Antiviral effect of the nucleoside analogue cidofovir in the context of sexual transmission of a gammaherpesvirus in mice

Antiviral effect of the nucleoside analogue cidofovir in the context of sexual transmission of a... Abstract Objectives To investigate the efficacy of cidofovir to block gammaherpesvirus replication in the context of sexual transmission. Methods A luciferase-expressing strain of murid herpesvirus 4 (MuHV-4) was used to monitor genital virus excretion from infected female BALB/c mice and sexual transmission to naive males. The efficiency of cidofovir to block genital excretion from infected females or replication and host colonization of naive males after sexual contact was tested by treating infected females (either once daily or at a single timepoint), naive males before exposure (either once daily or at a single timepoint) or males 24 h post-exposure. Results We showed that daily treatment of infected females can reduce MuHV-4 genital shedding by 75%. Similarly, daily preventive treatment of naive males was sufficient to block viral replication and latency establishment in males. In contrast, a single administration of cidofovir to infected females at day 14 post-infection or to naive males 2 to 6 days before contact with MuHV-4-excreting females was not sufficient to significantly reduce viral shedding from females or infection of males, respectively. Interestingly, a single administration of cidofovir to males 24 h after contact with MuHV-4-infected females excreting the virus in the genital tract significantly reduced virus replication in males and seroconversion. Conclusions Altogether, our results show that cidofovir can significantly reduce gammaherpesvirus replication, excretion and colonization of the naive partner in the context of sexual transmission. Such treatments could therefore be recommended in some specific conditions where gammaherpesvirus infections could be deleterious. Introduction Gammaherpesviruses (γHVs) are important pathogens that are ubiquitous in both human and animal populations. Thus, Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) infect, respectively, up to 90% and 30% of humans worldwide and are associated with several human malignancies such as Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinoma, Kaposi’s sarcoma and post-transplant lymphoproliferative disease.1–3 Efficient control of these infections is therefore of major interest, particularly in immunocompromised people.4 The very high infection prevalence of these infections is due to efficient transmission from carriers to new hosts5 and reflects the fact that these viruses have evolved to coexist with immune responses. Indeed, γHVs establish persistent, productive infections, with virus carriers both making antiviral immune responses that protect against disease and excreting infectious virions. While interrupting transmission is the sine qua non of infection control, strategies based on vaccination have therefore been mainly unsuccessful to reach this goal. For example, vaccination with the EBV gp350, to block virion binding to B cells, failed to reduce infection rates while protecting against infectious mononucleosis.6,7 Other approaches to block transmission are therefore needed. Antiviral drugs are another way to fight viral infections.8 No antiviral drugs are currently licensed for treatment of KSHV or EBV infections. However, several antiherpesvirus drugs have been shown to block these infections in vitro, especially those targeting the viral DNA polymerase or viral DNA replication, such as aciclovir, ganciclovir, foscarnet and cidofovir.8–12 Cidofovir, also called HPMPC {for (S)-1-[3-hydroxy-2-(phosphomethoxy)propyl]cytosine}, is a nucleotide analogue that needs to be phosphorylated by cellular kinases to its diphosphate form to become biologically active and block viral DNA polymerase.13 When evaluated in an in vivo model, cidofovir proved to be very efficacious in protecting mice from γHV-induced disease whereas aciclovir, ganciclovir and foscarnet had little or no effect.12 While cidofovir and related molecules are promising compounds for future clinical development to fight γHV infection, their use in the context of viral transmission has never been investigated. Analysing EBV and KSHV transmission has proved difficult as these viruses have no well-established in vivo infection models. Related animal viruses, such as murid herpesvirus 4 (MuHV-4), provide another way to