Production of antiviral "OP7 chimera" defective interfering particles free of infectious virus.

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.

Generation of "OP7 chimera" defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice.

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.

Patch formation driven by stochastic effects of interaction between viruses and defective interfering particles.

Defective interfering particles (DIPs) are virus-like particles that occur naturally during virus infections. These particles are defective, lacking essential genetic materials for replication, but they can interact with the wild-type virus and potentially be used as therapeutic agents. However, the effect of DIPs on infection spread is still unclear due to complicated stochastic effects and nonlinear spatial dynamics. In this work, we develop a model with a new hybrid method to study the spatial-temporal dynamics of viruses and DIPs co-infections within hosts. We present two different scenarios of virus production and compare the results from deterministic and stochastic models to demonstrate how the stochastic effect is involved in the spatial dynamics of virus transmission. We compare the spread features of the virus in simulations and experiments, including the formation and the speed of virus spread and the emergence of stochastic patchy patterns of virus distribution. Our simulations simultaneously capture observed spatial spread features in the experimental data, including the spread rate of the virus and its patchiness. The results demonstrate that DIPs can slow down the growth of virus particles and make the spread of the virus more patchy.

Defective Interfering Particles of Influenza Virus and Their Characteristics, Impacts, and Use in Vaccines and Antiviral Strategies: A Systematic Review.

Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.

Defective RNA Particles of Plant Viruses-Origin, Structure and Role in Pathogenesis.

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 cis-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.

Evolution of naturally arising SARS-CoV-2 defective interfering particles.

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.

Levels of Influenza A Virus Defective Viral Genomes Determine Pathogenesis in the BALB/c Mouse Model.

Defective viral genomes (DVGs), which are generated by the viral polymerase in error during RNA replication, can trigger innate immunity and are implicated in altering the clinical outcome of infection. Here, we investigated the impact of DVGs on innate immunity and pathogenicity in a BALB/c mouse model of influenza virus infection. We generated stocks of influenza viruses containing the internal genes of an H5N1 virus that contained different levels of DVGs (indicated by different genome-to-PFU ratios). In lung epithelial cells, the high-DVG stock was immunostimulatory at early time points postinfection. DVGs were amplified during virus replication in myeloid immune cells and triggered proinflammatory cytokine production. In the mouse model, infection with the different virus stocks produced divergent outcomes. The high-DVG stock induced an early type I interferon (IFN) response that limited viral replication in the lungs, resulting in minimal weight loss. In contrast, the virus stock with low levels of DVGs replicated to high titers and amplified DVGs over time, resulting in elevated levels of proinflammatory cytokines accompanied by rapid weight loss and increased morbidity and mortality. Our results suggest that the timing and levels of immunostimulatory DVGs generated during infection contribute to H5N1 pathogenesis. IMPORTANCE Mammalian infections with highly pathogenic avian influenza viruses (HPAIVs) cause severe disease associated with excessive proinflammatory cytokine production. Aberrant replication products, such as defective viral genomes (DVGs), can stimulate the antiviral response, and cytokine induction is associated with their emergence in vivo. We show that stocks of a recombinant virus containing HPAIV internal genes that differ in their amounts of DVGs have vastly diverse outcomes in a mouse model. The high-DVG stock resulted in extremely mild disease due to suppression of viral replication. Conversely, the stock that contained low DVGs but rapidly accumulated DVGs over the course of infection led to severe disease. Therefore, the timing of DVG amplification and proinflammatory cytokine production impact disease outcome, and these findings demonstrate that not all DVG generation reduces viral virulence. This study also emphasizes the crucial requirement to examine the quality of virus preparations regarding DVG content to ensure reproducible research.

Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties.

Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication.

DVGfinder: A Metasearch Tool for Identifying Defective Viral Genomes in RNA-Seq Data.

The generation of different types of defective viral genomes (DVG) is an unavoidable consequence of the error-prone replication of RNA viruses. In recent years, a particular class of DVGs, those containing long deletions or genome rearrangements, has gain interest due to their potential therapeutic and biotechnological applications. Identifying such DVGs in high-throughput sequencing (HTS) data has become an interesting computational problem. Several algorithms have been proposed to accomplish this goal, though all incur false positives, a problem of practical interest if such DVGs have to be synthetized and tested in the laboratory. We present a metasearch tool, DVGfinder, that wraps the two most commonly used DVG search algorithms in a single workflow for the identification of the DVGs in HTS data. DVGfinder processes the results of ViReMa-a and DI-tector and uses a gradient boosting classifier machine learning algorithm to reduce the number of false-positive events. The program also generates output files in user-friendly HTML format, which can help users to explore the DVGs identified in the sample. We evaluated the performance of DVGfinder compared to the two search algorithms used separately and found that it slightly improves sensitivities for low-coverage synthetic HTS data and DI-tector precision for high-coverage samples. The metasearch program also showed higher sensitivity on a real sample for which a set of copy-backs were previously validated.

Harnessing defective viruses to fight infections.

One of the most pressing challenges in medicine is to develop fast and effective strategies to combat infections. Xiao et al.1 report proof-of-concept experiments for the use of defective viral genomes as broad-spectrum antivirals by harnessing their ability to stimulate the innate immune response.

Virus-like Particles: Measures and Biological Functions.

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.

Library-based analysis reveals segment and length dependent characteristics of defective influenza genomes.

Found in a diverse set of viral populations, defective interfering particles are parasitic variants that are unable to replicate on their own yet rise to relatively high frequencies. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during population expansion reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir.

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

Evidence that two instead of one defective interfering RNA in influenza A virus-derived defective interfering particles (DIPs) does not enhance antiviral activity.

Influenza A virus (IAV) infection constitutes a significant health threat. Defective interfering particles (DIPs) can arise during IAV infection and inhibit spread of wild type (WT) IAV. DIPs harbor defective RNA segments, termed DI RNAs, that usually contain internal deletions and interfere with replication of WT viral RNA segments. Here, we asked whether DIPs harboring two instead of one DI RNA exert increased antiviral activity. For this, we focused on DI RNAs derived from segments 1 and 3, which encode the polymerase subunits PB2 and PA, respectively. We demonstrate the successful production of DIPs harboring deletions in segments 1 and/or 3, using cell lines that co-express PB2 and PA. Further, we demonstrate that DIPs harboring two instead of one DI RNA do not exhibit increased ability to inhibit replication of a WT RNA segment. Similarly, the presence of two DI RNAs did not augment the induction of the interferon-stimulated gene MxA and the inhibition of IAV infection. Collectively, our findings suggest that the presence of multiple DI RNAs derived from genomic segments encoding polymerase subunits might not result in increased antiviral activity.

Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice.

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.

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture.

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.

Intra- and Inter-cellular Modeling of Dynamic Interaction between Zika Virus and Its Naturally Occurring Defective Viral Genomes.

Here, we examine in silico 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. IMPORTANCE 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.

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

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.

Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides.

DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.

Sustained Replication of Synthetic Canine Distemper Virus Defective Genomes In Vitro and In Vivo.

Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.