Productivity, apoptosis, and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants

Isken, Britta; Genzel, Yvonne; Reichl, Udo

doi:10.1016/j.vaccine.2012.05.065

Cited by 20 publications

(24 citation statements)

References 39 publications

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“…The comparison of different PR8 isolates and high-growth reassortants ( Supplementary Table S1; also used by Isken et al (2012)) confirmed that infections with seed virus preparations with low amounts of DIPs lead to higher virus yields (Supplementary Figure S1). Hence, for vaccine virus strains not only the nature of their HA and NA or point mutations in other segments affect the virus titer but also the presence of DIPs can have a strong impact on yields in vaccine manufacturing.…”

Section: Comparison Of Virus Yieldsmentioning

confidence: 65%

“…However, strong variations in virus yields were observed in these processes if no optimization of the MOI and harvest time was performed (Aggarwal et al 2011). One aspect that was speculated to cause these variations is the presence of DIPs (Aggarwal et al 2011;Isken et al 2012). In this study we examined the impact of DIPs on virus replication in MDCK cells which are an important cell substrate for the propagation of influenza viruses.…”

Section: Discussionmentioning

confidence: 99%

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Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

Frensing

Pflugmacher

Bachmann

et al. 2014

Appl Microbiol Biotechnol

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During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.

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Section: Comparison Of Virus Yieldsmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

Frensing

Pflugmacher

Bachmann

et al. 2014

Appl Microbiol Biotechnol

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“…Of note, all three families have been shown to play roles in apoptosis (42–44), and several publications have demonstrated the relevance of apoptosis to IAV infection (17, 45–47). We therefore tested whether there might be an association between strain-specific miRNA expression and apoptosis.…”

Section: Resultsmentioning

confidence: 99%

Host Genetic Background Strongly Affects Pulmonary microRNA Expression before and during Influenza A Virus Infection

2017

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BackgroundExpression of host microRNAs (miRNAs) changes markedly during influenza A virus (IAV) infection of natural and adaptive hosts, but their role in genetically determined host susceptibility to IAV infection has not been explored. We, therefore, compared pulmonary miRNA expression during IAV infection in two inbred mouse strains with differential susceptibility to IAV infection.ResultsmiRNA expression profiles were determined in lungs of the more susceptible strain DBA/2J and the less susceptible strain C57BL/6J within 120 h post infection (hpi) with IAV (H1N1) PR8. Even the miRNomes of uninfected lungs differed substantially between the two strains. After a period of relative quiescence, major miRNome reprogramming was detected in both strains by 48 hpi and increased through 120 hpi. Distinct groups of miRNAs regulated by IAV infection could be defined: (1) miRNAs (n = 39) whose expression correlated with hemagglutinin (HA) mRNA expression and represented the general response to IAV infection independent of host genetic background; (2) miRNAs (n = 20) whose expression correlated with HA mRNA expression but differed between the two strains; and (3) remarkably, miR-147-3p, miR-208b-3p, miR-3096a-5p, miR-3069b-3p, and the miR-467 family, whose abundance even in uninfected lungs differentiated nearly perfectly (area under the ROC curve > 0.99) between the two strains throughout the time course, suggesting a particularly strong association with the differential susceptibility of the two mouse strains. Expression of subsets of miRNAs correlated significantly with peripheral blood granulocyte and monocyte numbers, particularly in DBA/2J mice; miR-223-3p, miR-142-3p, and miR-20b-5p correlated most positively with these cell types in both mouse strains. Higher abundance of antiapoptotic (e.g., miR-467 family) and lower abundance of proapoptotic miRNAs (e.g., miR-34 family) and those regulating the PI3K-Akt pathway (e.g., miR-31-5p) were associated with the more susceptible DBA/2J strain.ConclusionSubstantial differences in pulmonary miRNA expression between the two differentially susceptible mouse strains were evident even before infection, but evolved further throughout infection and could in part be attributed to differences in peripheral blood leukocyte populations. Thus, pulmonary miRNA expression both before and during IAV infection is in part determined genetically and contributes to susceptibility to IAV infection in this murine host, and likely in humans.

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“…In consequence, they may be more prone to lysis upon infection due to the shear stress in a stirred system. In addition, high viral loads (high MOI) may cause a faster progression of virus-induced cell death, which shortens the productive phase of the cells, as shown for influenza viruses (Isken et al 2012). This effect was reduced by using a lower MOI of CAV-2 to infect MDCK.SUS2 cells.…”

Section: Discussionmentioning

confidence: 99%

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

Fernandes

Laske

Sousa

et al. 2015

Appl Microbiol Biotechnol

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The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

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Productivity, apoptosis, and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants

Cited by 20 publications

References 39 publications

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

Host Genetic Background Strongly Affects Pulmonary microRNA Expression before and during Influenza A Virus Infection

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells

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