In vitro and in vivo characterization of a novel HSV-1 H129 neurotracer recombinant reveals dose-dependent replication and spread dynamics

Figures

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  Fig 1. Construction of the new H129 recombinants. (A) H129 genome map showing the insert site (UL23) of mNG-P2A-recombinase (Cre or FlpO). (B) Simplified overview for homologous recombination and plaque purification of H129 recombinants using Vero-cre or Vero cells.

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  Fig 2. Verification of the novel H129 recombinants. (A) PCR amplification of the mNG (top) or lox-stop-lox region (bottom) using H129 recombinants or H129 wild-type virus as templates. (B) Western blots showing the presence or lack of HSV TK (top) expression. β-actin was used as a loading control (bottom). (C) Poly-HSV antibody was used to compare the structural protein expression levels of the recombinant viruses to wt H129. (D) Neurons were infected with the recombinant and wild-type H129 viruses to confirm mNG expression. Images were taken at 24 hpi. Scale bar: 100 µm.

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  Fig 3. Replication efficiency of the new recombinants in Vero cells and primary CNs (n = 3). (A) Virus yields were determined in Vero cells infected at 1 MOI 24 hpi. SN = Supernatant; solid bars, CA = cell associated; checkered bars. (B) Virus yields in CNs at 1 MOI 48 hpi. Statistical significance was analyzed via Student’s t test, P-value of 0.004 is indicated with **, and P-values >0.05 are marked as not significant (ns).

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  Fig 4. Retrograde infection efficiency of the new H129 recombinants in primary SCG neurons. (A) Diagram of the experimental setup. SCGs in tri-chambers were infected in the axonal (N-) compartment with wt and H129-BF viruses at 1, 10, and 100 MOIs. S-compartments were harvested at 72 hpi. (B) Virus yields were determined by titer assay on Vero cells. Statistical significance was analyzed via Student’s t test P-value of 0.016 is indicated with *, and P-values >0.05 are marked as not significant (ns).

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  Fig 5. Anterograde transport and spread assay in primary SCG neurons. (A) Diagram of experimental setup. SCG cell bodies in the S-compartments were infected with the wt and H129-BF at 1, 10, and 100 MOIs. Vero cells were seeded on axons in the N-compartments. (B) S- and (C) N- compartments were harvested at 72 hpi, and virus yields were determined. (D) H129-BF-infected SCGs in the S-compartments and Vero cells in N-compartments were imaged at 24, 48, and 72 hpi. Scale bar: 1 mm. P-values of <0.05 are indicated with *, and P-values >0.05 are marked not significant.

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  Fig 6. H129-dTK replication and anterograde transsynaptic transport in vivo. (A) Schematic workflow for the assessment of the H129-dTK transsynaptic transport in vivo. Emx1-Cre mice were crossbred to Ai65D mice to generate double transgenic Ai65D; Emx1-Cre mice. Emx1-Cre mice express Cre recombinase in a subpopulation of excitatory neurons under the Emx1 promoter. Expression of tdTomato is dependent on the expression of both Flp and Cre recombinases. Stereotaxic injection of Ai65D; Emx1-Cre at primary visual cortex (V1) with 0.2 µL (1 × 105 pfu) of H129-BF (one group of mice was injected with the AAV-TK; AAV-DIO-TK-EF1a-TK-T2A-iRFP, 10 days prior to H129-BF infection). Five days post-H129 injection, mice were perfused, and brains were fixed and removed for histology (n = 2 mice per condition). Created with BioRender.com. (B) Injection sites were shown in all three experimental conditions. Injection and contralateral sites are shown in (C) H129-BF plus AAV, (D) H129-BF alone, and (E) no virus injection control. (scale bars are 500 µm). Dashed circles show dual color neurons at the injection site, arrowheads point to single tdT expressing neurons, and stars show dual color neurons at the…

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  Fig 7. H129-dTK replication and transsynaptic transport increase with dose in vivo. (A) Schematic workflow: Ai65F mice express tdTomato (tdT) dependent on the expression of Flp recombinase. Stereotaxic injection of Ai65F mice at a primary visual cortex (V1) with H129-BF. Five days post-H129 injection, mice were perfused, and brains were fixed and removed for histology. Created with BioRender.com. (B) Injection sites were shown in two different doses of H129-BF infection. H129-BF replication and transsynaptic transport when injected at 1 × 105 pfu (0.2 µL) (C) and 2 × 105 pfu (0.4 µL) (D) in comparison to no virus infection control (E). (scale bars are 500 µm). Cells expressing both tdT and mNG appear yellow in the merge channel and are denoted by stars.