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Defective interfering viral particles in acute dengue infections.

ABSTRACT: While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

SUBMITTER: Li D

PROVIDER: S-EPMC3084866 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Defective interfering viral particles in acute dengue infections.

Li Dongsheng D Lott William B WB Lowry Kym K Jones Anita A Thu Hlaing Myat HM Aaskov John J

PloS one 20110429 4

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles h ...[more]

PMID: 21559384

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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.

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Defective interfering viral particles in acute dengue infections.

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Defective interfering viral particles in acute dengue infections.

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