Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

Two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, were shown to be the crucial players in transcriptional induction of IFN genes (1). IRF-3, which is expressed constitutively, is activated by virus-induced kinases (2, 3) and participates to the transcriptional induction of IFN-␤ and IFN-␣4 genes. The immediate-early IFNs produced by these genes signal through the IFN receptor in an autocrine and͞or paracrine fashion and up-regulate the transcription of many IFN-stimulated genes, notably of IRF-7. Upon viral infection of cells primed by IFN, IRF-7 and IRF-3, which are activated by the same kinases (2, 3), cooperate to induce the transcription of the other IFN-␣ genes (namely late IFNs) (4).In vitro, virtually any nucleated cell type can synthesize both IFN-␣ and IFN-␤. In vivo, however, for both humans (5, 6) and mice (7-9), the major IFN-producing cells were identified as being the plasmacytoid dendritic cells (pDCs). Large amounts of IFN are produced by human and mouse pDCs in response to a wide range of viruses, parasites, and bacteria.Many previous investigations focused on IFN-producing DCs in the periphery. In the central nervous system (CNS), however, DCs are reportedly limited to perivascular cells of peripheral origin (10), and pDCs were reported to be absent from the brain (11). Few data are available on cells responsible for IFN production in the CNS. In vitro experiments in primary cell cultures agree that astrocytes and microglia can produce type I IFNs but are conf licted regarding possible IFN production by neurons (12,13). A recent study showed that postmitotic neurons differentiated in vitro from the human NT2-N cell line were able to produce IFN-␤ in response to rabies virus infection (14). In vivo, very few studies tried to identify the IFN-producing cells in the CNS (15-18), and no general conclusion was reached.In this work, we used two neurotropic viruses, Theiler's virus, a murine picornavirus, and La Crosse virus, a bunyavirus, to investigate type I IFN production and response in the CNS in vivo. Theiler's virus (or Theiler's murine encephalomyelitis virus, TMEV) strains are divided into two subgroups according to the disease they produce. The neurovirulent strain (GDVII) causes an acute lethal encephalomyelitis, whereas the persistent strain (DA) causes a mild transient encephalitis that resolves and is followed by viral persistence in the spinal cord white matter (19). La Crosse virus (LACV) massively infects neurons and causes fulminant encephalitis. A mutant of LACV lacking a functional NSs gene (LACVdelNSs) was used in this study (20). Because the NSs gene product is an IFN antagonist (21), LACVdelNSs induces high amounts of IFN in infected cells. We used in situ hybridization (ISH) and double immunostaining to identify IFN-producing cells in the CNS. Our data show that IFN is largely produced by infected resident cells of the CNS. Interestingly, neurons accounted for a substantial proportion of IFN-producing cells. Neurons also responded to IFN by expressing Mx ...

La Crosse virus (LACV) is a mosquito-transmitted member of the

No abstract

La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3 ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10 8 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSsexpressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.