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Journal of Virology Oct 1994Previous studies showed that B cells and CD4+ T cells are required for induction of a murine retrovirus-induced immunodeficiency syndrome, murine AIDS. Using B6 mice...
Previous studies showed that B cells and CD4+ T cells are required for induction of a murine retrovirus-induced immunodeficiency syndrome, murine AIDS. Using B6 mice deficient in mature B cells as a result of a knockout of the transmembrane exon of the immunoglobulin M gene, we found that spleen and other tissues from murine AIDS virus-infected mice did not express the defective virus (BM5def) required for induction of disease, even though helper viruses were readily detectable and BM5def proviral DNA was present. This indicates that the B-lineage cells are the primary targets for infection and expression of the defective virus and that in the absence of mature B cells, there is inefficient infection of T cells and macrophages.
Topics: Actins; Animals; B-Lymphocytes; Base Sequence; Crosses, Genetic; DNA Primers; DNA, Viral; Defective Viruses; Exons; Genes, Immunoglobulin; Immunoglobulin M; Leukemia Virus, Murine; Macrophages; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Molecular Sequence Data; Murine Acquired Immunodeficiency Syndrome; Polymerase Chain Reaction; Proviruses; T-Lymphocytes; Time Factors; Transcription, Genetic; Virus Replication
PubMed: 8084011
DOI: 10.1128/JVI.68.10.6767-6769.1994 -
PLoS Computational Biology Sep 2021Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs...
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.
Topics: Animals; Antiviral Agents; Cell Culture Techniques; Defective Viruses; Dogs; Influenza A virus; Madin Darby Canine Kidney Cells; Models, Biological; Orthomyxoviridae Infections; RNA, Viral; Virus Replication
PubMed: 34491996
DOI: 10.1371/journal.pcbi.1009357 -
Journal of Virology Feb 2013Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs...
Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4(+) T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.
Topics: CD4-Positive T-Lymphocytes; Cells, Cultured; DNA, Viral; Defective Viruses; Gene Deletion; Herpesvirus 4, Human; Humans; Membrane Proteins; Vaccines, Virus-Like Particle; Viral Proteins; Virus Assembly
PubMed: 23236073
DOI: 10.1128/JVI.02533-12 -
Journal of Virology Jul 1975We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious... (Comparative Study)
Comparative Study
We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.
Topics: Carcinoma, Squamous Cell; Cell Line; Centrifugation, Density Gradient; DNA Restriction Enzymes; DNA, Viral; Defective Viruses; Electrophoresis, Polyacrylamide Gel; Formaldehyde; Humans; Laryngeal Neoplasms; Microscopy, Electron; Nucleic Acid Denaturation; Peptide Biosynthesis; Simplexvirus; Viral Interference; Viral Proteins; Virus Replication
PubMed: 166202
DOI: 10.1128/JVI.16.1.153-167.1975 -
MBio May 2024Defective viral genomes (DVGs) are truncated derivatives of their parental viral genomes generated during an aberrant round of viral genomic replication. Distinct... (Review)
Review
Defective viral genomes (DVGs) are truncated derivatives of their parental viral genomes generated during an aberrant round of viral genomic replication. Distinct classes of DVGs have been identified in most families of both positive- and negative-sense RNA viruses. Importantly, DVGs have been detected in clinical samples from virally infected individuals and an emerging body of association studies implicates DVGs in shaping the severity of disease caused by viral infections in humans. Consequently, there is growing interest in understanding the molecular mechanisms of DVG generation, how DVGs interact with the innate immune system, and harnessing DVGs as novel therapeutics and vaccine adjuvants to attenuate viral pathogenesis. This minireview focuses on single-stranded RNA viruses (excluding retroviridae), and summarizes the current knowledge of DVG generation, the functions and diversity of DVG species, the roles DVGs play in influencing disease progression, and their application as antivirals and vaccine adjuvants.
Topics: Humans; Genome, Viral; Defective Viruses; Virus Replication; Animals; RNA Viruses; Immunity, Innate; Virus Diseases
PubMed: 38567955
DOI: 10.1128/mbio.00692-24 -
Emerging Microbes & Infections 2019Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic...
Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.
