Fig 1.
Characterization of the low-dose neonatal HSV infection model.
(A) Schematic of the timeline of infection and behavioral assessment. One-day-old C57BL/6 mice were intranasally (i.n.) infected with escalating doses of strain 17 (st17) HSV-1 or virus-free lysate “mockulum”. At 4-5 mpi, nHSV and mockulum-treated mice were assayed for memory and cognitive performance deficits using the novel object recognition (NOR) task, the novel object location (NOL) task, the modified Barnes maze (MBM), and the different paired-associate learning (dPAL) task. (B) Weights of pups i.n. infected with 100 PFU HSV-1 (24) or mock-infected (27). (C) Survival of neonatal mice i.n. infected with mockulum, 100 PFU, 500 PFU, or 1000 PFU HSV-1. (D) Representative brain slice of a 5 dpi 1000 PFU HSV-1 infected mouse stained with DAPI (white) and antibody against HSV glycoprotein C (gC; red). Scale bar = 1 mm. (E) Normalized average staining intensity of gC staining in brains of 5 dpi 1000 PFU HSV-1 infected mice. (F) HSV genome copy number in the midbrain of 5 dpi mock-, 100 PFU-, 500 PFU-, and 1000 PFU-infected mice, relative to mouse single-copy adipsin gene. (G) HSV genome copy number in the midbrain of 6 mpi 100 PFU HSV-1-infected mice, relative to mouse single-copy adipsin gene. (H) LAT quantification in the midbrain of 6 mpi 100 PFU HSV-1-infected mice, relative to mouse single-copy adipsin gene. Statistical significance was determined by comparing infected groups to mock-infected animals using two-way ANOVA (B), log-rank (Mantel-Cox) tests (C), and one-way ANOVA (E, F). ns = not significant, ***p < 0.001, ns = not significant. Error bars represent standard error (C, E; SEM) and standard deviation from the geometric mean (F,G). Fig graphics created in BioRender. Leib, D. (2025) https://BioRender.com/s18a614.
Fig 2.
Low-dose nHSV infection leads to impaired novelty recognition in adult mice.
(A) Schematic of novel object recognition (NOR) task (top), and performance of mock-infected (n = 18) and strain 17 (st17) nHSV-infected (n = 20) mice on the NOR task at 5 mpi (below). Bar graph shows the mean discrimination index for the novel object. (B) Schematic of novel object location (NOL) task (top) and performance of 5 mpi mockulum-treated mice (n = 29), 5 mpi st17 nHSV-infected mice (n = 16), and 7-month-old uninfected female 5xFAD mice (n = 6) on the NOL task (below). Bar graph shows the mean discrimination index for the object in a novel location. Discrimination index = (Timenovel − Timefamiliar)/Timetotal. Statistical significance was determined by unpaired t-tests. *p < 0.05, **p < 0.01. Error bars represent standard error (SEM). Fig graphics created in BioRender. Leib, D. (2025) https://BioRender.com/k15h873.
Fig 3.
Low-dose nHSV infection leads to impaired behavioral flexibility in adult mice.
(A) Schematic of modified Barnes maze (MBM) test. Over the course of 4 days of 3 trials per day, mock-infected and 100 PFU HSV-1 strain 17-infected mice were trained in a MBM arena to locate, and exit through, an escape hole using visual cues (“W”, “S”, “O”, and “T”) for orientation. Following training, the location of the exit hole was reversed, and mice were tested for their ability to locate the reversal phase exit. (B) Mean latency to escape per day during the training phase of the MBM. (C) Mean latency to escape per trial during the reversal testing phase of the MBM. (D) Mean latency to escape on the first trial of reversal testing in the MBM. (E) Mean distance traveled in centimeters (cm) during the first trial of reversal testing in the MBM. (F) Mean errors per trial committed during the first trial of reversal testing in the MBM. (G) Quadrant designations (left) for the location of the escape hole during training (I) and reversal (III) phases of the MBM. Mean number of errors per quadrant (right) committed during the first reversal trial on day one of reversal testing. Statistical significance was determined by two-way ANOVA with a repeated measures design (B, C) and unpaired t-tests (D-G). ns = not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001. Error bars represent standard error (SEM). Created in BioRender. Leib, D. (2025) https://BioRender.com/c44t589 (A) and https://BioRender.com/d82i762 (F).
Fig 4.
Low-dose nHSV infection leads to impaired associative memory in adult mice.
(A) Schematic of a mouse completing the different paired-associate learning (dPAL) paradigm in a Bussey box chamber (top), and a diagram representing all possible combinations of test stimuli and correct responses (orange; bottom). (B) Average performance (% correct on first attempt) of mock-infected (20) and strain 17 (st17) nHSV-infected (26) mice during the first 5 trial blocks. 1 block = 3 testing sessions = 108 trials completed across 3 days. (C) Average number of correction trials per testing block during the first 5 blocks. (D) Average performance (% correct on first attempt) on day 1 of dPAL testing. (E) Average number of correction trials on day 1 of dPAL testing. (F) Average number of total trials completed on day 1 of dPAL testing. Statistical significance was determined by two-way ANOVA with repeated measures design and post-hoc Sidak multiple comparisons tests (B, C) and unpaired t-tests (D-F). ns = not significant, *p < 0.05. Error bars represent standard error (SEM). Fig graphics created in BioRender. Leib, D. (2025) https://BioRender.com/v77i905 (A, top), https://BioRender.com/t51l472 (A, bottom).
Fig 5.
Maternal vaccination protects against HSV CNS infection and nHSV-induced behavioral morbidity.
(A) Schematic showing the timeline of dam treatment, neonatal infection, and behavioral assessment using the NOR task. (B) Representative brain slice of a 5 dpi 1000 PFU nHSV-1 infected mouse pup from a dl5-29 vaccinated dam stained with DAPI (white) and antibody against HSV glycoprotein C (gC; red). Scale bar = 1 mm. (C) HSV genome copy number in the midbrain of 5 dpi 100 PFU nHSV-1 infected mice from naïve and dl5-29 vaccinated dams, relative to mouse single-copy adipsin gene. (D) Average discrimination index of mock-infected and strain 17 (st17) nHSV-infected offspring of naïve or dl5-29 vaccinated dams at 5 mpi on the NOR task. (E) Average discrimination index of mice neonatally infected with replication-deficient HSV vaccine strain dl5-29 at 5 mpi on the NOR task compared to mice neonatally infected with 100 PFU HSV-1 or mockulum (Vero cell lysate). Data are shown as average discrimination index for the novel object during the testing phase of the NOR task. Discrimination index = (Timenovel − Timefamiliar)/Timetotal. Statistical significance determined by unpaired t-test (C) or one-way ANOVA with Dunnett’s multiple comparisons post-hoc tests (D-E). ns = not significant, *p < 0.05, **p < 0.01. Error bars represent standard deviation from the geometric mean (C) and standard error (D, E; SEM). Fig graphics created in BioRender. Leib, D. (2025) https://BioRender.com/m43o918 (A).