RNase L Mediated Protection from Virus Induced Demyelination

IFN-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. However, the IFN-α/β dependent mechanisms underlying innate anti-viral functions within the CNS are poorly understood. The role of RNase L in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the IFN-α/β pathway through RNA degradation intermediates. Infection of RNase L deficient (RL−/−) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. However, RNase L deficiency did not affect overall control of infectious virus, or diminish IFN-α/β expression in the CNS. Furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the CNS. The unique phenotype of infected RL−/− mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. Increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. These data demonstrate a novel protective role for RNase L in viral induced CNS encephalomyelitis, which is not reflected in overall viral control or propagation of IFN-α/β mediated signals. Protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination

Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2−/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1−/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2−/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2−/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2−/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2−/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material

Memory Effector Cells but Not Effector Cells Derived from Naive T Cells Can Utilize a Non-Perforin and Non-FasL Pathway To Inhibit Allogeneic Progenitor Cell Function Ex-Vivo

Abstract We are interested in understanding the effector pathways used by host T cells to inhibit engraftment following allogeneic HCT. Previous work in MHC matched models found that mice deficient in either perforin or fasl continue to mediate resistance, however, the numbers of BMCs necessary to override the host barrier is less than in cytotoxically normal mice. These findings suggest both cytotoxic pathways are likely to contribute to the barrier. Notably, in the combined absence of both pathways, resistance is virtually absent. In contrast, animals which contain memory CD8 T cells can resist large numbers of BMCs regardless of the absence of both perforin and fasl pathways. The present studies examined the use of these pathways by T cell populations with respect to the inhibition of progenitor cell (PC)activity (CFU-IL3) from BMC ex-vivo to determine the relative contributions of perforin and/or fasL in inhibition mediated by effector cells derived from naive vs. memory cell populations. Effector cells were generated from unsensitized BALB/c and BALB/c-perf−/− mice as well as mice immunized against H2b alloantigens. Primary effectors from unsensitized mice and memory effectors were generated following 5 and 2–3 day MLC cultures, respectively. Effector cells were co-cultured up to 48 hrs. with BMC and then plated with rmIL-3 to elicit CFU formation. Effector cells from unsensitized BALB/c-wt mice inhibited allogeneic B6-wt CFU (>80%) as well as B6-lpr CFU (>70%) formation. Syngeneic BM was not inhibited. These data demonstrate that the perforin dependent pathway is sufficient for inhibition, consistent with in vivo resistance observed in FasL defective recipients. Effector cells generated from unsensitized BALB/c-perf−/− also effectively inhibited B6-wt CFU (>80%), consistent with a fas mediated pathway. However, in contrast to effectors generated from unsensitized BALB/C-wt mice, effector cells generated from BALB/c-perf−/−did not inhibit CFU from B6-lpr BM, suggesting that: 1) both perforin and fasl effector pathways can be utilized to inhibit CFU formation ex-vivo and, 2) in the absence of both pathways, these primary effector cells could not mediate such inhibition. As anticipated, memory effector cells from BALB/c-wt mice exhibited the same pattern as the "naive" effectors (i.e.>90% inhibition) against both B6-wt and B6-lpr BM. This finding is consistent with inhibition by perforin and/or additional effector pathways. However, memory effector cells from cytotoxically deficient BALB/c-perf−/− mice failed to mediate the same pattern of inhibition as "naive" effectors from this strain. In contrast to the inability of primary effectors to inhibit B6-lpr CFU activity, the memory effectors efficiently (i.e.>90% inhibition) inhibited CFU generation from B6-lpr BM. These findings demonstrate that unlike effector cells generated from naive T cells, memory effectors can use a non-perforin/fasL dependent pathway to inhibit allogeneic PC activity. The results are consistent with the inhibition of engraftment in vivo following conditioning and transplant into cytotoxically double (perforin + fasL) defective mice and support the notion that memory - but not primary effectors utilize a non-perforin/fasl pathway to inhibit allogeneic CFU PC function. Current experiments are examining the kinetics of CFU inhibition and precise characterization of the effector T cells