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Viral-deletion mutants that conditionally replicate and inhibit wild-type virus (i.e., Defective Interfering Particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., Therapeutic Interfering Particles–TIPs) must conditionally spread between cells with R0>1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0>1, and inhibit viral replication 10–100 fold. Inhibition occurs via competition for viral replication machinery and, a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100 fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof-of-concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants., A defective viral particle derived from SARS-CoV-2 competes with the full virus for resources to replicate, showing therapeutic potential by inhibiting viral proliferation in culture, and reducing viral load and pathology in animal models for infection.

Viral defective interfering particles (DIPs) with more than 90% of the genomic RNA (gRNA, ∼11 000 nucleotides) deleted have been detected in sera from dengue patients. The DIP RNA contains stem-loop structures in the 5' and 3' end, which may permit RNA replication in the same manner as dengue virus (DENV) gRNA. Transfection of DENV2 infected human hepatoma cells with DIP RNA (DIP-296) resulted in significant inhibition of virus replication. DIP-296 RNA inhibited DENV replication in a dose-dependent manner in several cell lines tested. The mechanism of inhibition by DIP RNA is unclear; however, our studies imply that the retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) mediated innate immune antiviral signaling pathways and direct interactions of DIP RNA with viral replication proteins may be involved. The latter is supported by

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory ddPCR of urine samples taken from immunosuppressed individuals, we show clinical BKPyV and JCPyV strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were universally derived from type I subtype BKPyV. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCEDefective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens -- no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes with the majority affecting genes required for viral replication. Longitudinal analysis showed these DVGs can persist during an infection, but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests these parasitic sequences exist across the many classes of viruses capable of causing human disease.

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., Author summary Influenza viruses replicate inside their hosts after infection. Along with the release of wild-type standard virus (STV), they can also generate specific kinds of particles that have deletions in their genome. These so-called “defective interfering particles” (DIPs) are unable to replicate on their own. However, during co-infection with STV they can interfere with the viral life cycle suppressing STV production and instead release a large number of progeny DIPs. Recent studies have shown promising results regarding their potential as antiviral agents. To characterize the interactions between STVs and DIPs during co-infections, we infected animal cell cultures using 12 different multiplicities of infection for STVs and DIPs, respectively. We measured intra- and extracellular infection dynamics and show that STV replication can be suppressed completely using high amounts of DIPs. Then, we developed a mathematical model that describes co-infection dynamics closely using a single set of parameters. We used this model to predict optimal dosing ratios for DIPs to suppress STV infections and for cell culture-derived production of DIPs for antiviral therapy.

Viruses thrive by exploiting the cells they infect but must also produce viral proteins to replicate and infect other cells. As a consequence, they are also susceptible to exploitation by defective versions of themselves that do not produce such proteins. A defective viral genome with deletions in protein-coding genes could still replicate in cells coinfected with full-length viruses, and even replicate faster due to its shorter size, interfering with the replication of the virus. We have created a synthetic defective interfering version of SARS-CoV-2, the virus causing the recent Covid-19 pandemic, assembling parts of the viral genome that do not code for any functional protein but enable it to be replicated and packaged. This synthetic defective genome replicates three times faster than SARS-CoV-2 in coinfected cells, and interferes with it, reducing the viral load of a cell by half in 24 hours. The synthetic genome is transmitted as efficiently as the full-length genome, confirming the location of the putative packaging signal of SARS-CoV-2. A version of such a synthetic construct could be used as a self-promoting antiviral therapy: by enabling replication of the synthetic genome, the virus promotes its own demise. Graphic summary

Abstract The novel influenza A virus (IAV) defective interfering particle “OP7” inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated. Key points • OP7 efficacy strongly depended on the multiplicity of infection used for production • Purification by steric exclusion chromatography increased OP7 efficacy • OP7-treated mice were protected against a lethal infection with IAV

All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication., Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

