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Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D

Abstract

Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever—YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.

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Fig. 1: Controlled anterograde transneuronal spreading of YFV-17D.
Fig. 2: Anterograde-only tracing by inducible replication of YFV∆NS1.
Fig. 3: Dual fluorescence tracing of parallel circuits.
Fig. 4: Transneuronal genetic control by YFV∆NS1-Cre.
Fig. 5: Mapping monosynaptic projectomes with YFV∆CME.
Fig. 6: Monosynaptic transneuronal genetic control with YFV∆CMENS1.

Data availability

The original data in this study include images of brain sections, calcium imaging data of freely moving mice and sequences of plasmids. The sequences of the plasmids have been deposited to GenBank as follows: pAAV-DIO-NS1 (MZ708030), pAAV-Syn-NS1-p2A-NLSdTomato (MZ695807), pAAV-rtTA (MZ708018), pAAV-SynaptoTAG2 (MZ708019), pAAV-TRE-C-prM-E-NS1 (MZ708020), pAAV-TRE-NS1-dTomato (MZ708021), pAAV-TRE-NS1NF (MZ708022), pAAV-tTA (MZ708023), pFUW-C-prM-E-NS1 (MZ708024), pYFVdelCME-mVenus (MZ708025), pYFVdeltaCMENS1-Cre (MZ708026), pYFVdeltaNS1-Cre (MZ708027), pYFVdeltaNS1-mCherry (MZ708028) and pYFVdeltaNS1-mVenus (MZ708029). The plasmids have been deposited to Addgene as follows: pAAV-DIO-NS1 (plasmid 175273), pAAV-Syn-NS1-p2A-NLSdTomato (plasmid 175276), pAAV-rtTA (plasmid 175274), pAAV-SynaptoTAG2 (plasmid 175275), pAAV-TRE-C-prM-E-NS1 (plasmid 175277), pAAV-TRE-NS1-dTomato (plasmid 175278), pAAV-TRE-NS1NF (plasmid 175279), pAAV-tTA (plasmid 175280) and pFUW-C-prM-E-NS1 (plasmid 175281). The other plasmids or reagents can be requested from the corresponding authors. The raw imaging data are in large files (total >100 GB) and can be requested from the corresponding authors. Source data are provided with this paper.

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Acknowledgements

We thank E. Kavalali for critical comments and suggestions. This study was supported by Klingenstein-Simons Fellowship Awards in Neuroscience (to W.X.) and grants NIH/NINDS (no. NS104828 to W.X.), NIH/NIMH (no. MH099153 to W.X.) and NIH/NIAID (no. AI117922 to J.W.S.). UT BRAIN seed grants (no. 365231) and a Texas Institute for Brain Injury and Repair pilot grant provided funds to initiate this study. We thank D. Ramirez, J. Meeks and the Whole Brain Microscopy Facility at UT Southwestern and S. Yamazaki (Neuroscience Microscopy Facility at UT Southwestern) for help with imaging.

Author information

Authors and Affiliations

Authors

Contributions

W.X. initiated, designed and oversaw this study. W.X., J.W.S., T.R. and J.G. designed the experiments. E.L., J.G., S.J.O., Y.L., H.C.O., W.D., R.A., Y.K., Y.-T.C., J.E., D.-T.L., Y.L., T.R., J.W.S. and W.X. participated in conducting the experiments, analyzing the results and writing the paper.

Corresponding authors

Correspondence to John W. Schoggins or Wei Xu.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Methods thanks Esteban Engel, Liqun Luo and Jennifer Treweek for their contribution to the peer review of this work. Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Anterograde transport of YFV-17D in the PFC-striatum-SN pathway.

(a) Schematics showing the PFC-striatum-SN pathway. (b) Expression of mVenus after injection of YFV-mVenus into the PFC. Brains were collected 3, 6 or 9 days after injection. All images are tile scans of brain sections. The blue color is counterstaining with DAPI. (c) Quantification of images in b: Density of mVenus-positive neurons in each brain region on day 3, 6, and 9. The bars are mean±SEM, n=10-12 sections from 3 mice for each brain region.

Source data

Extended Data Fig. 2 Transneuronal spreading of YFV-17D in hippocampal pathways.