Topics: Animals; Bronchi; Defective Viruses; Disease Models, Animal; Epithelial Cells; Genome, Viral; Humans; Influenza A Virus, H7N9 Subtype; Influenza, Human; Mice; Nasopharynx; Orthomyxoviridae Infections; RNA, Viral; Sequence Analysis, DNA; Sequence Deletion
PubMed: 31084471
DOI: 10.1080/22221751.2019.1611346 -
Scientific Reports Nov 2016RNA recombination in non-segmented RNA viruses is important for viral evolution and documented for several virus species through in vitro studies. Here we confirm viral...
RNA recombination in non-segmented RNA viruses is important for viral evolution and documented for several virus species through in vitro studies. Here we confirm viral RNA recombination in vivo using an alphavirus, the SAV3 subtype of Salmon pancreas disease virus. The virus causes pancreas disease in Atlantic salmon and heavy losses in European salmonid aquaculture. Atlantic salmon were injected with a SAV3 6K-gene deleted cDNA plasmid, encoding a non-viable variant of SAV3, together with a helper cDNA plasmid encoding structural proteins and 6K only. Later, SAV3-specific RNA was detected and recombination of viral RNA was confirmed. Virus was grown from plasmid-injected fish and shown to infect and cause pathology in salmon. Subsequent cloning of PCR products confirming recombination, documented imprecise homologous recombination creating RNA deletion variants in fish injected with cDNA plasmid, corresponding with deletion variants previously found in SAV3 from the field. This is the first experimental documentation of alphavirus RNA recombination in an animal model and provides new insight into the production of defective virus RNA.
Topics: Alphavirus; Animals; Cell Line; Defective Viruses; Models, Animal; Oncorhynchus keta; Plasmids; RNA, Viral; Recombination, Genetic; Sequence Deletion
PubMed: 27805034
DOI: 10.1038/srep36317 -
Journal of Virology Aug 2016When viruses infect their host cells, they can make defective virus-like particles along with intact virus. Cells coinfected with virus and defective particles often...
UNLABELLED
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.
Topics: Animals; Cell Line; Cricetinae; Defective Viruses; Epithelial Cells; Microscopy, Fluorescence; Spatio-Temporal Analysis; Vesiculovirus; Viral Interference; Virus Replication
PubMed: 27279621
DOI: 10.1128/JVI.00807-16 -
Journal of Biotechnology Sep 2000Immunization of mucosal surfaces has become an attractive route of vaccine delivery because of its ability to induce mucosal immunity. Although various methods of... (Review)
Review
Immunization of mucosal surfaces has become an attractive route of vaccine delivery because of its ability to induce mucosal immunity. Although various methods of inducing mucosal immunity are being developed, our laboratory has focused on developing adenoviruses as replication-competent and replication-incompetent vectors. The present report will summarize our progress in sequencing the entire bovine adenovirus-3 genome and identifying regions which can be deleted and subsequently used as insertion sites for foreign genes in developing recombinant viral vaccines. Using these recombinant viruses, we demonstrated the 'proof-of-principle' in developing mucosal immunity and, more importantly, inducing protection against bovine herpes virus in a natural host-cattle. Finally, we demonstrated that immunity and protection occurred even in animals that had pre-existing antibodies to the vector.
Topics: Adenoviridae; Animals; Cattle; Defective Viruses; Genetic Vectors; Immunity, Mucosal; Mucous Membrane; Vaccines
PubMed: 11000466
DOI: 10.1016/s0168-1656(00)00314-x -
Microbiological Reviews Sep 1993Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis... (Comparative Study)
Comparative Study Review
Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.
Topics: Amino Acid Sequence; Bacteriophage P2; Base Sequence; Capsid; Coliphages; DNA Replication; Defective Viruses; Escherichia coli; Gene Expression Regulation, Viral; Genes, Viral; Genome, Viral; Helper Viruses; Lysogeny; Molecular Sequence Data; Morphogenesis; Nucleic Acid Conformation; Plasmids; RNA, Viral; Viral Interference; Viral Structural Proteins; Virus Integration; Virus Replication
PubMed: 8246844
DOI: 10.1128/mr.57.3.683-702.1993