Naturally occurring defective interfering RNAs have been found in 4 of 14 coronavirus species. They range in size from 2.2 kb to approximately 25 kb, or 80% of the 30-kb parent virus genome. The large DI RNAs do not in all cases appear to require helper virus for intracellular replication and it has been postulated that they may on their own function as agents of disease. Coronavirus DI RNAs appear to arise by internal deletions (through nonhomologous recombination events) on the virus genome or on DI RNAs of larger size by a polymerase strand-switching (copy-choice) mechanism. In addition to their use in the study of virus RNA replication and virus assembly, coronavirus DI RNAs are being used in a major way to study the mechanism of a high-frequency, site-specific RNA recombination event that leads to leader acquisition during virus replication (i.e., the leader fusion event that occurs during synthesis of subgenomic mRNAs, and the leader-switching event that can occur during DI RNA replication), a distinguishing feature of coronaviruses (and arteriviruses). Coronavirus DI RNAs are also being engineered as vehicles for the generation of targeted recombinants of the parent virus genome.

As the ongoing outbreak in the Democratic Republic of Congo illustrates, Ebola virus disease continues to pose a significant risk to humankind and this necessitates the continued development of therapeutic options. One option that warrants evaluation is that of defective genomes; these can potentially parasitize resources from the wild-type virus and may even be packaged for repeated co-infection cycles. Deletion and copy-back defective genomes have been identified and reported in the literature. As a crude, mixed preparation these were found to have limiting effects on cytopathology. Here we have used synthetic virology to clone and manufacture two deletion defective genomes. These genomes were tested with Ebola virus using in vitro cell culture and shown to inhibit viral replication; however, and against expectations, the defective genomes were not released in biologically significant numbers. We propose that EBOV might have yet unknown mechanisms to prevent parasitisation by defective interfering particles beyond the known mechanism that prevents sequential infection of the same cell. Understanding this mechanism would be necessary in any development of a defective interfering particle-based therapy.

The mechanisms and consequences of defective interfering particle (DIP) formation during influenza virus infection remain poorly understood. The development of next-generation sequencing (NGS) technologies has made it possible to identify large numbers of DIP-associated sequences, providing a powerful tool to better understand their biological relevance. However, NGS approaches pose numerous technical challenges, including the precise identification and mapping of deletion junctions in the presence of frequent mutation and base-calling errors, and the potential for numerous experimental and computational artifacts. Here, we detail an Illumina-based sequencing framework and bioinformatics pipeline capable of generating highly accurate and reproducible profiles of DIP-associated junction sequences. We use a combination of simulated and experimental control data sets to optimize pipeline performance and demonstrate the absence of significant artifacts. Finally, we use this optimized pipeline to reveal how the patterns of DIP-associated junction formation differ between different strains and subtypes of influenza A and B viruses and to demonstrate how these data can provide insight into mechanisms of DIP formation. Overall, this work provides a detailed roadmap for high-resolution profiling and analysis of DIP-associated sequences within influenza virus populations. IMPORTANCE Influenza virus defective interfering particles (DIPs) that harbor internal deletions within their genomes occur naturally during infection in humans and during cell culture. They have been hypothesized to influence the pathogenicity of the virus; however, their specific function remains elusive. The accurate detection of DIP-associated deletion junctions is crucial for understanding DIP biology but is complicated by an array of technical issues that can bias or confound results. Here, we demonstrate a combined experimental and computational framework for detecting DIP-associated deletion junctions using next-generation sequencing (NGS). We detail how to validate pipeline performance and provide the bioinformatics pipeline for groups interested in using it. Using this optimized pipeline, we detect hundreds of distinct deletion junctions generated during infection with a diverse panel of influenza viruses and use these data to test a long-standing hypothesis concerning the molecular details of DIP formation.