(a)The dentate gyrus (DG)-CA3-CA1-subiculum (SUB)-striatum pathway. (b) Expression of mVenus after injection of YFV-mVenus into the DG. Brains were collected 3, 6 or 10 days after injection. (c-e) YFV-mVenus spread from the CA1 to SUB and dorsal striatum along polysynaptic pathways. mVenus expression after YFV-mVenus injection into dorsal CA1. Brains were collected 6 (c), 9 (d) or 11 (e) days after injection. (e) Images of brain sections containing the striatum from a mouse 11 days after YFV-mVenus injection at dorsal CA1 that were immunostained with MOR, a marker for striosomes. The experiments were repeated 3 times with similar results. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 3 mVenus co-localizes with YFV viral proteins.

(a) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for YFV protein E. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate cells positive for E but not mVenus. (b) Quantification of mVenus-positive cells expressing E, and E-positive cells expressing mVenus. (c) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for NS1. The arrowheads indicate cells positive for NS1 but not mVenus. (d) Quantification of mVenus-positive cells expressing NS1, or NS1-positive cells expressing mVenus. n=14 brain sections from 3 mice for ‘E’ and n=14 brain sections from 4 mice for ‘NS1’. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Source data

Extended Data Fig. 4 Preferential infection of neurons by YFV-mVenus.

Expression of the neuronal marker NeuN in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate a non-neuronal cell expressing mVenus. n=22 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Source data

Extended Data Fig. 5 Immunostaining of NG2 on YFV-mVenus-infected brain sections.

(a) NG2 (Neuron-glial antigen 2) expression in brain sections from mice infected with YFV-mVenus. NG2 is a marker of NG2 cells (also referred to as oligodendrocyte precursor cells). The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the areas indicated by arrowheads in a, showing a NG2-positive cell next to a mVenus-positive cell. (n=25 brain sections from 3 mice). The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 6 Immunostaining of Olig2 on YFV-mVenus-infected sections.

(a) Olig2 (a marker of oligodendrocytes) expression in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the cropped areas in a. CC: corpus callosum. n=26 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 7 Broad tropism of YFV-17D.

(a) Expression of mVenus in multiple brain regions after injection of YFV-mVenus into the PFC and fixation of the brains 15 days later. (b) Expression of mVenus in a cerebellar Purkinje cell. The experiments were repeated 6 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 8 Delayed retrograde transport of YFV∆NS1-mVenus.

Expression of mVenus and tdTomato after injection of AAVDJ-Syn-NS1 into the PFC and the striatum, and injection of YFV∆NS1-mVenus into the striatum. The brains were fixed 6 (a) or 12 (b) days after YFV∆NS1-mVenus injection. The experiments were repeated 3 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 9 Efficiency of YFV∆NS1-Cre without post-synaptic NS1.

(a) The experimental design was the same as in Fig. 4b–d except that no AAVs for NS1 expression were injected into the striatum. We fixed the brains 15 days after YFV∆NS1-Cre injections. (b) jGCaMP7f-positive neurons in the striatum. The image is a tile scan of the brain section. The blue color is counterstaining with DAPI. (c) The density of labeled cells in mice with or without NS1 in the striatum (n=15 sections from 4 mice for ‘with NS1’ and n=14 sections from 4 mice ‘without NS1’, *** P=0.000000026, Mann–Whitney test, two tailed). The data of the group ‘with NS1’ were also shown in Fig. 4d.

Source data

Extended Data Fig. 10 YFV∆CME-mVenus tracing from pontine nuclei to cerebellum.

(a) Image of a brain section after injection of AAV-tTA, AAV-TRE-C-prM-E-NS1 and YFV∆CME-mVenus into pontine nucleus and adjacent reticular tegmental nucleus. (b) mVenus-positive cells in the granular layer of the cerebellar cortex. The sections were counterstained with a marker for Purkinje cells, PCP4 (red), and DAPI (Blue). The experiments were repeated 4 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Table 1 and step-by-step protocol.

Reporting Summary

Supplementary Video 1

Calcium activities of traced striatal neurons in a mouse undergoing open-field test 42 days after YFV∆NS1-Cre injection. Video is played at 5× speed.

Supplementary Video 2

Calcium activities of traced striatal neurons in a mouse undergoing open-field test 49 days after YFV∆NS1-Cre injection. Video is played at 5× speed.

Source data

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 9

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Li, E., Guo, J., Oh, S.J. et al. Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D. Nat Methods 18, 1542–1551 (2021). https://doi.org/10.1038/s41592-021-01319-9

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