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.

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.

Semi-continuous Propagation of Influenza A Virus and Its Defective Interfering Particles: Analyzing the Dynamic Competition To Select Candidates for Antiviral Therapy.

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

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.

Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity.

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

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 show only limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.

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