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

When viruses infect their host cells, they can make defective virus-like particles along with intact virus. Cells coinfected with virus and defective particles often exhibit interference with virus growth caused by the competition for resources by defective genomes. Recent reports of the coexistence and cotransmission of such defective interfering particles (DIPs) in vivo , across epidemiological length and time scales, suggest a role in viral pathogenesis, but it is not known how DIPs impact infection spread, even under controlled culture conditions. Using fluorescence microscopy, we quantified coinfections of vesicular stomatitis virus (VSV) expressing a fluorescent reporter protein and its DIPs on BHK-21 host cell monolayers. We found that viral gene expression was more delayed, infections spread more slowly, and patterns of spread became more “patchy” with higher DIP inputs to the initial cell. To examine how infection spread might depend on the behavior of the initial coinfected cell, we built a computational model, adapting a cellular automaton (CA) approach to incorporate kinetic data on virus growth for the first time. Specifically, changes in observed patterns of infection spread could be directly linked to previous high-throughput single-cell measures of virus-DIP coinfection. The CA model also provided testable hypotheses on the spatial-temporal distribution of the DIPs, which remain governed by their predator-prey interaction. More generally, this work offers a data-driven computational modeling approach for better understanding of how single infected cells impact the multiround spread of virus infections across cell populations. IMPORTANCE Defective interfering particles (DIPs) compete with intact virus, depleting host cell resources that are essential for virus growth and infection spread. However, it is not known how such competition, strong or weak, ultimately affects the way in which infections spread and cause disease. In this study, we address this unmet need by developing an integrated experimental-computational approach, which sheds new light on how infections spread. We anticipate that our approach will also be useful in the development of DIPs as therapeutic agents to manage the spread of viral infections.

Influenza A viruses (IAV) are commonly used to infect animal cell cultures for research purposes and vaccine production. Their replication is influenced strongly by the multiplicity of infection (MOI), which ranges over several orders of magnitude depending on the respective application. So far, mathematical models of IAV replication have paid little attention to the impact of the MOI on infection dynamics and virus yields. To address this issue, we extended an existing model of IAV replication in adherent MDCK cells with kinetics that explicitly consider the time point of cell infection. This modification does not only enable the fitting of high MOI measurements, but also the successful prediction of viral release dynamics of low MOI experiments using the same set of parameters. Furthermore, this model allows the investigation of defective interfering particle (DIP) propagation in different MOI regimes. The key difference between high and low MOI conditions is the percentage of infectious virions among the total virus particle release. Simulation studies show that DIP interference at a high MOI is determined exclusively by the DIP content of the seed virus while, in low MOI conditions, it is predominantly controlled by the de novo generation of DIPs. Overall, the extended model provides an ideal framework for the prediction and optimization of cell culture-derived IAV manufacturing and the production of DIPs for therapeutic use., Author summary Influenza is a contagious respiratory disease that severely affects several million people each year. Vaccination can provide protection against the infection, but vaccine composition has to be adjusted regularly to remain effective against this fast evolving pathogen. While influenza vaccines are mostly produced in embryonated chicken eggs, cell culture-based vaccine production is developing as an alternative providing controlled process conditions in closed systems, better scalability, and a short response time in case of pandemic outbreaks. Here, we employ a computational model to describe underlying mechanisms during the IAV infection in adherent MDCK cells. Special attention was paid on the influence of the MOI on virus spread in cell populations. Although dynamics between infections with high and low amounts of infecting virions differ significantly, our model closely captures both scenarios. Furthermore, our results provide insights into IAV-induced apoptosis and the switch from transcription to replication in intracellular IAV replication. Additionally, model simulations indicate how virus particle release is regulated, and what impact defective interfering particles have on virus replication in different infection conditions. Taken together, we developed a computational model that enables detailed analyses of IAV replication dynamics in animal cell culture.

