There has never been a wholesale way of identifying neurons that are monosynaptically connected either to some other cell group or, especially, to a single cell. The best available tools, transsynaptic tracers, are unable to distinguish weak direct connections from strong indirect ones. Furthermore, no tracer has proven potent enough to label any connected neurons whatsoever when starting from a single cell. Here we present a transsynaptic tracer that crosses only one synaptic step, unambiguously identifying cells directly presynaptic to the starting population. Based on rabies virus, it is genetically targetable, allows high-level expression of any gene of interest in the synaptically coupled neurons, and robustly labels connections made to single cells. This technology should enable a far more detailed understanding of neural connectivity than has previously been possible.

We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.A fundamental question regarding brain organization is how the various structures are connected to each other. A standard means of addressing it has been to inject into a region of interest some 'retrograde tracer': a substance taken up by the axon terminals of neurons that project to the injection site and which allows either visualization of their anatomy or, in the case of fluorescent tracers, their targeting for physiological study 1,2 . The rise of molecular biology has allowed the use of retrogradely infectious viruses expressing genetically encoded fluorophores 3,4 . In no published cases, however, has the resulting fluorescence been bright enough to consistently provide detailed anatomical information without immunohistochemical amplification and therefore to permit high-resolution identification of live labeled neurons for subsequent physiological study. Here we present a newly created virus that, because of several unique characteristics, does achieve these goals.Rabies virus, which has been used as a trans-synaptic tracer 5 , infects neurons through axon terminals and spreads between synaptically coupled neurons in an exclusively retrograde direction. In earlier work, K.K.C. and colleagues produced a virus that had the envelope glycoprotein gene deleted from its genome but that was grown in complementing cells so that the glycoprotein itself was incorporated into the viral particles'membranes despite the lack of its coding sequence in the viral genome 6 . Such a virus can infect contacted cells normally and, because the glycoprotein plays no role in transcription and replication, can still express its remaining genes and proliferate the viral core within initially infected cells. However, with no means of synthesizing glycoprotein in these cells, the newly created progeny are unable to infect other cells 6,7 , transforming the virus into a first-order retrograde tracer instead of a transsynaptic one. To reveal detailed morphology, we substituted the gene for enhanced green COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Methods website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ Note: Supplementary information is available on the Nature Methods website. (Fig. 1a). The result should be a virus that can infect initially contacted cells, replicate its core to high copy number and express high levels of EGFP, but be unable to spread beyond these initially infected cells. We termed this virus, which ...

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that allcis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

Rabies is a life-threatening neglected tropical disease: tens of thousands of cases are reported annually in endemic countries (mainly in Africa and Asia), although the actual numbers are most likely underestimated. Rabies is a zoonotic disease that is caused by infection with viruses of the Lyssavirus genus, which are transmitted via the saliva of an infected animal. Dogs are the most important reservoir for rabies viruses, and dog bites account for >99% of human cases. The virus first infects peripheral motor neurons, and symptoms occur after the virus reaches the central nervous system. Once clinical disease develops, it is almost certainly fatal. Primary prevention involves dog vaccination campaigns to reduce the virus reservoir. If exposure occurs, timely post-exposure prophylaxis can prevent the progression to clinical disease and involves appropriate wound care, the administration of rabies immunoglobulin and vaccination. A multifaceted approach for human rabies eradication that involves government support, disease awareness, vaccination of at-risk human populations and, most importantly, dog rabies control is necessary to achieve the WHO goal of reducing the number of cases of dog-mediated human rabies to zero by 2030.