Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Effect of interferons on the replication of alcelaphine herpesvirus-1 of malignant catarrhal fever

Four isolates of alcelaphine herpesvirus-1 of malignant catarrhal fever (MCF) were tested for their inducibility of and sensitivity to various interferons. Viral isolates from an Indian gaur (Bos gaurus), a greater kudu (Tragelaphus strepsiceros) and two wildebeest (Connochaetes gnou) calves did not induce measurable interferon (IFN) in bovine fetal kidney cells. However, these low passages of each virus were all highly cell-associated and viral replication was inhibited at these passages by IFN at 14 IFN units/0.05 ml recovered from NDV-infected MDBK cells and at 7.6 IFN units/0.05 ml of IFN from NDV-infected bovine macrophages. The herpesvirus from the Indian gaur and greater kudu and high passages (greater than 50) of the cell-free WC-11 strain of alcelaphine herpesvirus-1 also were inhibited in their replication by recombinant IFN of bovine and human origins as determined by a fluorescent focus unit (FFU) reduction assay. The concentrations of IFN required to produce a 50% reduction in herpesvirus-produced FFU ranged between 6.4 and 480 IFN units. These findings promote the use of IFN as part of the regimens of treatment of captive endangered ruminant species with clinical MCF.     

蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用

【目的】在细胞水平上研究黄酮类化合物flavopiridol的抗流感病毒效果,初步探索了其抗流感病毒的机制。【方法】首先用Western blot初步检测了在蛋白激酶抑制剂flavopiridol处理下流感病毒NP和M1蛋白的水平,然后通过免疫荧光实验观察了宿主细胞中流感病毒vRNP的合成,又利用噬斑实验检测了flavopiridol对病毒复制的影响,最后通过检测flavopiridol处理的宿主细胞内RNA聚合酶Ⅱ的磷酸化状态和病毒各种RNA的合成量,探究了flavopiridol抑制流感病毒复制的机理。【结果】结果表明,flavopiridol在细胞水平上可以显著抑制流感病毒蛋白质和vRNP的合成及病毒的复制,同时flavopiridol也可以抑制宿主RNA聚合酶Ⅱ大亚基CTD结构域七肽重复序列中的2位丝氨酸的磷酸化来抑制聚合酶的转录延伸活性,显著地减少病毒vRNA的合成。【结论】Flavopiridol可以通过抑制宿主细胞RNA聚合酶Ⅱ的转录延伸活性有效地抑制流感病毒的复制。