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Journal of Virology Aug 2009The dynamic changes in the temporal appearance and quantity of a new class of influenza virus, noninfectious cell-killing particles (niCKP), were compared to defective...
The dynamic changes in the temporal appearance and quantity of a new class of influenza virus, noninfectious cell-killing particles (niCKP), were compared to defective interfering particles (DIP). After a single high-multiplicity passage in MDCK cells of an egg-derived stock that lacked detectable niCKP or DIP, both classes of particles appeared in large numbers (>5 x 10(8)/ml), and the plaque-forming particle (PFP) titer dropped approximately 60-fold. After two additional serial high-multiplicity passages the DIP remained relatively constant, the DIP/niCKP ratio reached 10:1, and the PFP had declined by about 10,000-fold. Together, the niCKP and DIP subpopulations constituted ca. 20% of the total hemagglutinating particle population in which these noninfectious biologically active particles (niBAP) were subsumed. DIP neither killed cells nor interfered with the cell-killing (apoptosis-inducing) activity of niCKP or PFP (infectious CKP), even though they blocked the replication of PFP. Relative to the UV-target of approximately 13,600 nucleotides (nt) for inactivation of PFP, the UV target for niCKP was approximately 2,400 nt, consistent with one of the polymerase subunit genes, and that for DIP was approximately 350 nt, consistent with the small DI-RNA responsible for DIP-mediated interference. Thus, niCKP and DIP are viewed as distinct particles with a propensity to form during infection at high multiplicities. These conditions are postulated to cause aberrations in the temporally regulated replication of virus and its packaging, leading to the production of niBAP. DIP have been implicated in the virulence of influenza virus, but the role of niCKP is yet unknown.
Topics: Animals; Cell Line; Chick Embryo; Defective Viruses; Dogs; Orthomyxoviridae; Orthomyxoviridae Infections; Viral Plaque Assay
PubMed: 19494019
DOI: 10.1128/JVI.02680-08 -
Virus Research May 2019Viruses frequently spread among cells or hosts in groups, with multiple viral genomes inside the same infectious unit. These collective infectious units can consist of...
Viruses frequently spread among cells or hosts in groups, with multiple viral genomes inside the same infectious unit. These collective infectious units can consist of multiple viral genomes inside the same virion, or multiple virions inside a larger structure such as a vesicle. Collective infectious units deliver multiple viral genomes to the same cell simultaneously, which can have important implications for viral pathogenesis, antiviral resistance, and social evolution. However, little is known about why some viruses transmit in collective infectious units, whereas others do not. We used a simple evolutionary approach to model the potential costs and benefits of transmitting in a collective infectious unit. We found that collective infectious units could be favoured if cells infected by multiple viral genomes were significantly more productive than cells infected by just one viral genome, and especially if there were also efficiency benefits to packaging multiple viral genomes inside the same infectious unit. We also found that if some viral sequences are defective, then collective infectious units could evolve to become very large, but that if these defective sequences interfered with wild-type virus replication, then collective infectious units were disfavoured.
Topics: Defective Viruses; Evolution, Molecular; Genome, Viral; Models, Theoretical; RNA, Viral; Virion; Virus Assembly; Virus Diseases; Virus Replication
PubMed: 30894320
DOI: 10.1016/j.virusres.2019.03.013 -
PloS One 2019Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV...
Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy.
Topics: Animals; Chlorocebus aethiops; Defective Viruses; Dogs; HEK293 Cells; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza, Human; Madin Darby Canine Kidney Cells; Vero Cells
PubMed: 30822349
DOI: 10.1371/journal.pone.0212757 -
Journal of Virology Feb 1983Direct sequencing of nine Sendai virus defective interfering RNA species revealed two kinds of 3'-terminal sequences. Six RNA species had 3' termini identical to the...
Direct sequencing of nine Sendai virus defective interfering RNA species revealed two kinds of 3'-terminal sequences. Six RNA species had 3' termini identical to the virus genome (negative strand), confirming that internal deletions are a frequent cause of Sendai virus defectiveness. The other three RNA species had 3'-terminal sequences identical to that described as the complement of the 5' terminus of the virus genome (R. A. Lazzarini, J. D. Keene, and M. Schubert, Cell 26:145-154, 1981), indicating that they are of the copy-back type. Extensive homology between these two types of 3' sequences evidently accounts for the ability of the copy-back sequence to function as an initiation signal for viral RNA replication. There may not be a selective advantage of one type of terminus over the other, since one defective interfering strain possessed two RNA species, one of which had the genomic 3' terminus and the other copy-back type.
Topics: Base Sequence; Defective Viruses; Genes, Viral; Parainfluenza Virus 1, Human; RNA, Viral; Virus Replication
PubMed: 6300428
DOI: 10.1128/JVI.45.2.659-664.1983 -
Virology Sep 2004A yellow fever (YFV) 17D virus variant, which causes persistent infection of mouse neuroblastoma cells associated with defective cell penetration and small plaque size,...
