HIV-1 integration landscape during latent and active infection.

The barrier to curing HIV-1 is thought to reside primarily in CD4(+) T cells containing silent proviruses. To characterize these latently infected cells, we studied the integration profile of HIV-1 in viremic progressors, individuals receiving antiretroviral therapy, and viremic controllers. Clonally expanded T cells represented the majority of all integrations and increased during therapy. However, none of the 75 expanded T cell clones assayed contained intact virus. In contrast, the cells bearing single integration events decreased in frequency over time on therapy, and the surviving cells were enriched for HIV-1 integration in silent regions of the genome. Finally, there was a strong preference for integration into, or in close proximity to, Alu repeats, which were also enriched in local hotspots for integration. The data indicate that dividing clonally expanded T cells contain defective proviruses and that the replication-competent reservoir is primarily found in CD4(+) T cells that remain relatively quiescent.

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.

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.

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.

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.

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.

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.

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.

Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes.

Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods, that is, next-generation sequencing (NGS) technologies and related bioinformatics algorithms. Although many algorithms were developed, their sensitivity and reproducibility were mostly assessed on simulated data. Here, we introduce DG-seq, a time-efficient pipeline for DVG detection and quantification, and a set of biological controls to assess the performance of not only our bioinformatics algorithm but also the upstream NGS steps. Using these tools, we provide the first rigorous comparison of the two commonly used sample processing methods for RNA-seq, with or without a PCR preamplification step. Our data show that preamplification confers a limited advantage in terms of sensitivity and introduces size- but also sequence-dependent biases in DVG quantification, thereby providing a strong rationale to favor preamplification-free methods. We further examine the features of DVGs produced by wild-type and transcription-defective (PA-K635A or PA-R638A) influenza viruses, and show an increased diversity and frequency of DVGs produced by the PA mutants compared to the wild-type virus. Finally, we demonstrate a significant enrichment in DVGs showing direct, A/T-rich sequence repeats at the deletion breakpoint sites. Our findings provide novel insights into the mechanisms of influenza virus DVG production.

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.

Identification and Characterization of Defective Viral Genomes in Ebola Virus-Infected Rhesus Macaques.

Ebola virus (EBOV), of the family Filoviridae, is an RNA virus that can cause a hemorrhagic fever with a high mortality rate. Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including in vitro EBOV infection, and have previously been associated with viral persistence and immunostimulatory activity. As DVGs have been detected in cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in filovirus-infected hosts. Therefore, we interrogated sequence data from serum and tissue samples using a bioinformatics tool in order to identify the presence of DVGs in nonhuman primates (NHPs) infected with EBOV, Sudan virus (SUDV), or Marburg virus (MARV). Multiple 5' copy-back DVGs (cbDVGs) were detected in NHP serum during the acute phase of filovirus infection. While the relative abundance of total DVGs in most animals was low, serum collected during acute EBOV and SUDV infections, but not MARV infections, contained a higher proportion of short trailer sequence cbDVGs than the challenge stock. This indicated an accumulation of these DVGs throughout infection, potentially due to the preferential replication of short DVGs over the longer viral genome. Using reverse transcriptase PCR (RT-PCR) and deep sequencing, we also confirmed the presence of 5' cbDVGs in EBOV-infected NHP testes, which is of interest due to EBOV persistence in semen of male survivors of infection. This work suggests that DVGs play a role in EBOV infection in vivo and that further study will lead to a better understanding of EBOV pathogenesis. IMPORTANCE The study of filovirus pathogenesis is critical for understanding the consequences of infection and for the development of strategies to ameliorate future outbreaks. Defective viral genomes (DVGs) have been detected during EBOV infections in vitro; however, their presence in in vivo infections remains unknown. In this study, DVGs were detected in samples collected from EBOV- and SUDV-infected nonhuman primates (NHPs). The accumulation of these DVGs in the trailer region of the genome during infection indicates a potential role in EBOV and SUDV pathogenesis. In particular, the presence of DVGs in the testes of infected NHPs requires further investigation as it may be linked to the establishment of persistence.

Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.

Targeting Conserved Sequences Circumvents the Evolution of Resistance in a Viral Gene Drive against Human Cytomegalovirus.

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wild-type viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. In this study, we analyzed in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wild-type population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionarily conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. IMPORTANCE The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells-a therapeutic strategy known as viral interference-has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction. In this study, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we showed that after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show, however, that targeting evolutionarily conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

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.

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.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BACKGROUND: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential. RESULTS: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived. CONCLUSION: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Epstein-Barr virus (EBV) deletions as biomarkers of response to treatment of chronic active EBV.

Chronic active Epstein-Barr virus (CAEBV) disease is a rare condition characterised by persistent EBV infection in previously healthy individuals. Defective EBV genomes were found in East Asian patients with CAEBV. In the present study, we sequenced 14 blood EBV samples from three UK patients with CAEBV, comparing the results with saliva CAEBV samples and other conditions. We observed EBV deletions in blood, some of which may disrupt viral replication, but not saliva in CAEBV. Deletions were lost overtime after successful treatment. These findings are compatible with CAEBV being associated with the evolution and persistence of EBV+ haematological clones that are lost on successful treatment.

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes.

Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.