Figure 1.
In vivo infection by luciferase-expressing MHV-68.
Female mice were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every days. A. Images show a representative mouse at days 0, 7, 14 and 21 post-infection (p.i.). B. Specific signal from the genital region was highlighted in an equivalent mouse. The scale bar (photons sec−1 cm−2 steradian−1) shows the color scheme for signal intensity. The same scale bar is used in A and B. C. Temporal progression of the genital signal in different mice (each curve represents one mouse). For the reliable comparison of signal intensities, the signal intensities were measured from equivalent regions of interest after subtraction of individual backgrounds measured in the right abdominal region.
Figure 2.
Luciferase signal and MHV-68 antigen detection in isolated genital tract after intranasal infection.
A. A mouse equivalent to those in Figure 1 was dissected and its genital tract imaged ex vivo. The images are representative of data from at least 10 mice, and show either a standard photograph (Photo) or that photograph overlaid with the luciferase signal (Photo+Luc). The region with the highest signal was isolated and processed for histological analysis. The scale bar (photons sec−1 cm−2 steradian−1) shows the color scheme for signal intensity. Ov, ovary; UtH, uterine horn; Bd, body of the uterus; Cx, cervix; Vg, Vagina. B. The piece of vagina isolated in A. was fixed in formaldehyde and organ slices were either stained with hematoxylin-eosin (panels i to iii) or processed for immunohistochemistry with anti-MHV-68 rabbit polyserum (panels iv to vi) as described in the Materials and Methods section. Rectangles indicate regions highlighted in the following panels. Arrows indicate focal recruitment of leukocytes at the periphery of MHV-68 antigen detection. Lu, lumen; SqEp, stratified squamous epithelium; LaPr, lamina propria; Mi, Muscularis; StCo, stratum corneum; StSp, stratum spinosum; Stba, stratum basale.
Figure 3.
Quantification of MHV-68 infection in female genital tract after intranasal infection.
A. Female mice were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia. 23 days post-infection, luciferase signal in the genital region was assessed and mice were categorized as IVIS- (white dots) and IVIS+ (black dots). For the reliable comparison of signal intensities, the signal intensities were measured from equivalent regions of interest after subtraction of individual backgrounds measured in the right thoracic region. B–D. Individual genital tracts were removed at day 23 p.i. and assayed individually for the presence of MHV-68 by infectious center assay (B), infections virus titration (C) and viral genome quantification (D). Groups were compared by student t-test (****P<0.0001). E. Vaginal flush samples collected before euthanasia were tested for the presence of infectious virus. Samples from mock infected mice (grey dots) were used as controls.
Figure 4.
Influence of estrous cycle on genital MHV-68 excretion after intranasal infection.
A. Control female mice and ovariectomized mice, implanted or not with slow-release hormonal pellets (progesterone and/or estrogen), were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia. Presence of genital signal was monitored between days 14 and 32 post-infection and percentages of positive observations were recorded individually. For the reliable comparison of signal intensities, the signal intensities were measured from equivalent regions of interest after subtraction of individual backgrounds measured daily in the right thoracic region. Each point shows percentage of positive observation for one animal. Groups were compared by ANOVA1 and Bonferroni post-test (***P<0.001). B–C. Stimulation of MHV-68 reactivation from persistently infected cells with 17β-Estradiol was tested. MHV-68 persistently infected A20 cells (B) or bulk splenocytes (C), obtained 14 days following MHV-68 intranasal inoculation (104 PFU), were analyzed for the frequency of cells reactivating virus with and without increasing concentrations of 17β-Estradiol as described in the Materials and Methods section. The data presented are the average for triplicate measurements +/− standard error of the mean and were analyzed by 1way ANOVA and Bonferroni post-tests, no statistically significant difference was observed upon treatment.
Figure 5.
Vertical and non sexual transmissibility of MHV-68 from virus-excreting mice.
A–D. Female mice were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every day. At the time of the first observation of genital signal, infected females were mated with uninfected males. Mock infected female mice were used as controls. Effect of MHV-68 infection on litter size (A), mortality/litter (B) and gestation length (C) was then monitored. The data presented are the average for 20 (infected) and 11 (Mock) pregnancies +/− standard error of the mean and were analyzed by 1way ANOVA and Bonferroni post-tests, no statistically significant difference was observed. Transmission to the progeny (n≥20 per group) was assessed by infectious center assays performed on isolated organs taken from newborn or at 3 or 6 weeks after birth (C). Data are plotted individually. E. Female mice (n = 10) were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every day. At the time of the first observation of genital signal, infected females were co-housed with 3 uninfected females. Potential MHV-68 transmission was monitored 45 days later by detection of anti-MHV-68 specific antibodies. The dashed line indicates the mean value obtained with sera from 3 uninfected mice taken as controls.
Figure 6.
Sexual transmission of MHV-68 from virus-excreting female mice.
A–B. Female mice were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every day. At the time of the first observation of genital signal, infected females (3 per cages) were mated with uninfected males (3 per cages). MHV-68 infection was monitored at the indicated times by detection of anti-MHV-68 specific antibodies (A) or quantification of viral genomes in male spleens performed after at least 20 days post-contact (B). The dashed line shows the lower limit of the assay sensitivity. Dpi, days post-infection; dpc, days post-contact.
Figure 7.
Spatial and temporal progression of MHV-68 infection after sexual transmission to male mice.
Female mice were infected intranasally (104 PFU) with WT luciferase+ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every day. At the time of the first observation of genital signal, infected females were mated with uninfected males. The males were then injected with luciferin and imaged every day. Images show a representative mouse over time. The day post-contact with the infected female (e.g., d4 is day 4 post-contact) is shown at the top of each image.
Figure 8.
Luciferase signal and MHV-68 antigen detection in isolated male genital tract after sexual transmission.
A. A mouse equivalent to that in Figure 7 was dissected and its genital tract imaged ex vivo at day 10 post-contact with the infected female. The images are representative of data from at least 5 mice, and show either a standard photograph (Photo) or that photograph overlaid with the luciferase signal (Photo+Luc). The region with the highest signal was isolated and processed for histological analysis. The scale bar (photons sec−1 cm−2 steradian−1) shows the color scheme for signal intensity. MUMP, male urogenital mating protuberance. B. The piece of penis isolated in A. was fixed in formaldehyde and organ slices were either stained with hematoxylin-eosin (panel i) or processed for immunohistochemistry with anti-MHV-68 rabbit polyserum (panels ii to v) as described in the Materials and Methods section. Rectangles indicate regions highlighted in the following panels. Filled and open arrows indicate detection of MHV-68 antigens in superficial regions of the penis and in deeper region of the Corpus cavernosum, respectively. Ur, urethra; EpPe, Epithelium of the penis; BoMa, Bone marrow; OsPe, Os penis; CoCa, Corpus cavernosum; FiPa, Filiform papilla.
Figure 9.
Direct visualization of lymph node colonization after sexual transmission of MHV-68 infection.
A mouse equivalent to those in Figure 8 and 9 was dissected 2 weeks post-contact with the infected female. Representative images show either a standard photograph (Photo) or that photograph overlaid with the luciferase signal (Photo+Luc). The entire body (panels i and iv) and the pelvis region (panels ii and v) are shown after displacement of the genital tract. Panels iii and vi show isolated lymph nodes. The scale bar (photons sec−1 cm−2 steradian−1) shows the color scheme for signal intensity. SCLN, superficial cervical lymph nodes; Ax, axillary lymph nodes; LbAo, lumbar aortic lymph nodes; IlMd, medial iliac lymph nodes; SbIl, subiliac lymph nodes.