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1.
J Virol ; 95(24): e0117421, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1691429

ABSTRACT

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. IMPORTANCE Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro, and we suggest them for effective antiviral therapy.


Subject(s)
Antiviral Agents/pharmacology , Drug Design/methods , Influenza A virus , Influenza, Human/virology , RNA, Viral , Animals , Cell Culture Techniques , Cell Line , Defective Viruses/genetics , Dogs , Gene Deletion , Genome, Viral , Humans , Immunity, Innate/drug effects , Madin Darby Canine Kidney Cells , Oscillometry , Real-Time Polymerase Chain Reaction , Viral Load/drug effects , Virus Replication/drug effects
2.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1481965

ABSTRACT

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.


Subject(s)
Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , RNA, Viral/administration & dosage , Replicon , Viral Vaccines/administration & dosage , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Defective Viruses/genetics , Defective Viruses/immunology , Female , Gene Deletion , Genes, env , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral/genetics , RNA, Viral/immunology , Vaccines, DNA , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics , Virulence/immunology
3.
PLoS Comput Biol ; 17(9): e1009357, 2021 09.
Article in English | MEDLINE | ID: covidwho-1470651

ABSTRACT

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.


Subject(s)
Defective Viruses , Influenza A virus , Models, Biological , Orthomyxoviridae Infections/virology , Virus Replication/physiology , Animals , Antiviral Agents , Cell Culture Techniques , Defective Viruses/chemistry , Defective Viruses/genetics , Defective Viruses/pathogenicity , Dogs , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/pathogenicity , Madin Darby Canine Kidney Cells , RNA, Viral/genetics
4.
J Virol ; 95(24): e0117421, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1434894

ABSTRACT

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. IMPORTANCE Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro, and we suggest them for effective antiviral therapy.


Subject(s)
Antiviral Agents/pharmacology , Drug Design/methods , Influenza A virus , Influenza, Human/virology , RNA, Viral , Animals , Cell Culture Techniques , Cell Line , Defective Viruses/genetics , Dogs , Gene Deletion , Genome, Viral , Humans , Immunity, Innate/drug effects , Madin Darby Canine Kidney Cells , Oscillometry , Real-Time Polymerase Chain Reaction , Viral Load/drug effects , Virus Replication/drug effects
5.
Commun Biol ; 4(1): 557, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1387494

ABSTRACT

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.


Subject(s)
Defective Viruses , Dengue Vaccines/therapeutic use , Dengue Virus/growth & development , Dengue/prevention & control , Virus Replication , Animals , Cell Line, Tumor , Chlorocebus aethiops , Defective Viruses/genetics , Defective Viruses/metabolism , Dengue/virology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , HEK293 Cells , Host-Pathogen Interactions , Humans , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Vero Cells , Viral Load
6.
Emerg Microbes Infect ; 10(1): 894-904, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1209882

ABSTRACT

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Ammonium Chloride/pharmacology , Animals , Antigen-Antibody Reactions , Blotting, Western , COVID-19/blood , Chlorocebus aethiops , Convalescence , Defective Viruses/genetics , Genes, Reporter , Genetic Vectors/immunology , HEK293 Cells , HIV-1/genetics , Humans , Immunoglobulin G/immunology , Lentivirus/genetics , Mutagenesis, Site-Directed , Pandemics , Point Mutation , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
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