Herpes simplex virus type 1 infection via the bloodstream with apolipoprotein E dependence in the gonads is influenced by gender

Abstract

Herpes simplex virus type 1 (HSV-1) causes disease in humans and animals. Infection usually occurs via the neural route and possibly occurs via the hematogenous route. The latter, however, is the main route by which immunosuppressed individuals and neonates are infected. Gender-dependent differences in the incidence and severity of some viral infections have been reported. To detect differences between the sexes with respect to HSV-1 colonization and disease, the characteristics of both acute and latent infections in hematogenously infected male and female mice were compared. In acute infection, the female mice had a poorer outcome: HSV-1 colonization was more effective, especially in the gonads and brain. In the encephalon, the midbrain had the highest viral load. In latent infection, brain viral loads were not significantly different with respect to sex. Significant differences were seen, however, in the blood and trigeminal ganglia: HSV-1 seroprevalence was observed in females, with no virus detected in males. In brain dissections, only the cerebral cortex of the females had viral loads statistically higher than those observed in the males. The spread of the virus to several organs of interest during acute infection was examined immunohistochemically. Female mice showed greater viral immunostaining, especially in the adrenal cortex, gonads, and midbrain. In male mice, HSV-1 was detected predominantly in the adrenal cortex. It was also found that apolipoprotein E promotes virus colonization of the ovaries, the APOE gene dose being directly related to viral invasiveness.

FIG. 1.

Quantification of viral loads with respect to the viral doses injected into several organs (the viral load in the brain is the sum of those in the midbrain, ventricles, cortex, and cerebellum). A total of 33 female mice that were 14 weeks old were inoculated i.p. with HSV-1 suspensions ranging from 10³ to 10⁷ PFU, sacrificed, and dissected at 5.7 dpi, when an acute infection was established. The bar graph represents the viral load detected in each organ, expressed on a logarithmic scale. Values are means and standard errors of the means (SEMs) for the quantity of viral DNA, expressed as PFU equivalents, and normalized with respect to the quantity of mouse genomes, expressed as nanograms of amplified β-actin housekeeping gene. Fisher's exact test was used to compare the viral loads achieved with the different doses (always compared to a dose of 10⁶ PFU) (an asterisk indicates a P value of <0.01). (Left inset) Dissected areas of the brain analyzed in this study. Values are means and SEMs on a logarithmic scale. (Right inset) Clinical disease scores related to the various injected doses, expressed as percentages of animals with disease.

FIG. 2.

Viral loads in plasma and cellular fractions of whole blood. A total of 11 female mice that were 14 weeks old were inoculated i.p. with 10⁶ PFU of HSV-1 and bled at 5.7 dpi, when an acute infection was established. Blood was fractionated by centrifugation at 1,870 × g for 25 min at 4°C to leave a cell pellet and the plasma supernatant. DNA from both fractions was extracted, and HSV-1 was detected; viral loads were expressed as PFU equivalents of virus normalized with respect to the number of mouse genomes (expressed in nanograms of amplified β-actin housekeeping gene) (black bars) or with respect to the number of cells counted in each fraction (white bars). The plasma supernatant showed significantly higher viral loads than the cell pellet, when normalized with respect to both the number of mouse genomes (16.2 ± 3.8 compared to 2.8 ± 1.0 PFU/ng in the cellular fraction) and the number of cells (2.8 ± 0.9 compared to 0.0 ± 0.0 PFU/cell in the cellular fraction). Values are means and standard errors of the means. Fisher's exact test was used to compare the viral loads in the two fractions (an asterisk indicates a P value of <0.01).

FIG. 3.

Quantification of viral loads in several organs by gender in acute and latent infections. Totals of 28 female and 16 male mice that were 14 weeks old were inoculated i.p. with a suspension of 10⁶ PFU of HSV-1, sacrificed, and dissected at 5.7 dpi (acute infection) or at 37 dpi (latent infection). The bar graphs represent the viral load detected in each organ, expressed on a logarithmic scale. Values are means and standard errors of the means for the quantity of viral DNA, expressed as PFU equivalents, and normalized with respect to the quantity of mouse genomes, expressed as nanograms of amplified β-actin housekeeping gene.

FIG. 4.

Quantification of viral loads in dissected areas of the brain (midbrain, ventricles, cortex, and cerebellum) by gender in acute and latent infections. Totals of 28 female and 16 male mice that were 14 weeks old were inoculated i.p. with a suspension of 10⁶ PFU of HSV-1, sacrificed, and dissected at 5.7 dpi (acute infection) or at 37 dpi (latent infection). The bar graphs represent the viral load detected in each brain region, expressed on a logarithmic scale. Values are means and standard errors of the means for the quantity of viral DNA, expressed as PFU equivalents, and normalized with respect to the quantity of mouse genomes, expressed as nanograms of amplified β-actin housekeeping gene. Fisher's exact test was used to compare the values for the two genders (an asterisk indicates a P value of <0.01).

FIG. 5.

Immunohistochemical detection of HSV-1 in several organs in acute infection. A total of 10 mice that were 14 weeks old were inoculated i.p. with a suspension of 10⁶ PFU of HSV-1, sacrificed, and dissected at 5.7 dpi. The organs were embedded in paraffin wax and serially sectioned. Immunodetection was performed with anti-tegument viral protein VP16 and a red chromogenic substrate as detailed in Material and Methods. (A) Immunohistochemical analysis of an adrenal gland from a female mouse, showing many foci of infection preferentially located in the zona fasciculata and zona reticularis and with involvement of the corticomedullary junction. (B) Immunohistochemical analysis of an adrenal gland from a male mouse, with a pattern of expression of HSV-1 similar to that of the female mouse but with fewer foci of infection. (C) Immunodetection of HSV-1 in ovaries, showing VP16 staining in the stroma and in some follicles at different stages of development. None of the oocytes observed showed HSV-1 infection. (D) Immunohistochemical analysis of the testis, showing no reaction to HSV-1. (E) Sagittal section of female brain showing HSV-1 staining at some foci of infection in the midbrain. (Inset) Intense detection of the virus in neurons of the mesencephalic trigeminal nucleus. (F) Negative staining of the midbrain from a male mouse. Insets show higher magnifications of a region of each organ. Bars: A, 100 μm; inset, 20 μm.

FIG. 6.

Time course of HSV-1 infection in the ovaries in relation to APOE gene dose. Totals of 48 wild-type, 47 APOE knockout, and 19 APOE hemizygote female mice that were 14 weeks old were inoculated i.p. with a suspension of 10⁶ PFU of HSV-1, sacrificed, and dissected at intervals ranging from 18 h to 10 dpi. Solid lines represent the wild-type group; broken lines represent the APOE knockout group. Values are expressed on a logarithmic scale as means and standard errors of the means for the quantity of viral DNA, expressed as PFU equivalents, and normalized with respect to the quantity of mouse genomes, expressed as nanograms of amplified β-actin housekeeping gene. Fisher's exact test was used to compare the values for the different APOE gene dose groups (an asterisk indicates a P value of <0.001). (Inset) Viral loads in ovaries of wild-type, APOE knockout, and APOE hemizygote mice at a selected time point (day 4).