Type I interferon signaling does not alter mortality or control viral replication in the neonatal mouse brain after HSV infection.

<p>(A) Survival of 7-day-old (neonatal) WT mice inoculated with 10³ PFU HSV-1 F (n = 5), neonatal IFNAR KO mice (n = 9) inoculated with 10³ PFU of HSV-1 F, or neonatal WT mice inoculated with 100 PFU HSV-1 F (n = 6). Results shown represent survival data combined from 6 independent inoculations. Log-rank analysis showed no difference in time to mortality between groups of mice inoculated with 10³ PFU HSV-1 (<i>P</i> = 1.00) or between groups of WT mice inoculated with different amounts of virus (<i>P</i> = 0.17, median survival = 3 days in all groups). (B) HSV-1 titer in brain tissue of neonatal WT (10³ or 100 PFU) or IFNAR KO (10³ PFU) mice at death or post-inoculation day 14. Log-transformed mean titer at death did not differ between IFNAR KO and WT mice inoculated with 10³ PFU of HSV-1 (10^7.2 PFU/g vs 10^7.1 PFU/g, respectively, <i>P</i> = 0.67, <i>t</i> test). Log-transformed mean titer at death in brain homogenates from WT neonates inoculated with 100 PFU were statistically higher than those inoculated with 10³ PFU (10^7.3 PFU/g vs 10^7.1 PFU/g, respectively, <i>P</i> = 0.01).</p

Activation of autophagy and apoptotic cell death in a human case of neonatal HSV encephalitis.

<p>H&E staining of the thalamus in case of neonatal HSV encephalitis demonstrates cytopathic effect (CPE) pathognomonic of HSV infection (top, middle left). In serial sections, these same cells were positive for HSV-antigen. Markers of activated autophagy (LC3 and p62) were present in multinucleated cells with CPE characteristic of HSV infection (top row). Infected cells were positive for TUNEL staining (far right). Control thalamic tissue from a neonate without HSV infection was negative for HSV, LC3, and p62 and demonstrated only scantly positive TUNEL staining (bottom row).</p

Autophagy is activated during HSV infection independent of type I interferon signaling in the newborn, but not the adult, murine CNS.

<p>(A) Survival of 7-day-old IFNAR KO littermates inoculated IC with 10³ PFU of either the dBBD mutant virus or its marker rescue d68HR (n = 11 in each group). Results shown represent survival data combined from 4 independent inoculations. Log-rank analysis showed no statistically significant difference in time to mortality between d68HR and dBBD-inoculated IFNAR KO neonatal mice (<i>P</i> = 0.25, median survival  = 4 days in both groups) (B) Titer of dBBD or d68HR in the brain tissue of neonatal IFNAR KO mice at death or post-inoculation day 14. Of the mice that died from HSV disease, no differences in log-transformed mean titer were identified between dBBD and d68HR-infected mice (10^8.1 PFU/g vs 10^8.2 PFU/g, respectively, <i>P</i> = 0.5). (C) Representative immunohistochemical analysis (original magnification: x400) of neonatal IFNAR KO mice inoculated IC with 10³ PFU of either d68HR (top row) or dBBD (bottom row). Mice were followed until neurologic symptoms developed on day 3. The representative images of d68HR and dBBD-infected regions are positive for markers of autophagy on serial sections. (D) Survival of adult IFNAR KO mice inoculated IC with 10⁴ PFU of either dBBD or d68HR (n = 10 in each group). Results shown represent survival data combined from 6 independent inoculations. Log-rank analysis showed no statistically significant difference in time to mortality after inoculation with dBBD (<i>P</i> = 0.1, median survival  = 3 days in both groups). (E) Titer of dBBD or d68HR in the brain tissue of adult IFNAR KO mice at death or post-inoculation day 14. Log-transformed mean titer was not significantly different between adult IFNAR KO mice infected with dBBD or d68HR (10^7.6 PFU/g vs 10^7.4 PFU/g, respectively, <i>P</i> = 0.5). (F) Representative immunohistochemical analysis (original magnification: x200) of adult IFNAR KO mice inoculated IC with 10⁴ PFU of either d68HR (top row) or dBBD (bottom row). Mice were followed until neurologic symptoms developed on day 3. The representative images of d68HR and dBBD-infected regions are negative for markers of autophagy on serial sections.</p

Inhibition of autophagy through beclin 1 binding is dispensable for HSV-1 pathogenesis in the neonatal mouse brain.

