Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88.

Alpha/beta interferons (IFN-alpha/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-alpha/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-alpha/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-alpha/beta in mDCs while fusion-defective virus did not induce IFN-alpha/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-alpha/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-alpha/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

Photodynamic inactivation of enveloped viruses by buckminsterfullerene.

Photodynamic reactions induced by singlet oxygen-generating agents are known to inactivate enveloped viruses. In this report we demonstrate that the water-insoluble photosensitizer buckminsterfullerene (C60) can be used to mediate the inactivation of enveloped viruses. Viruses from two different families, Semliki Forest virus (SFV, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae) were used as model systems. Buffered solutions containing C60 plus either of these viruses were illuminated with visible light for up to 5 h, resulting in a loss of infectivity of more than 7 log10/ml (TCID50). Furthermore, it was demonstrated that this viral inactivation was oxygen-dependent and equally efficient in solutions containing protein. C60 yields singlet oxygen in very high amounts and is completely inert to photo-oxidative destruction. In addition, it can be easily removed and recycled from aqueous solutions. For these reasons, it may prove useful in the inactivation of viruses in biological systems.

Inactivation of viruses during ultraviolet light treatment of human intravenous immunoglobulin and albumin.

A comparison of ultraviolet (UV) irradiation of two wavelength ranges UVB (280-320 nm) and UVC (lower than 280 nm) showed that UVC in particular could very effectively inactivate, in intravenous immunoglobulin (IVIG) and albumin preparations, non-enveloped and non-acid labile model viruses (i.e., Polio 2 and T4 phage) and dry heat-resistant viruses (vaccinia and T4 phage). This effective virucidal treatment (5 min, 5,000 J/m2 dose) was achieved before an unacceptable level of IVIG aggregates occurred. The use of UV irradiation to inactivate infectious agents could add safety and supplement current methods, e.g. solvent/detergent, low pH, which do not inactivate non-enveloped, non-acid labile or dry-heat-resistant viruses at present.

Assay of defective-interfering semliki forest virus by the inhibition of synthesis of virus-specified RNAs.

We 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.

Prevention of death in Semliki Forest virus-infected mice by administration of defective-interfering Semliki Forest virus.

Adult mice inoculated with Semliki Forest virus (SFV) were protected from a lethal infection of the central nervous system by intranasal administration of defective-interfering (DI) SFV. DI SFV was prepared by eight passages at high m.o.i. in BHK 21 cells. Mice were treated with unpurified, unconcentrated tissue culture fluid which had been u.v.-irradiated to inactivate the infective virus present. Prevention of death was maximal when the DI virus was administered simultaneously with the infecting inoculum, and under the same conditions multiplication of infective virus in the brains of treated mice was reduced by 10(5)-fold. It was shown that DI SFV was propagated in mouse brains followed intranasal inoculation and it was concluded that protection was brought about through the intrinsic interfering capacity of the DI virus.

Synthesis of alphavirus-specified RNA.

UV irradiation of chicken fibroblasts infected with Semliki Forest or Sindbis virus has been used to investigate the mechanism of synthesis of 42S and 26S RNA, the major plus-strand virus-specified RNAs formed during the multiplication of standard virus particles. From an analysis of the kinetics of UV inactivation of the synthesis of these two RNAs, we conclude (i) that 26S RNA is formed by internal transcriptive initiation from a point about two-thirds of the way from the 3' end of the 42S negative-strand template; (ii) that there exists a population of plus-strand synthesizing complexes whose members are each capable of synthesizing both 42S and 26S RNA; and (iii) that, on a time-averaged basis, each complex in wild-type virus-infected cells contains one virus polymerase mediating 42S RNA synthesis and three mediating 26S RNA synthesis. The RNA phenotypes of 15 RNA(-)ts mutants of Sindbis virus have been examined after temperature shift to the restrictive temperature. Under these conditions, cells infected with three mutants, N2, N7, and E268, synthesized four to six times as much 42S RNA (relative to 26S RNA) as wild-type virus-infected cells. These studies were extended by examining, in detail, the RNA and polypeptide phenotypes of mutants N2 and E268. These experiments showed that, in N2- and E268-infected cells, one of the virus-specified nonstructural (NS) polypeptides (NS p89; H. Brzeski and S. I. T. Kennedy, J. Virol. 22:420-429, 1977) is thermolabile after shift up to restrictive temperature. This finding, together with the observation that, after shift, the 26S/42S RNA ratio in N2-infected cells changes markedly in favor of 42S RNA synthesis, leads us to conclude that, of the three NS polypeptides, NS p89 modulates 26S RNA synthesis.