Defective interfering particles (DIPs) are mutated versions of viruses that are characterized by carrying internal deletions in their genome. These deletions are introduced randomly during virus replication and the truncated genomes interfere with the propagation of their standard virus (STV) leading to reduced infectious virus titers. Therefore, DIPs were recently proposed to be used for antiviral therapy which increased the demand for a reliable production process and a better understanding of the interference mechanism. The infection dynamics were analysed by a deterministic modelling approach, however, the impact of stochastic effects introduced by cell-to-cell variability, different coinfection scenarios and an independent genome segment replication remain largely elusive. Hence, we developed a stochastic model of influenza A virus and DIP replication which considers the influence of these random effects on STV and DIP release. We found that the viral nucleoprotein (NP), which is essential for encapsidation of the naked viral RNA (vRNA), is strongly affected by fluctuations and three distinct sub-populations emerged in our model. Furthermore, simulations performed with one DIP and one STV infecting the cell resulted in mostly non-productive simulations, mainly caused by failures during the endocytosis of particles and by the random degradation of vRNAs. Moreover, the optimal DIP production was achieved when STV enters nucleus first and the DIP entry is delayed between 1.5 and 3 hours. Lastly, we demonstrate that the implementation of a two-step packaging process, which separates the formation of genome complexes and the assembly of all required proteins for release, is crucial to achieve a substantial DIP advantage over STV production. Overall, our simulations suggest that a combination of various random effects influences the replication of STVs and DIPs inducing a broad distribution of progeny particle release. The stochastic model developed in this thesis provides an ideal basis for the analysis of these effects and their impact on DIP interference and production.

The mechanisms and consequences of defective interfering particle (DIP) formation during influenza virus infection remain poorly understood. The development of next generation sequencing (NGS) technologies has made it possible to identify large numbers of DIP-associated sequences, providing a powerful tool to better understand their biological relevance. However, NGS approaches pose numerous technical challenges including the precise identification and mapping of deletion junctions in the presence of frequent mutation and base-calling errors, and the potential for numerous experimental and computational artifacts. Here we detail an Illumina-based sequencing framework and bioinformatics pipeline capable of generating highly accurate and reproducible profiles of DIP-associated junction sequences. We use a combination of simulated and experimental control datasets to optimize pipeline performance and demonstrate the absence of significant artifacts. Finally, we use this optimized pipeline to generate a high-resolution profile of DIP-associated junctions produced during influenza virus infection and demonstrate how this data can provide insight into mechanisms of DIP formation. This work highlights the specific challenges associated with NGS-based detection of DIP-associated sequences, and details the computational and experimental controls required for such studies.

The baculovirus expression vector system (BEVS) is an emerging tool for the production of recombinant proteins, vaccines and bio-pesticides. However, a system-level understanding of the complex infection process is important in realizing large-scale production at a lower cost. The entire baculovirus infection process is summarized as a combination of various modules and the existing mathematical models are discussed in light of these modules. This covers a systematic review of the present understanding of virus internalization, viral DNA replication, protein expression, budded virus (BV) and occlusion-derived virus (ODV) formation, few polyhedral (FP) and defective interfering particle (DIP) mutant formation, cell cycle modification and apoptosis during the viral infection process. The corresponding theoretical models are also included. Current knowledge regarding the molecular biology of the baculovirus/insect cell system is integrated with population balance and mass action kinetics models. Furthermore, the key steps for simulating cell and virus densities and their underlying features are discussed. This review may facilitate the further development and refinement of mathematical models, thereby providing the basis for enhanced control and optimization of bioreactor operation.

Classical swine fever virus (CSFV), the causative agent of classical swine fever, belongs to the family Flaviviridae and genus Pestivirus. Some pestiviruses exhibit cytopathic effect in cell culture but exact phenomenon is unknown. Over expression of NS2-3 gene, presence of defective interfering particle and exaltation of Newcastle disease virus (END) phenomenon could be the reasons of cytopathogenicity. In the present study, a CSFV isolate exhibiting cytopathic effect (CPE) in Madin-Darby Canine Kidney (MDCK) cell line was characterized. To characterize cytopathogenicity of such isolate, END test was carried out. Interference of Newcastle disease virus (NDV) in MDCK adapted CSFV was confirmed by RT-PCR and virus neutralization test. Absence of CPE and NDV specific nucleic acid after neutralization confirmed the induction of CPE by NDV. Further, identity of the CSFV isolate in MDCK cell line by immunoperoxidase test, immunoblotting and RT-PCR post NDV neutralization established the virus replication without CPE (non-cytopathic isolate). Findings suggest that, there could be a chance of mixed infection of both CSFV and NDV in the piglet from which the sample was collected for virus isolation.