<p>(A) Diagram of the HSV-1 γ34.5 protein indicating domain deleted to create the dBBD virus. (B) Survival of 7-day-old WT littermates inoculated IC with 10³ PFU of either the HSV-1 mutant deleted in the 20 amino acid sequence of γ34.5 required for beclin 1 binding (dBBD, n = 9), or its marker rescue (d68HR, n = 8). Results shown represent survival data combined from 5 independent inoculations. Log-rank analysis showed a statistically significant delay in time to mortality after inoculation with d68HR (median survival  = 4 days) compared to dBBD (<i>P</i> = 0.04, median survival  = 3 days). (C) Titer of dBBD or d68HR in the brain tissue of neonatal WT mice at death or post-inoculation day 14. Of those that died from HSV disease, no differences in log-transformed mean titer were identified between the two groups (10^8.1 PFU/g for dBBD and 10^8.4 PFU/g for d68HR, <i>P</i> = 0.19). (D) Representative beclin 1 immunoblots (top) and densitometry (bottom, mean values ± standard deviation) of whole-brain homogenates from uninfected 7-day-old neonatal and 10-week-old adult mice (n = 4 in each group). (E) Survival of adult WT mice inoculated IC with 10⁴ PFU of either dBBD (n = 14) or d68HR (n = 14). Results shown represent survival data combined from 5 independent inoculations. Log-rank analysis showed a statistically significant delay in time to mortality after inoculation with dBBD (median survival  =  undefined) compared to d68HR (<i>P</i> = 0.005, median survival  = 6 days). (F) Titer of dBBD or d68HR in the brain tissue of adult WT mice at death or post-inoculation day 14. No differences in log-transformed mean titer between the two groups were identified among animals that that died from HSV disease (10^6.7 PFU/g for dBBD and 10^6.9 PFU/g for d68HR, <i>P</i> = 0.64).</p

The HSV-1 F γ34.5 protein is important for mortality and viral replication in the CNS of both neonatal and adult mice.

<p>(A) Survival of 7-day-old WT (n = 11–13) or IFNAR KO (n = 12–13) littermates after IC inoculation with 10³ PFU of either the γ34.5-null HSV-1 F-strain mutant (R3616) or its marker rescue (HSV-1(F)R). Results shown represent survival data combined from 9 independent inoculations. Log-rank analysis showed a statistically significant delay in time to mortality for WT newborns (<i>P</i> = 0.0015), or for IFNAR KO newborns (<i>P</i> = 0.003), after inoculation with R3616 as compared with HSV-1(F)R (median survival  = 4 days (WT) and 3 days (IFNAR KO)). (B) Titer of R3616 or HSV-1(F)R in the brain tissue of neonatal WT or IFNAR KO mice at death or post-inoculation day 14. Titers at death for R3616 in IFNAR KO neonates were significantly lower than for HSV-1(F)R (10^6.6 PFU/g vs 10^7.7 PFU/g, respectively, <i>P</i> = 0.005). (C) Survival of adult WT (n = 8 in each group) or IFNAR KO (n = 6–7) mice inoculated IC with 10⁴ PFU of either R3616 or HSV-1(F)R. Results shown represent survival data combined from 7 independent inoculations. Log-rank analysis showed a statistically significant delay in time to mortality for WT adults (<i>P</i> = 0.025) or IFNAR KO adults (<i>P</i> = 0.0004); after inoculation with R3616 as compared with HSV-1(F)R (median survival  = 10 days (WT) and 3.5 days (IFNAR KO)). (D) Titer of R3616 or HSV-1(F)R in the brain tissue of adult WT or IFNAR KO mice at death or post-inoculation day 14. R3616 was not detected at day 14 after infection in brain homogenates from WT adult mice, and titers at death for R3616 in IFNAR KO adults were significantly lower than for HSV-1(F)R (10^5.4 PFU/g vs 10^8.1 PFU/g, respectively, <i>P</i> = 0.003).</p

Infected regions with activated autophagy in the newborn mouse brain are associated with markers of apoptotic cell death.

<p>(A) Representative immunohistochemical analysis of neonatal murine brain infected with WT HSV-1 F (original magnification: 200x). The HSV antiserum panel from Fig. 5 is repeated for orientation. The WT HSV-infected neonatal brain is positive for TUNEL staining and caspase-3 activation (CC-3) at the site of infection (top). Representative sections from a neonatal murine brain infected with the dBBD virus also demonstrate positive TUNEL staining (middle) and cleaved caspase-3 (middle, right) in infected regions of the brain. The control uninfected neonatal brain is negative for markers of apoptosis (bottom). (B) Serial sections of brain from an adult mouse infected with HSV-1 F demonstrate scant TUNEL staining and few detectable cleaved caspase-3 positive cells (top). The dBBD-infected adult murine brain is also scantly positive for TUNEL staining (middle), and cleaved caspase-3 was positive in serial sections. Control uninfected adult mouse brain sections are shown below (original magnification: 200x).</p