-
MBio Jan 2020Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the...
Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection. Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.
Topics: A549 Cells; Bronchi; Cells, Cultured; Defective Viruses; Epithelial Cells; Gene Expression Profiling; Genome, Viral; Host Microbial Interactions; Humans; Influenza A virus; RNA, Viral; Sequence Analysis, RNA; Single-Cell Analysis; Virus Replication
PubMed: 31937643
DOI: 10.1128/mBio.02880-19 -
Viruses Dec 2022The genomes of RNA viruses may be monopartite or multipartite, and sub-genomic particles such as defective RNAs (D RNAs) or satellite RNAs (satRNAs) can be associated... (Review)
Review
The genomes of RNA viruses may be monopartite or multipartite, and sub-genomic particles such as defective RNAs (D RNAs) or satellite RNAs (satRNAs) can be associated with some of them. D RNAs are small, deletion mutants of a virus that have lost essential functions for independent replication, encapsidation and/or movement. D RNAs are common elements associated with human and animal viruses, and they have been described for numerous plant viruses so far. Over 30 years of studies on D RNAs allow for some general conclusions to be drawn. First, the essential condition for D RNA formation is prolonged passaging of the virus at a high cellular multiplicity of infection (MOI) in one host. Second, recombination plays crucial roles in D RNA formation. Moreover, during virus propagation, D RNAs evolve, and the composition of the particle depends on, e.g., host plant, virus isolate or number of passages. Defective RNAs are often engaged in transient interactions with full-length viruses-they can modulate accumulation, infection dynamics and virulence, and are widely used, i.e., as a tool for research on -acting elements crucial for viral replication. Nevertheless, many questions regarding the generation and role of D RNAs in pathogenesis remain open. In this review, we summarise the knowledge about D RNAs of plant viruses obtained so far.
Topics: Animals; Humans; RNA, Viral; Plant Viruses; RNA Viruses; RNA, Satellite; Virus Replication; Defective Viruses
PubMed: 36560818
DOI: 10.3390/v14122814 -
Journal of Virology Oct 2021Here, we examine the infection dynamics and interactions of two Zika virus (ZIKV) genomes: one is the full-length ZIKV genome (wild type [WT]), and the other is one of...
Here, we examine the infection dynamics and interactions of two Zika virus (ZIKV) genomes: one is the full-length ZIKV genome (wild type [WT]), and the other is one of the naturally occurring defective viral genomes (DVGs), which can replicate in the presence of the WT genome, appears under high-MOI (multiplicity of infection) passaging conditions, and carries a deletion encompassing part of the structural and NS1 protein-coding region. Ordinary differential equations (ODEs) were used to simulate the infection of cells by virus particles and the intracellular replication of the WT and DVG genomes that produce these particles. For each virus passage in Vero and C6/36 cell cultures, the rates of the simulated processes were fitted to two types of observations: virus titer data and the assembled haplotypes of the replicate passage samples. We studied the consistency of the model with the experimental data across all passages of infection in each cell type separately as well as the sensitivity of the model's parameters. We also determined which simulated processes of virus evolution are the most important for the adaptation of the WT and DVG interplay in these two disparate cell culture environments. Our results demonstrate that in the majority of passages, the rates of DVG production are higher inC6/36 cells than in Vero cells, which might result in tolerance and therefore drive the persistence of the mosquito vector in the context of ZIKV infection. Additionally, the model simulations showed a slower accumulation of infected cells under higher activation of the DVG-associated processes, which indicates a potential role of DVGs in virus attenuation. One of the ideas for lessening Zika pathogenicity is the addition of its natural or engineered defective virus genomes (DVGs) (have no pathogenicity) to the infection pool: a DVG is redirecting the wild-type (WT)-associated virus development resources toward its own maturation. The mathematical model presented here, attuned to the data from interplays between WT Zika viruses and their natural DVGs in mammalian and mosquito cells, provides evidence that the loss of uninfected cells is attenuated by the DVG development processes. This model enabled us to estimate the rates of virus development processes in the WT/DVG interplay, determine the key processes, and show that the key processes are faster in mosquito cells than in mammalian ones. In general, the presented model and its detailed study suggest in what important virus development processes the therapeutically efficient DVG might compete with the WT; this may help in assembling engineered DVGs for ZIKV and other flaviviruses.
Topics: Aedes; Animals; Chlorocebus aethiops; Defective Viruses; Host Microbial Interactions; Vero Cells; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 34468175
DOI: 10.1128/JVI.00977-21 -
PloS One 2019Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that...
Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.
Topics: Algorithms; Animals; Chromosome Mapping; DNA Primers; Datasets as Topic; Defective Viruses; Genome, Viral; High-Throughput Nucleotide Sequencing; Humans; Molecular Typing; Mumps virus; Parainfluenza Virus 5; Real-Time Polymerase Chain Reaction; Sendai virus; Sensitivity and Specificity
PubMed: 31100083
DOI: 10.1371/journal.pone.0216944 -
BMC Biology May 2021Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides...
BACKGROUND
Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.
RESULTS
In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1 mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.
CONCLUSION
In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.