Defective interfering particles (DIPs) have been found for many important viral pathogens and it is believed that most viruses generate DIPs. This article reviews the current knowledge of the generation and amplification of DIPs, which possess deletions in the viral genome but retain the ability to replicate in the presence of a complete helper virus. In addition, mechanisms are discussed by which DIPs interfere with the replication of their helper virus leading to the production of mainly progeny DIPs by coinfected cells. Even though DIPs cannot replicate on their own, they are biologically active and it is well known that they have a huge impact on virus replication, evolution, and pathogenesis. Moreover, defective genomes are potent inducers of the innate immune response. Yet, little attention has been paid to DIPs in recent years and their impact on biotechnological products such as vaccines and viral vectors remains elusive in most cases. With a focus on influenza virus, this review demonstrates that DIPs are important for basic research on viruses and for the production of viral vaccines and vectors. Reducing the generation and/or amplification of DIPs ensures reproducible results as well as high yields and consistent product quality in virus production.

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages., Graphical abstract Image, graphical abstract

Although virus release from host cells and tissues propels the spread of many infectious diseases, most virus particles are not infectious; many are defective, lacking essential genetic information needed for replication. When defective and viable particles enter the same cell, the defective particles can multiply while interfering with viable particle production. Defective interfering particles (DIPs) occur in nature, but their role in disease pathogenesis and spread is not known. Here, we engineered an RNA virus and its DIPs to express different fluorescent reporters, and we observed how DIPs impact viral gene expression and infection spread. Across thousands of host cells, co-infected with infectious virus and DIPs, gene expression was highly variable, but average levels of viral reporter expression fell at higher DIP doses. In cell populations spatial patterns of infection spread provided the first direct evidence for the co-transmission of DIPs with infectious virus. Patterns of spread were highly sensitive to the behavior of initial or early co-infected cells, with slower overall spread stemming from higher early DIP doses. Under such conditions striking patterns of patchy gene expression reflected localized regions of DIP or virus enrichment. From a broader perspective, these results suggest DIPs contribute to the ecological and evolutionary persistence of viruses in nature.

Defective interfering (DI) virus is simply defined as a spontaneously generated virus mutant from which a critical portion of the virus genome has been deleted. At least one essential gene of the virus is deleted, either in its entirety, or sufficiently to make it non-functional. The resulting DI genome is then defective for replication in the absence of the product(s) of the deleted gene(s), and its replication requires the presence of the complete functional virus genome to provide the missing functions. In addition to being defective DI virus suppresses production of the helper virus in co-infected cells, and this process of interference can readily be observed in cultured cells. In some cases, DI virus has been observed to attenuate disease in virus-infected animals. In this article, we review the properties of DI virus, potential mechanisms of interference and progress in using DI virus (in particular that derived from influenza A virus) as a novel type of antiviral agent.

