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A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

ABSTRACT: Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

SUBMITTER: Smith CM

PROVIDER: S-EPMC4997599 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

Smith Claire M CM Scott Paul D PD O'Callaghan Christopher C Easton Andrew J AJ Dimmock Nigel J NJ

Viruses 20160822 8

Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides a ...[more]

PMID: 27556481

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Project description:Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. KEY POINTS: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers.

Project description:A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90-99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.

Project description:Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.

Dataset Information

A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

Publications

A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

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