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Journal of Virology May 2014Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging... (Review)
Review
Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging signals, are synthesized preferentially over infectious genomes, and are packaged as DI virus particles which can be transmitted to susceptible cells. Their ability to interfere with the replication of infectious virus in cell culture and their potential as antivirals in the clinic have long been known. However, until now, no realistic formulation has been described. In this review, we consider the early evidence of antiviral activity by DI viruses and, using the example of DI influenza A virus, outline developments that have led to the production of a cloned DI RNA that is highly active in preclinical studies not only against different subtypes of influenza A virus but also against heterologous respiratory viruses. These data suggest the timeliness of reassessing the potential of DI viruses as a novel class of antivirals that may have general applicability.
Topics: Animals; Antiviral Agents; Biological Products; Biomedical Research; Defective Viruses; Disease Models, Animal; Orthomyxoviridae; Orthomyxoviridae Infections; Viral Interference
PubMed: 24574404
DOI: 10.1128/JVI.03193-13 -
Applied Microbiology and Biotechnology Dec 2024Defective 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"...
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 log(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.
Topics: Defective Viruses; Influenza A virus; Virus Replication; Antiviral Agents; Salicylates
PubMed: 38229300
DOI: 10.1007/s00253-023-12959-6 -
Communications Biology Oct 2022Defective interfering (DI) particles arise during virus propagation, are conditional on parental virus for replication and packaging, and interfere with viral expansion....
Defective interfering (DI) particles arise during virus propagation, are conditional on parental virus for replication and packaging, and interfere with viral expansion. There is much interest in developing DIs as anti-viral agents. Here we characterize DI particles that arose following serial passaging of SARS-CoV-2 at high multiplicity of infection. The prominent DIs identified have lost ~84% of the SARS-CoV-2 genome and are capable of attenuating parental viral titers. Synthetic variants of the DI genomes also interfere with infection and can be used as conditional, gene delivery vehicles. In addition, the DI genomes encode an Nsp1-10 fusion protein capable of attenuating viral replication. These results identify naturally selected defective viral genomes that emerged and stably propagated in the presence of parental virus.
Topics: Humans; Defective Viruses; SARS-CoV-2; Defective Interfering Viruses; RNA, Viral; COVID-19
PubMed: 36302891
DOI: 10.1038/s42003-022-04058-5 -
Journal of Virology Nov 2018Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is...
Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When -complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIV) that could initiate single-round infection. The -complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIV production, with titers of >10 infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIV elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIV vaccine. The technology could be applicable to vaccine development for other viral pathogens. Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens.
Topics: Animals; Antibodies, Neutralizing; Defective Viruses; Dengue; Dengue Vaccines; Dengue Virus; Female; Humans; Male; Mice; Mutation; RNA, Viral; Viral Nonstructural Proteins; Virus Assembly; Virus Replication
PubMed: 30111567
DOI: 10.1128/JVI.01002-18 -
Virologica Sinica Apr 2019Since the publication of the DRiP (defective ribosomal product) hypothesis in 1996, numerous studies have addressed the contribution of DRiPs to generating viral... (Review)
Review
Since the publication of the DRiP (defective ribosomal product) hypothesis in 1996, numerous studies have addressed the contribution of DRiPs to generating viral antigenic peptides for CD8 T cell immunosurveillance. Here, we review studies characterizing the generation of antigenic peptides from influenza A virus encoded DRiPs, discuss the many remaining mysteries regarding the nature of their co-translational generation, and speculate on where the future might lead.
Topics: Animals; Antigens, Viral; CD8-Positive T-Lymphocytes; Defective Viruses; Histocompatibility Antigens Class I; Humans; Influenza A virus; Influenza, Human; Mice; Monitoring, Immunologic; Peptides
PubMed: 30456657
DOI: 10.1007/s12250-018-0061-y -
Journal of Virology Oct 2011The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a...
The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Bluetongue; Bluetongue virus; Cell Culture Techniques; Defective Viruses; Female; Male; Reassortant Viruses; Sheep; Viral Vaccines; Viremia
PubMed: 21795358
DOI: 10.1128/JVI.05412-11 -
PLoS Pathogens May 2020
Review
Topics: Animals; Defective Viruses; Humans; Influenza, Human; Orthomyxoviridae; Orthomyxoviridae Infections; RNA, Viral; Virion; Virus Replication
PubMed: 32437428
DOI: 10.1371/journal.ppat.1008436 -
Virology Journal Sep 2010Viruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI...
BACKGROUND
Viruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI particles can cause virus populations to rise and fall, reflecting predator-prey interactions between DI and virus particles. The levels of virus and DI particles in each population passage can be determined experimentally by plaque and yield-reduction assays, respectively.
RESULTS
To better understand DI and virus particle interactions we measured vesicular stomatitis virus and DI particle production during serial-passage culture on BHK cells. When the multiplicity of infection (MOI, or ratio of infectious virus particles to cells) was fixed, virus yields followed a pattern of progressive decline, with higher MOI driving earlier and faster drops in virus level. These patterns of virus decline were consistent with predictions from a mathematical model based on single-passage behavior of cells co-infected with virus and DI particles. By contrast, the production of virus during fixed-volume passages exhibited irregular fluctuations that could not be described by either the steady-state or regular oscillatory dynamics of the model. However, these irregularities were, to a significant degree, reproduced when measured host-cell levels were incorporated into the model, revealing a high sensitivity of virus and DI particle populations to fluctuations in available cell resources.
CONCLUSIONS
This study shows how the development of mathematical models, when guided by quantitative experiments, can provide new insight into the dynamic behavior of virus populations.
Topics: Animals; Cells, Cultured; Cricetinae; Defective Viruses; Models, Theoretical; Serial Passage; Vesiculovirus; Viral Load; Viral Plaque Assay; Virus Cultivation
PubMed: 20920247
DOI: 10.1186/1743-422X-7-257 -
Virus Research Nov 2015The host interferon (IFN) response represents one of the first barriers that influenza viruses must surmount in order to establish an infection. Many advances have been... (Review)
Review
The host interferon (IFN) response represents one of the first barriers that influenza viruses must surmount in order to establish an infection. Many advances have been made in recent years in understanding the interactions between influenza viruses and the interferon system. In this review, we summarise recent work regarding activation of the type I IFN response by influenza viruses, including attempts to identify the viral RNA responsible for IFN induction, the stage of the virus life cycle at which it is generated and the role of defective viruses in this process.
Topics: Defective Viruses; Host-Pathogen Interactions; Humans; Immunity, Innate; Interferon Type I; Orthomyxoviridae
PubMed: 25678267
DOI: 10.1016/j.virusres.2015.02.003 -
PloS One 2012Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In...
Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.
Topics: Animals; Defective Viruses; Ferrets; Influenza A virus; Influenza Vaccines; Orthomyxoviridae Infections; Pandemics
PubMed: 23251341
DOI: 10.1371/journal.pone.0049394