Comparative study of Murid gammaherpesvirus 4 infection in mice and in its natural host, the bank voles.

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. They are host-range specific and establish persistent, productive infections of immunocompetent hosts. Thus, infected individuals simultaneously both elicit antiviral protective immune response and secrete infectious virions. The best studied gammaherpesviruses are Human herpesvirus 4 and Human herpesvirus 8. As these viruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4), a virus that has originally been isolated from bank voles (Myodes glareolus). Although MuHV-4 has not been isolated from house mice (Mus musculus), infection of inbred laboratory mouse strains is commonly accepted as a good model for studying gammaherpesviruses in vivo. It has however never been possible to monitor viral reexcretion and virus transmission in this species suggesting that this model could be imperfect. In this study, we therefore characterized MuHV-4 infection in its natural host, the bank voles, through classical virological methods but also through global luciferase imaging for an anatomical complete view of the infection. Results obtained show that, after intra-nasal infection, the natural route of infection is similar in mice and voles. Following nasal productive infection, the virus spreads to the lung where the infection is accompanied by massive cellular infiltrates. By opposition to extensive viral replication observed in mice, the different analyses indicated that the viral replication was ~1000 fold lower in bank voles. This lower replication did however not affect colonization of latency sites in superficial cervical lymph nodes and spleen as measured by real-time PCR quantification of viral genomes in these organs. In conclusion, this study revealed that MuHV-4 can experimentally infect bank voles, the supposed natural host, but with a lower replicative power. As, gammaherpesvirus epidemiology indicates that transmission correlates with the latent load, our results suggest that gammaherpesviruses may have evolved to infect their hosts without extensive lytic spread. In the future, establishment of experimental transmission in a population of Myodes glareolus should help us to better understand mechanisms used by gammaherpesviruses to evade immune response

Genital re-excretion of Murid gammaherpesvirus 4 following intranasal infection

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4) in inbred laboratory mouse strains which are commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. Surprisingly, we detected transient viral replication in mice genital tract at various times after latency establishment. Ex vivo imaging, quantitative PCR and immunohistochemistry revealed that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Moreover, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. As this ephemeral viral reexcretion could reveal a link with reproductive cycle, we compared reexcretion in normal and ovariectomized mice. Interestingly, no viral reactivation was observed in absence of hormonal cycle. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop strategies that could prevent the spread of these viruses in natural populations

Direct visualization of lymph node colonization after sexual transmission of MHV-68 infection.

<p>A mouse equivalent to those in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#ppat-1003292-g008" target="_blank">Figure 8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#ppat-1003292-g009" target="_blank">9</a> 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.</p

Luciferase signal and MHV-68 antigen detection in isolated male genital tract after sexual transmission.

<p><b>A.</b> A mouse equivalent to that in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#ppat-1003292-g007" target="_blank">Figure 7</a> was dissected and its genital tract imaged <i>ex vivo</i> 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>B.</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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#s4" target="_blank">Materials and Methods</a> 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.</p

<i>In vivo</i> infection by luciferase-expressing MHV-68.

<p>Female mice were infected intranasally (10⁴ PFU) with WT luciferase⁺ MHV-68 under general anaesthesia, and then injected with luciferin and imaged every days. <b>A.</b> Images show a representative mouse at days 0, 7, 14 and 21 post-infection (p.i.). <b>B.</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. <b>C.</b> 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.</p

Luciferase signal and MHV-68 antigen detection in isolated genital tract after intranasal infection.

<p><b>A.</b> A mouse equivalent to those in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#ppat-1003292-g001" target="_blank">Figure 1</a> was dissected and its genital tract imaged <i>ex vivo</i>. 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>B.</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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#s4" target="_blank">Materials and Methods</a> 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.</p

Spatial and temporal progression of MHV-68 infection after sexual transmission to male mice.

<p>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.</p

Sexual transmission of MHV-68 from virus-excreting female mice.

<p><b>A–B.</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.</p

Influence of estrous cycle on genital MHV-68 excretion after intranasal infection.

<p><b>A.</b> 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 (***<i>P</i><0.001). <b>B–C.</b> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003292#s4" target="_blank">Materials and Methods</a> 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.</p