Parasites and hosts can experience oscillatory cycles, where the densities of these interacting species dynamically fluctuate through time. Viruses with different replication strategies can also interact to produce cyclical dynamics. Frequent cellular co-infection can select for defective-interfering particles (DIPs): “cheater” viruses with shortened genomes that interfere with intracellular replication of full-length (ordinary) viruses. DIPs are positively selected when rare because they out-replicate ordinary viruses during co-infection, but DIPs are negatively selected when common because ordinary viruses become unavailable for intracellular exploitation via cheating. Here, we tested whether oscillatory dynamics of ordinary viruses were similar across independently evolved populations of vesicular stomatitis virus (VSV). Results showed identical cyclical dynamics across populations in the first 10 experimental passages, which transitioned to repeatable dampened oscillations by passage 20. Genomic analyses revealed parallel molecular substitutions across populations, particularly novel mutations that became dominant by passage 10. Our study showed that oscillatory dynamics and molecular evolution of interacting viruses were highly repeatable in VSV populations passaged under frequent co-infection. Furthermore, our data suggested that frequent co-infection with DIPs caused lowered performance of full-length viruses, by reducing their population densities by orders of magnitude compared to reproduction of ordinary viruses during strictly clonal infections.

During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198–207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increase the likelihood of co-infection by a DI and wt RUB during serial passage thus enhancing maintenance of the DI. Cluster formation and sustaining infected cell percentage were found to be due to a combination of attenuated cytopathogenicity of DIs that express the C-E1 fusion protein and cell-to-cell movement of the DI. In infected cells, the C-GFP-E1 fusion protein was localized to potentially novel vesicular structures that appear to originate from ER–Golgi transport vacuoles. This species of DI expressing a C-E1 fusion protein that exhibits attenuated cytopathogenicity and the ability to increase the number of infected cells through cell-to-cell movement could be the basis for development of an attractive vaccine vector.

In order to control HIV pandemic, many vaccines are invented. Although none first verified its efficacy in clinic, we hypothesize that HIV vaccine based on poliovirus is potential to develop the promising one, because it can elicit the broad immune response including the main mucosal, humoral and cellular reaction. However, the viral neural virulence is one major concern. The attenuated Sabin strain is a better candidate. While partial poliovirus genes are replaced by HIV antigen genes, the defective interfering particle will fail to produce progeny virions, which may further ensure its security. Although the vaccinal immune efficacy was verified in some similar animal experiments based on poliovirus to express the exogenous genes, more animal and clinical immune trials about HIV-poliovirus chimeric minireplicons are to be carried out and the hypotheses are to be validated.

Like many other viral pathogens, influenza A viruses can form defective interfering particles (DIPs). These particles carry a large internal deletion in at least one of their genome segments. Thus, their replication depends on the co-infection of cells by standard viruses (STVs), which supply the viral protein(s) encoded by the defective segment. However, DIPs also interfere with STV replication at the molecular level and, despite considerable research efforts, the mechanism of this interference remains largely elusive. Here, we present a mechanistic mathematical model for the intracellular replication of DIPs. In this model, we account for the common hypothesis that defective interfering RNAs (DI RNAs) possess a replication advantage over full-length (FL) RNAs due to their reduced length. By this means, the model captures experimental data from yield reduction assays and from studies testing different co-infection timings. In addition, our model predicts that one important aspect of interference is the competition for viral proteins, namely the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) and the viral nucleoprotein (NP), which are needed for encapsidation of naked viral RNA. Moreover, we find that there may be an optimum for both the DI RNA synthesis rate and the time point of successive co-infection of a cell by DIPs and STVs. Comparing simulations for the growth of DIPs with a deletion in different genome segments suggests that DI RNAs derived from segments which encode for the polymerase subunits are more competitive than others. Overall, our model, thus, helps to elucidate the interference mechanism of DI RNAs and provides a novel hypothesis why DI RNAs derived from the polymerase-encoding segments are more abundant in DIP preparations.

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV Ind ) are capable of interfering with the replication of both homotypic VSV Ind and heterotypic New Jersey serotype (VSV NJ ) standard virus. In contrast, DI particles from VSV NJ do not interfere with the replication of VSV Ind standard virus but do interfere with VSV NJ replication. The differences in the interfering activities of VSV Ind DI particles and VSV NJ DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV Ind DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV NJ DI particles could assemble only with homotypic VSV NJ viral proteins, although the genomic RNAs of VSV NJ DI particles could be replicated by using heterotypic VSV Ind N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.

