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RNA (New York, N.Y.) Oct 2018Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral...
Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed , a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. confirmed the presence of 5' cb genomes in cells infected with measles virus (MV). also identified a novel 5' cb genome, as well as a variety of 3' cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of We propose as a universal tool for the unbiased detection of DI viral genomes, including 5' cb/sb DI genomes, in NGS data.
Topics: Cell Line; Computational Biology; Defective Viruses; Genes, rRNA; Genome, Viral; Genomics; High-Throughput Nucleotide Sequencing; Humans; RNA, Viral; Reproducibility of Results; Sensitivity and Specificity; Software; Virus Replication
PubMed: 30012569
DOI: 10.1261/rna.066910.118 -
Microbiology and Immunology May 2019Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have...
Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus-based antivirals.
Topics: Animals; Antiviral Agents; Defective Viruses; Dogs; HEK293 Cells; Humans; Influenza A virus; Influenza, Human; Madin Darby Canine Kidney Cells; Orthomyxoviridae Infections; RNA, Viral; RNA-Dependent RNA Polymerase; Viral Proteins; Virus Replication
PubMed: 30997933
DOI: 10.1111/1348-0421.12681 -
Journal of Virology Feb 1977A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is...
A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.
Topics: Animals; Cell Line; Defective Viruses; Mice; RNA, Viral; Rabies virus; Viral Interference; Viral Proteins; Virus Replication
PubMed: 833940
DOI: 10.1128/JVI.21.2.626-635.1977 -
Scientific Reports Nov 2023Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the...
Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP "OP7" that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness.
Topics: Animals; Mice; Humans; Influenza, Human; Coinfection; Defective Viruses; Influenza A virus; Virus Replication; Antiviral Agents
PubMed: 38017026
DOI: 10.1038/s41598-023-47547-1 -
Virology May 1998Mice inoculated with the murine AIDS (MAIDS)-defective virus develop severe B and T cell dysfunctions. The primary event in the development of this disease is the...
Mice inoculated with the murine AIDS (MAIDS)-defective virus develop severe B and T cell dysfunctions. The primary event in the development of this disease is the infection and polyclonal expansion of the target cells of this defective virus, which have been reported to belong to the B cell lineage. To further study the central role that these cells play in the development of MAIDS, we attempted to establish MAIDS-defective virus-infected B cell lines in vitro. We succeeded in establishing two cell lines, SD1 and CSTB5, from the enlarged organs of C57BL/6 mice inoculated with helper-free stocks of the MAIDS-defective virus. Both cell lines are not transplantable in syngeneic C57BL/6 mice or in nude or CD8-/- mice and are apparently not malignant. They both belong to the B lineage, as their immunoglobulin (Ig) genes, but not the T cell receptor (TcR) beta locus, are rearranged, suggesting that they are relatively mature B cells. However, analysis of cell surface marker expression by FACS revealed a surface phenotype similar to that of pre-B cells (MHC I+, MHC II+, B7.2+, sIgM-, sIgG-, kappa-, B220-, CD5-, Thy1.2-, TcR-, CD3-, CD4-, CD8-, Mac-1-, 33D1-). Additionally, the CSTB5 cells express CD40 and the SD1 cells express CD43. Both cell lines contain the MAIDS-defective provirus and express the expected 4.2-kb viral RNA and the corresponding Pr60gag protein. The CSTB5 cells are nonproducer, while the SD1 cell line produces what appears to be an endogenous MuLV. The phenotype of these cell lines is very similar to what is known about the target B cells of this virus in vivo. These new established cell lines are likely to be useful in elucidating the mechanism(s) by which the MAIDS-defective virus causes its target B cells to proliferate and induce T cell anergy in infected animals.
Topics: Animals; B-Lymphocytes; Cell Line; Defective Viruses; Gene Products, gag; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Leukemia Virus, Murine; Lymphocyte Activation; Lymphoid Tissue; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Mice, SCID; Murine Acquired Immunodeficiency Syndrome; Proviruses; Viral Proteins
PubMed: 9601499
DOI: 10.1006/viro.1998.9112 -
Journal of Virology Feb 1967Electron microscopic particle counting of the defective adeno-satellite virus (ASV), by use of pseudoreplication and negative staining with phosphotungstic acid, was...
