H1N1H7N9,PCNA表达水平依次为H7N9H1N1H5N1。结论 H7N9病毒感染后宿主炎症反应较小,感染后小鼠肺组织自我修复能力较强;H5N1病毒感染BALB/c小鼠后的机体反应最为强烈,感染后恢复能力差,致死率高。     

H1N1流感病毒在微载体培养MDCK细胞上增殖的研究

目的探索MDCK细胞在微载体上的培养条件,并研究H1N1型流感病毒在MDCK细胞上的增殖条件。方法在微载体上培养好MDCK细胞上用H1N1型流感病毒在不同的病毒感染复数(MOI)、胰酶浓度两个关键的病毒增殖条件进行流感病毒在细胞上的增殖研究。结果微载体质量浓度为6 g/L时,MDCK细胞培养密度可以达到4.5×106cells/mL。在MOI为0.05接种流感病毒,胰酶质量浓度4μg/mL,流感病毒在MDCK细胞上可获得较高的滴度。结论 MDCK细胞用微载体培养可以达到较高的细胞密度,可以作为规模化生产新型流感病毒疫苗的主要细胞基质进行进一步的研究。     

H7N9和H1N1流感病毒感染BV2细胞的差异表达miRNA初步分析

筛选鉴定H7N9病毒感染BV2细胞的特异聚类microRNA(miRNA)并初步探讨这些miRNA的可能致病机制。H7N9和H1N1流感病毒感染BV2细胞,分别收集12 h、24 h和48 h的细胞并提取总RNA。并利用高通量测序技术进行miRNA测序,同时比较鉴定不同病毒特异性miRNA。筛选出了10个H7N9病毒特异聚类miRNA,其中有3个miRNA被miRBase数据库所收录。这些特异聚类miRNA调控诸多信号通路的信号传导,比如Ras信号通路、PI3K-Akt信号通路、MAPK信号通路、轴突导向和癌症相关基因等。该研究为miRNA调控H7N9禽流感病毒的致病机制提供了科学基础。     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

Plasma proteomic analysis of patients infected with H1N1 influenza virus

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1‐infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano‐ultra performance LC‐MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1‐infected group as compared with the control group. Of 14 up‐ and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine‐rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.     

Mast cell-induced lung injury in mice infected with H5N1 influenza virus

Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.     

Growth determinants for H5N1 influenza vaccine seed viruses in MDCK cells

H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.     

Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2)

Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS‐based proteomics approach. Moreover, O‐sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus–host interaction.     

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium

Background

Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.

Principal Finding

The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×10⁶ cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10⁸ pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.

Conclusions

The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.