No cytopathic effect (CPE) can be observed on classical swine fever virus (CSFV) infected cell culture in vitro. This brings an obstacle to the researches on reciprocity between CSFV and host cells. Based on the construction of full-length genomic infectious cDNA clone of Chinese CSFV standard virulent Shimen strain, partial deletion is introduced into genomic cDNA to obtain a 7.5 kb subgenomic cDNA. A new subgenomic CSFV is derived from transfection with the subgenomic cDNA on PK-15 cells pre-infected by CSFV Shimen virus. Typical CPE induced by this subgenomic virus is observed on PK-15 cells. Coexistence of wildtype and subgenomic virus in cytopathic cell culture is demonstrated by RT-PCR detection in cytopathic cells. For conclusion, the construction of cytopathic cell model exploited a new way for researches on the molecular mechanism of CSFV pathogenesis.

Background Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR). Results To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions. Conclusions BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0269-5) contains supplementary material, which is available to authorized users.

Defective interfering (DI) RNAs associated with members of the genus Tombusvirus are among the most extensively characterized. These molecules, which represent replicable deletion mutants of viral genomes, are able to accumulate to high levels in viral infections and have the effect of suppressing virus accumulation and attenuating virus-induced symptoms in host plants. Numerous studies have led to a greater understanding of how these molecules are formed and what factors influence their accumulation. Here an overview is provided of what is currently known about the structure, formation and evolution of this important class of sub-viral replicon.

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.

Defective interfering particles (DIPs) are viral deletion mutants lacking essential transacting or packaging elements and must be complemented by wild-type virus to propagate. DIPs transmit through human populations, replicating at the expense of the wild-type virus and acting as molecular parasites of viruses. Consequently, engineered DIPs have been proposed as therapies for a number of diseases, including human immunodeficiency virus (HIV). However, it is not clear if DIP-based therapies would face evolutionary blocks given the high mutation rates and high within-host diversity of lentiviruses. Divergent evolution of HIV and DIPs appears likely since natural DIPs have not been detected for lentiviruses, despite extensive sequencing of HIVs and simian immunodeficiency viruses (SIVs). Here, we tested if the apparent lack of lentiviral DIPs is due to natural selection and analyzed which molecular characteristics a DIP or DIP-based therapy would need to maintain coadaptive stability with HIV-1. Using a well-established mathematical model of HIV-1 in a host extended to include its replication in a single cell and interference from DIP, we calculated evolutionary selection coefficients. The analysis predicts that interference by codimerization between DIPs and HIV-1 genomes is evolutionarily unstable, indicating that recombination between DIPs and HIV-1 would be selected against. In contrast, DIPs that interfere via competition for capsids have the potential to be evolutionarily stable if the capsid-to-genome production ratio of HIV-1 is >1. Thus, HIV-1 variants that attempt to “starve” DIPs to escape interference would be selected against. In summary, the analysis suggests specific experimental measurements that could address the apparent lack of naturally occurring lentiviral DIPs and specifies how therapeutic approaches based on engineered DIPs could be evolutionarily robust and avoid recombination.

A defective interfering particle (DIP) in the context of influenza A virus is a virion with a significantly shortened RNA segment substituting one of eight full-length parent RNA segments, such that it is preferentially amplified. Hence, a cell co-infected with DIPs will produce mainly DIPs, suppressing infectious virus yields and affecting infection kinetics. Unfortunately, the quantification of DIPs contained in a sample is difficult because they are indistinguishable from standard virus (STV). Using a mathematical model, we investigated the standard experimental method for counting DIPs based on the reduction in STV yield (Bellett & Cooper, 1959, Journal of General Microbiology 21 , 498–509 ( doi:10.1099/00221287-21-3-498 )). We found the method is valid for counting DIPs provided that: (i) an STV-infected cell's co-infection window is approximately half its eclipse phase (it blocks infection by other virions before it begins producing progeny virions), (ii) a cell co-infected by STV and DIP produces less than 1 STV per 1000 DIPs and (iii) a high MOI of STV stock (more than 4 PFU per cell) is added to perform the assay. Prior work makes no mention of these criteria such that the method has been applied incorrectly in several publications discussed herein. We determined influenza A virus meets these criteria, making the method suitable for counting influenza A DIPs.