Electron microscopic particle counting of the defective adeno-satellite virus (ASV), by use of pseudoreplication and negative staining with phosphotungstic acid, was shown to be a reproducible quantitative assay procedure. Particles of satellite type 4 that were counted in fluids from infected cultures had the same morphology as particles that banded at a buoyant density of 1.43 g/cc in cesium chloride. Other satellite virus serotypes examined in the same manner had a buoyant density of 1.37 to 1.38 g/cc. A comparison of satellite titers obtained by complement fixation and by particle counting demonstrated that an increase in satellite particles resulted in a corresponding increase in CF titers; however, electron microscopy was at least 10 times more sensitive than complement fixation for detecting satellite virus. Growth cycle studies of satellite virus in cells co-infected with adenovirus, as assayed by particle counting, indicated that the kinetics of satellite virus production closely followed the kinetics of its helper adenovirus production, with an eclipse period of 12 to 16 hr. The eclipse period of the satellite remained the same when cultures were preinfected with satellite 24 hr prior to adenovirus inoculation. However, when cultures were infected with adenovirus 12 hr before satellite virus, the eclipse period of the satellite was shortened to between 4 and 6 hr. Thus, satellite virus replication seems dependent upon a relatively late event in the adenovirus replication cycle. When cells were co-infected with adenovirus and its defective satellite, the yield of adenovirus was markedly reduced from that obtained in cells singly infected with adenovirus.
Topics: Adenoviridae; Animals; Centrifugation, Density Gradient; Complement Fixation Tests; Culture Techniques; Defective Viruses; Haplorhini; Hemolytic Plaque Technique; Kidney; Kinetics; Microscopy, Electron; Virus Cultivation
PubMed: 4990036
DOI: 10.1128/JVI.1.1.171-180.1967 -
Journal of Virology Jun 2020During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus...
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. Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both and 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.
Topics: A549 Cells; Animals; Base Sequence; Cell Line; Chlorocebus aethiops; Cytoplasm; Defective Viruses; Genome, Viral; High-Throughput Nucleotide Sequencing; Humans; Immunity, Innate; Interferons; Parainfluenza Virus 5; RNA, Viral; Vero Cells; Virion; Virus Diseases; Virus Replication
PubMed: 32295916
DOI: 10.1128/JVI.00246-20 -
Viruses May 2020The host-vector shuttle and the bottleneck in dengue transmission is a significant aspect with regard to the study of dengue outbreaks. As mosquitoes require 100-1000...
The host-vector shuttle and the bottleneck in dengue transmission is a significant aspect with regard to the study of dengue outbreaks. As mosquitoes require 100-1000 times more virus to become infected than human, the transmission of dengue virus from human to mosquito is a vulnerability that can be targeted to improve disease control. In order to capture the heterogeneity in the infectiousness of an infected patient population towards the mosquito population, we calibrate a population of host-to-vector virus transmission models based on an experimentally quantified infected fraction of a mosquito population. Once the population of models is well-calibrated, we deploy a population of controls that helps to inhibit the human-to-mosquito transmission of the dengue virus indirectly by reducing the viral load in the patient body fluid. We use an optimal bang-bang control on the administration of the defective virus (transmissible interfering particles (TIPs)) to symptomatic patients in the course of their febrile period and observe the dynamics in successful reduction of dengue spread into mosquitoes.
Topics: Aedes; Animals; Defective Viruses; Dengue; Dengue Virus; Humans; Models, Theoretical; Mosquito Vectors; Viral Load; Viremia
PubMed: 32443524
DOI: 10.3390/v12050558 -
Journal of Virology Apr 1976Eight genome RNA segments are present in both normal and von Magnustype influenza virus preparations and all species are transcribed by the virion-associated polymerase....
Eight genome RNA segments are present in both normal and von Magnustype influenza virus preparations and all species are transcribed by the virion-associated polymerase. Although the RNA polymerase activity and the amount of the three largest RNA segments are reduced in defective influenza virus preparations, these reductions do not appear to be great enough to account for the much greater loss of infectivity.
Topics: DNA-Directed RNA Polymerases; Defective Viruses; Orthomyxoviridae; RNA, Viral
PubMed: 1255876
DOI: 10.1128/JVI.18.1.365-369.1976 -
Journal of Virology Apr 1997Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy...
Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.
Topics: Amino Acid Sequence; Animals; Capsid; Cell Line; Cricetinae; Defective Viruses; Helper Viruses; Molecular Sequence Data; RNA, Viral; Recombination, Genetic; Replicon; Ross River virus; Sindbis Virus; Transfection; Viral Envelope Proteins; Virion; Virus Assembly
PubMed: 9060637
DOI: 10.1128/JVI.71.4.2819-2829.1997