A yellow fever (YFV) 17D virus variant, which causes persistent infection of mouse neuroblastoma cells associated with defective cell penetration and small plaque size, yielded plaque-revertant viruses from cells transfected with viral transcripts encoding the adaptive mutation (Gly360 in the E protein). Reconstruction of a plaque-purified revertant which contained Gly360 and additional substitutions (Asn for Lys303 and Val for Ala261) yielded a virus whose infectious center size, growth efficiency, and cell penetration rate similar to the parental YF5.2iv virus, whereas viruses with Asn303 or Val261 alone with Gly360 yielded either a small-plaque virus or a parental revertant. These data indicate that the YFV E protein is subject to suppression of mutations in domain III that are deleterious for viral entry and spread by a second-site mutation in domain II. Position 261 lies within the hydrophobic ligand-binding pocket at the domain I-II interface, a site believed to be involved in the hinge-like conformational change of domain II during activation of membrane fusion-activity. Results of this study provide genetic data consistent with findings on flavivirus structure and implicate domain III in functions beyond simply cell surface attachment.
Topics: Amino Acid Sequence; Animals; Chlorocebus aethiops; Defective Viruses; Models, Molecular; Molecular Sequence Data; Mutation; Suppression, Genetic; Vero Cells; Viral Envelope Proteins; Viral Plaque Assay; Yellow fever virus
PubMed: 15327896
DOI: 10.1016/j.virol.2004.06.015 -
Journal of Virological Methods Nov 2013Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and...
Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses.
Topics: Animals; Cell Line; Cricetinae; Defective Viruses; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Semliki forest virus; Viral Load; Viral Nonstructural Proteins; Virus Cultivation
PubMed: 23933080
DOI: 10.1016/j.jviromet.2013.07.058 -
Proceedings of the National Academy of... Oct 1996
Review
Topics: Animals; Chlorocebus aethiops; Defective Viruses; Gene Transfer Techniques; Genes, Viral; Genetic Vectors; Herpesvirus 1, Human; Humans; Promoter Regions, Genetic; Simplexvirus; Vero Cells; Virus Integration; Virus Replication
PubMed: 8876133
DOI: 10.1073/pnas.93.21.11319 -
Journal of Virology Aug 1992We 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...
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.
Topics: Animals; Cell Line; Chick Embryo; DNA-Directed RNA Polymerases; Defective Viruses; Genome, Viral; Humans; Nucleocapsid Proteins; Nucleoproteins; Parainfluenza Virus 1, Human; Phosphoproteins; Plasmids; RNA, Viral; Templates, Genetic; Transfection; Vaccinia virus; Viral Core Proteins; Viral Proteins
PubMed: 1321276
DOI: 10.1128/JVI.66.8.4901-4908.1992 -
Journal of Virology Oct 1976When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to...
When 2-day-old rats were inoculated subcutaneously with the R2 strain of reovirus type 3 or with a class B (352) or class C (447) temperature-sensitive (ts) mutant, 5 to 10% of the animals died from acute encephalitis within 12 days. Approximately half of the survivors recovered rapidly and grew normally, but the remainder became runted. Two phases of infection are distinguished in the animals: an acute phase during which infectious virus reaches a maximum titer in brain and other tissues by 10 days p.i. and thes runting of the rats and the slow disappearance of virus from their brains over a period of 2 months or so. Virus isolated from chronically infected brains generally retained the genetic character (ts or wild type) of the inoculated virus, but two exceptions to this are described. Defective virions lacking the L1 segment of the viral genome (L1 defectives) were generated in rat brains during the acute phase of infection. Defective virus was also generated during the chronic phase, but during this period defectives were found with multiple segments deleted from the genome in addition to L1 defectives. In another type of experiment defective virus exerted a marked protective effect when inoculated intracerebrally with R2 virus. In the absence of defectives all animals died, but in their presence 17 of 23 animals survived and 15 of 23 became runted and chronically infected. The formation and evolution of defective particles in the brains of these rats were similar to those found in rats chronically infected after subcutaneous inoculation of reovirus. We conclude that the formation of defective virus particles may play a role in the initiation and maintenance of chronic neutropic infections with reovirus.
Topics: Animals; Animals, Newborn; Brain; Defective Viruses; Mammalian orthoreovirus 3; Mutation; Rats; Reoviridae; Reoviridae Infections; Temperature; Viral Interference; Virus Replication
PubMed: 185414
DOI: 10.1128/JVI.20.1.234-247.1976 -
PloS One 2017Although virus release from host cells and tissues propels the spread of many infectious diseases, most virus particles are not infectious; many are defective, lacking...
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.
Topics: Animals; Cell Line; Cricetinae; Defective Viruses; Gene Expression Regulation, Viral; Models, Biological; RNA Virus Infections; RNA Viruses
PubMed: 28915264
DOI: 10.1371/journal.pone.0184029