Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCERegulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well. Rhabdoviridae) are neurotropic viruses that enter the central nervous system (CNS) by axonal transport along microtubules (1). The negative-sense genomic viral RNA encodes five proteins, of which the 240-kDa large enzymatic subunit L and the 35-kDa cofactor phosphoprotein P form the PL polymerase complex (2). The viral polymerase is a ribonucleoprotein (RNP)-dependent polymerase that only uses the genomic RNA as a template for RNA synthesis when it is encap...

Oral vaccination using attenuated and recombinant rabies vaccines has been proven a powerful tool to combat rabies in wildlife. However, clear differences have been observed in vaccine titers needed to induce a protective immune response against rabies after oral vaccination in different reservoir species. The mechanisms contributing to the observed resistance against oral rabies vaccination in some species are not completely understood. Hence, the immunogenicity of the vaccine virus strain, SPBN GASGAS, was investigated in a species considered to be susceptible to oral rabies vaccination (red fox) and a species refractory to this route of administration (striped skunk). Additionally, the dissemination of the vaccine virus in the oral cavity was analyzed for these two species. It was shown that the palatine tonsils play a critical role in vaccine virus uptake. Main differences could be observed in palatine tonsil infection between both species, revealing a locally restricted dissemination of infected cells in foxes. The absence of virus infected cells in palatine tonsils of skunks suggests a less efficient uptake of or infection by vaccine virus which may lead to a reduced response to oral vaccination. Understanding the mechanisms of oral resistance to rabies virus vaccine absorption and primary replication may lead to the development of novel strategies to enhance vaccine efficacy in problematic species like the striped skunk.

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.