Defective Interfering Particles of Rhabdoviruses

SUMMARYThis study demonstrates that populations of defective interfering Semliki Forest virus (DI SFV) are heterogeneous particularly in respect of their interference properties. Interference was quantified by two assays, one measuring inhibition of the yield of infectious progeny virus, and the other measuring reduction in virus-directed RNA synthesis; for 11 different DI SFV preparations a ratio of the two interference titres was calculated. These ratios varied up to 46-fold indicating that each DI virus preparation contained an interference activity that varied independently of the other. However, sister stocks made from the same parental inoculum had similar properties. The effects of different DI virus preparations on other parameters (virus polypeptide synthesis, yield of DI virus and yield of infectious virus) were investigated using inocula with interference titres standardized by either assay. Co-inoculation of L929 cells with 50 p.f.u. SFV showed that these parameters varied independently of each other and of the DI virus inoculum. There was no correlation with the number of undiluted passages each DI stock had received. Direct evidence of physical heterogeneity was demonstrated by metrizamide density gradient centrifugation. Although infecting virus sedimented as a narrow band, DI SFV was distributed over a broad region of the gradient. Its position on the gradient indicated that DI SFV has a higher nucleic acid : protein ratio than standard virus. DI virus progeny obtained by using fractions of the gradient as inoculum were as heterogeneous as the unfractionated parent, confirming that DI viruses retain heterogeneity on passage.

Section: Introductionmentioning

confidence: 99%

Defective Interfering Semliki Forest Virus Populations Are Biologically and Physically Heterogeneous

Barrett

Crouch

Dimmock

1984

“…Recently, Sekellick & Marcus (1978, 1979 have proposed that induction of interferon may occur by DI particles or by ts mutants, and that these dual factors may play a synchronous role in VSV persistence. The involvement of DI particles as a mediator of persistent infection has been demonstrated in model systems using rhabdoviruses (Reichmann & Schnitzlein, 1979;Andzhaparidze et al, 1981), influenza viruses (De & Nayak, 1980) and togaviruses (Stollar, 1979). Recently Roux & Holland (1979) showed that persistently infected BHK21 cells established with preparations of DI particle-rich Sendai virus contained defective RNA in amounts that were 100-fold greater than standard virus RNAs.…”

Section: Introductionmentioning

confidence: 99%

Chronic Production of Defective-interfering Particles by Hamster Embryo Cultures of Herpesvirus Persistently Infected and Oncogenically Transformed Cells

Dauenhauer

Robinson

O’Callaghan

1982

SUMMARYHighly passaged defective-interfering (DI) particle preparations of equine herpesvirus type 1 (EHV-1) were found to mediate the co-establishment of persistent infection and oncogenic transformation of permissive hamster embryo cells. Four cell lines, designated DI-1 to DI-4, were shown to possess biological properties typical of transformed cells and to induce the rapid formation of metastatic fibrosarcomas when injected into syngeneic LSH hamsters. Corresponding DI tumour cell lines, designated DI-1T to DI-4T, were found to be virus non-producing, to be transplantable in the hamster, and, like the four parent DI cell lines, to express EHV-1-coded antigens and to be resistant to superinfection with EHV-1 but not with a heterologous virus, vesicular stomatitis virus. All transformed cell lines, but not the tumour cell lines, contained a population of cells (2.6 to 9%) that continued to release infectious virus after 100 passages in culture. The production of interferon and selection of temperature-sensitive mutants did not appear to play a role in the maintenance of persistent infection. However, it was demonstrated that these persistently infected cells continued to release not only infectious virus but also DI particles after more than 2 years in culture. DI particles were shown to be present in released virus by: (i) detection of the defective virus DNA species (density 1-724 g/ml; standard EHV-1, density 1.716 g/ml) by CsCI analytical ultracentrifugation techniques; (ii) measurement of interference activity of virus released from DI-I to DI-4 cells against standard EHV-1 replication; (iii) the presence of the 35 megadalton BgllI fragment unique to the DI particle genome in DNA of released virus. These studies indicate that herpesvirus DI particles may function both in the initiation and maintenance of persistent infection and alter the cytocidal effects of infection to allow the establishment of oncogenic transformation and persistent infection.

“…All DI viruses have a portion of their genome deleted and can only replicate in the presence of standard (infectious) virus and this interferes with the multiplication of the standard virus. Although much is known about the structure of DI RNAs particularly of vesicular stomatitis virus (VSV) (Reichmann & Schnitzlein, 1979) and alphaviruses (Stollar, 1979), understanding of the molecular mechanisms of interference and generation, even for VSV, is incomplete (Faulkner & Lazzarini, 1980;Kang, 1980) and the possible role of DI virus in modulating infection is unknown. Investigation of these aspects has been handicapped by the lack of a suitable quantitative assay for biologically active DI virus.…”

Section: Introductionmentioning

confidence: 99%

Assay of Defective-interfering Semliki Forest Virus by the Inhibition of Synthesis of Virus-specified RNAs

Barrett

Crouch

Dimmock

1981

SUMMARYWe describe a simple, rapid and reproducible assay for defective-interfering Semliki Forest virus (DI SFV) which is based on the inhibition of synthesis of virus-specified RNAs in SFV-infected cells. Using the assay, we have been able to show that DI virus is generated by a single passage in baby hamster kidney (BHK) cells in an inoculum which contained no detectable DI virus and we have calculated the u.v. target size of the interfering activity.