Topics: Animals; Antiviral Agents; Cell Culture Techniques; Coinfection; Defective Viruses; Felodipine; Influenza A virus; Mice
PubMed: 33941189
DOI: 10.1186/s12915-021-01020-5 -
Clinical and Vaccine Immunology : CVI Jul 2006
Review
Topics: Adult; Animals; CD4-Positive T-Lymphocytes; Defective Viruses; HIV Infections; HIV-1; Humans; Infant; Lymphocyte Activation
PubMed: 16829607
DOI: 10.1128/CVI.00052-06 -
Journal of Virology Nov 2021Defective 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)...
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 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 interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. 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 , and we suggest them for effective antiviral therapy.
Topics: Animals; Antiviral Agents; Cell Culture Techniques; Cell Line; Defective Interfering Viruses; Defective Viruses; Dogs; Drug Design; Gene Deletion; Genome, Viral; Humans; Immunity, Innate; Influenza A virus; Influenza, Human; Madin Darby Canine Kidney Cells; Oscillometry; RNA, Viral; Real-Time Polymerase Chain Reaction; Viral Load; Virus Replication
PubMed: 34550771
DOI: 10.1128/JVI.01174-21 -
Journal of Virology Oct 1977Defective Friend spleen focus-forming virus (SFFV) is able to interfere with the ability of its naturally occurring leukemia-inducing helper virus (LLV-F) to induce XC...
Defective Friend spleen focus-forming virus (SFFV) is able to interfere with the ability of its naturally occurring leukemia-inducing helper virus (LLV-F) to induce XC plaque formation in several different strains of mouse embryo cells. This interference has been observed by using two different SFFV preparations, one contained in an NB-tropic stock of Friend virus (FV) complex, and the second present in a C57BL-adapted strain of FV complex containing an associated B-tropic LLV-F helper. The LLV-F in NB-tropic FV complex effectively induced XC plaques in C57BL/6 (Fv-1(bb); Fv-2(rr)) mouse embryo fibroblasts (MEF) only in the absence of coinfecting SFFV, indicating that Fv-2-associated resistance to SFFV-induced focus formation in vivo does not necessarily extend to the restriction of SFFV function(s) in vitro (i.e., in Fv-2(rr) C57BL MEF). SFFV interference appears to be an intracellular event since LLV-F can adsorb onto, penetrate, and rescue defective murine sarcoma virus (MSV) from transformed 3T3FL S(+)L(-) cells with equal efficiency in the presence and absence of SFFV. However, significantly fewer LLV-infected S(+)L(-) cells released LLV-F progeny if SFFV was present. These observations suggest that Friend SFFV may be classified as a defective, interfering (DI) particle. Further support for this conclusion has come from studies designed to investigate two physical properties of defective SFFV particles. SFFV layered onto a 0 to 20% sucrose sedimentation gradient was recovered as a symmetrical band of virus that sedimented more slowly than standard LLV-F particles. Pooled SFFV-containing gradient samples contained visualizable type C virus particles and occasionally small amounts of detectable LLV-F. In an attempt to determine the buoyant density of sedimentation gradient-purified SFFV, pooled SFFV samples were layered onto a 25 to 50% sucrose equilibrium density gradient and were centrifuged to equilibrium. Greater than 50% of the infectious SFFV originally layered onto this gradient was recovered and seen as a narrow symmetrical band with peak SFFV infectivity at a sucrose density of 1.14 g/ml. The observed difference between SFFV and LLV-F buoyant densities appears to be related to an inherent physical property of each virus. Mixtures of these two viruses express the buoyant density of that virus population which is in excess in fabricated FV complexes probably due to the formation of SFFV-LLV aggregates. Finally, gradient-purified SFFV failed to induce XC plaques in MEF and did not function to rescue MSV as expected since SFFV itself is replication defective.
Topics: Animals; Cell Line; Centrifugation, Density Gradient; Defective Viruses; Female; Friend murine leukemia virus; Helper Viruses; Male; Mice; Mice, Inbred Strains; Viral Interference
PubMed: 904028
DOI: 10.1128/JVI.24.1.383-396.1977 -
Effects of defective interfering viruses on virus replication and pathogenesis in vitro and in vivo.Advances in Virus Research 1991DI viruses and defective viruses generally are widespread in nature. Laboratory studies show that they can sometimes exert powerful disease-modulating effects (either... (Review)
Review
DI viruses and defective viruses generally are widespread in nature. Laboratory studies show that they can sometimes exert powerful disease-modulating effects (either attenuation or intensification of symptoms). Their role in nature remains largely unexplored, despite recent suggestive evidence for their importance in a number of systems.
Topics: Animals; Defective Viruses; Humans; Plants; Virus Physiological Phenomena; Virus Replication; Viruses
PubMed: 1957718
DOI: 10.1016/s0065-3527(08)60279-1 -
PLoS Pathogens Sep 2021During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of...
During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.
Topics: Coinfection; Defective Viruses; Humans; Models, Theoretical; Poliovirus; Virus Replication
PubMed: 34570820
DOI: 10.1371/journal.ppat.1009277