Serial, high multiplicity passage of equine herpesvirus 1 (EHV-1) leads to the generation of defective interfering particles (DIP). EHV-1 DIP inhibit and interfere with the replication of standard EHV-1, establishing a state of persistent infection. These DIP package severely truncated and rearranged forms of the standard viral genome. Contained within the DIP genome are only three genes: UL3, UL4, and a unique hybrid gene (Hyb). The hybrid gene forms through a recombination event that fuses portions of the early regulatory IR4 and UL5 genes and is essential for DIP-mediated interference. The UL4 gene is an early gene dispensable for lytic replication and inhibits viral and cellular gene expression. However, the contribution of the UL4 gene during DIP-mediated persistent infection is unknown. Here, we describe the generation of a completely deleted UL4 virus and its use to investigate the role of the UL4 gene in the generation of the defective genome. Deletion of the UL4 gene resulted in delayed virus growth at late times post-infection. Cells infected with a mutant EHV-1 that lacked expression of the UL4 protein due to an inserted stop codon in the UL4 gene produced defective particles, while cells infected with a mutant EHV-1 that had the complete UL4 gene sequence deleted were unable to produce DIP. These data suggest that the UL4 gene sequence, but not the UL4 protein, is critical for the generation of defective interfering particles.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

The use of the insect cell-baculovirus system has grown significantly over the past three decades. The present chapter elucidates the fundamentals, scientific developments, technologies, and hurdles encountered in this field. The common types of insects and insect lines used as well as their specific features and culture conditions are described. Additionally, the types of baculoviruses used and the methods for generating them are also revealed. Particular emphasis has been given to describing the construction of recombinant baculoviruses that are used in production of heterologous proteins with insect cells. A major advantage in using insect cells is their ability to perform many post-translational processing events on these proteins including glycosylation. The types of insect cell glycosylation and the differences in glycosylation patterns between insect and mammalian systems are detailed. The parameters that affect insect cell growth and baculovirus infection including bioreactor configuration, process monitoring and control, oxygen supply, sparging, and media formulations are described. In particular, cell culture processing methods including batch, fed-batch, perfusion, and semi-batch processes are illustrated. Additional factors affecting baculovirus infection including multiplicity, time of infection, and the generation of mutant viruses are also considered. A final section is devoted to the use of insect larvae in combination with baculovirus as a potentially alternative to cell culture for the low-cost generation of baculoviruses and heterologous proteins. With all the advances in molecular biology and engineering processes for the baculovirus-insect cell system, insect cell culture and its application to recombinant protein production, vaccines, pesticides, and other areas will continue to expand in the coming decades. Keywords: baculovirus; High Five™; polyhedrin; defective interfering particle; baculovirus expression vector system (BEVS)

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN-β promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome replication.

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.

A rabies virus-derived defective interfering particle (DI) was isolated and characterized. The DI genome contained an internal deletion of 6.4 kb spanning the 3′ moiety of the pseudogene region (Ψ) and most of the L gene. DI-specific monocistronic N, NS, and M mRNAs as well as a G/L fusion mRNA were transcribed in cells coinfected with DI and helper virus. In addition, polycistronic DI RNAs and standard virus RNAs with internal A stretches and intergenic regions were found. Superinfection experiments showed that heterologous rabies-related viruses (Lyssavirus serotypes 2, 3, and 4) can complement the L deficiency of the DI genome. The heterologous polymerase proteins recognize correctly the replicational and transcriptional signal sequences of the Lyssavirus serotype 1-derived DI.

During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.