address such questions.14 Indeed, using luciferase imaging of MuHV-4 infection,15 we recently observed genital MuHV-4 excretion following intranasal inoculation of female mice and transmission to naive males after sexual contact.16 Interestingly, that way of transmission also occurs for the KSHV transmission associated with HIV infection17 and may also apply to EBV.18 In this study, we therefore want to investigate whether cidofovir could block γHV replication in conditions of sexual transmission close to natural settings. Materials and methods Ethics statement and animals Experiments, maintenance and care of mice complied with the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The Committee on the Ethics of Animal Experiments of the University of Liège, Belgium approved the protocol (Permit Number: 1502). All efforts were made to limit animal suffering. BALB/c mice were purchased from ENVIGO Laboratories and were housed under conventional conditions in the Department of Infectious Diseases, University of Liège, Liège, Belgium. Female mice were used at the age of 8 weeks and their weight was 20 ± 1.5 g. Mice were infected intranasally with 104 pfu of MuHV-4 diluted in 30 μL of sterile PBS, under general anaesthesia with isoflurane. Animals were randomly allocated to experimental groups. Antiviral compound Mice were injected subcutaneously at different timepoints according to experimental design with 25 mg/kg/day cidofovir (Gilead Sciences) diluted in PBS. Viral strain All viruses were derived from a MuHV-4 bacterial artificial chromosome (BAC).19 We used a strain expressing luciferase under the control of the M3 promoter that has been described previously (WT Luc+ strain).15 The loxP-flanked BAC/eGFP cassette was removed by subsequent growths of the virus in NIH-3T3-CRE cells until eGFP+ cells were no longer visible.20 Virus stocks were grown in BHK-21 cells cultured in DMEM (Gibco) supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% FCS. Cell culture medium was cleared of cell debris by low-speed centrifugation (1000 g, 30 min). Viruses were then concentrated by high-speed centrifugation (100 000 g, 90 min) and titrated by plaque assay on BHK-21 cells as described elsewhere.21 In vivo imaging Mice were anaesthetized, injected intraperitoneally with luciferin (60 mg/kg) and imaged 10 min later with an IVIS Spectrum (Perkin Elmer). For quantitative comparisons, Living Image software (Perkin Elmer) was used to obtain the average radiance [photons/s/cm2/steradian (p/s/cm2/sr)] over each region of interest. For the reliable comparison of data, an average background measured in the right abdominal region was removed from the measured intensities. Animals were considered as positive if the genital signal was superior to the mean value of 10 uninfected mice + 3 standard deviations (represented by a broken line in the figures). Quantification of anti-MuHV-4-specific antibodies by ELISA MuHV-4 virions pelleted as described above were disrupted by addition to the viral preparation of Triton X-100 to a concentration of 0.1% (v/v). Nunc Maxisorp ELISA plates (Nalgene Nunc) were coated for 18 h at 4°C with Triton X-100-disrupted MuHV-4 virions (106 pfu equivalents/well), blocked in PBS/0.1% Tween 20/3% BSA, and incubated with mouse sera (diluted 1/200 in PBS/0.1% Tween 20). Bound antibodies were detected with alkaline-phosphatase-conjugated goat anti-mouse immunoglobulin (Ig) polyclonal antibody (Sigma–Aldrich). Washing was performed with PBS/0.1% Tween 20. p-Nitrophenylphosphate (Sigma–Aldrich) was used as substrate for colorimetry and absorbance was read at 405 nm using a Benchmark ELISA plate reader (Thermo). Sera were considered as positive when absorbance was superior to the mean value of mock-infected mice + 3 standard deviations (represented by a broken line in the figures). Viral genome quantification MuHV-4 genomic coordinates 40 264–44 385 were amplified as described (ORF25 gene, forward primer 5′-ATGGTATAGCCGCCTTTGTG-3′, reverse primer 5′-ACAAGTGGATGAAGGGTTGC-3′).22 The PCR products were quantified by hybridization with a TaqMan probe (genomic coordinates 43 088–43 117, 5′ 6-FAM-TTCATAAGTTTTATGCTGATCCAGTGGTTG-BHQ1 3′) and converted to genome copies by comparison with a standard curve of cloned plasmid template serially diluted in control mouse spleen DNA and amplified in parallel (iCycler, Bio-Rad). Cellular DNA was quantified by amplifying part of the mouse interstitial retinoid binding protein (IRBP) gene (forward primer 5′-ATCCCTATGTCATCTCCTACYTG-3′, reverse primer 5′-CCRCTGCCTTCCCATGTYTG-3′). The PCR products were quantified with Sybr green (Invitrogen), the copy number was calculated by comparison with a standard curve of cloned mouse IRBP template amplified in parallel. Amplified products were distinguished from paired primers by melting curve analysis and the correct sizes of the products confirmed by electrophoresis and staining with ethidium bromide. Statistical analyses Fisher’s exact test or unpaired, two-sided Student’s t-test was used for comparisons between two sets of data. If multiple groups of data were compared simultaneously, an ANOVA was used and Bonferroni post-tests were used to compare groups. A P value <0.05 was used for statistical significance. Results Daily cidofovir treatment of infected females leads to reduction of MuHV-4 genital excretion In order to test the effect of cidofovir treatment on MuHV-4 transmission, we firstly investigated if cidofovir could reduce MuHV-4 genital excretion from infected female mice. We infected 8-week-old BALB/c female mice with a MuHV-4 strain expressing luciferase under the control of the M3 lytic promoter (WT Luc strain).15 Indeed, using this strain, we previously showed that, following intranasal infection, a luciferase signal is observed in the genital tract of ∼80% of infected female mice, that this signal is associated with viral excretion and that this excretion occurs in most of the cases between days 17 and 21 post-infection.16 Moreover, we showed that excretion of infectious virus in the female genital tract allows sexual transmission to naive males (Figure S1, available as Supplementary data at JAC Online).16 Therefore, cidofovir was given on a daily basis from day 15 post-infection until day 22 post-infection (n = 20) (Figure 1a) while control mice received PBS. Mice were then followed by daily in vivo imaging between days 17 and 22 post-infection in order to detect the MuHV-4 genital signal. While ∼90% of untreated females showed MuHV-4 excretion during this period as expected, only 20% of females excreted the virus after treatment with cidofovir (Figure 1b and c and Figure S2). In contrast, cidofovir treatment did not reduce seroconversion to MuHV-4 (Figure 1d) or the level of latent genomes in spleens (Figure 1e). Altogether, these results show that once-daily cidofovir administration reduces MuHV-4 genital excretion from MuHV-4-infected female mice without reducing latency establishment. Figure 1. View largeDownload slide Effect of once-daily treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu). They were then injected daily with cidofovir (25 mg/kg, subcutaneously) from day 15 to day 22 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 10 randomly subsampled mice from groups of 20 mice, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). ***P < 0.001 and *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 1. View largeDownload slide Effect of once-daily treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu). They were then injected daily with cidofovir (25 mg/kg, subcutaneously) from day 15 to day 22 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 10 randomly subsampled mice from groups of 20 mice, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). ***P < 0.001 and *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. A single cidofovir treatment of infected females does not reduce MuHV-4 genital excretion Daily cidofovir treatment is usually used in mice.12 However, in humans a weekly administration is sufficient and allows the reduction of associated adverse effects. Therefore, in order to address the effect of a single cidofovir treatment, we repeated the same experiment by replacing the daily injections by a single injection of cidofovir performed at day 14 post-infection as genital excretion usually starts from day 17 (Figure 2a).16 At 19 days post-infection, only 2 females of 20 (10%) showed genital excretion after treatment while 8 females of 20 (40%) had displayed the genital signal in the control group (P < 0.05 by χ2 test; Figure 2b). However, at 21 days post-infection, there was no difference between the two groups (P > 0.05 by χ2 test) (Figure 2b and c and Figure S3). Again, cidofovir treatment did not reduce seroconversion to MuHV-4 (Figure 2d) or the level of latent genomes in spleens (Figure 2e). Altogether, these results show that a single cidofovir administration at day 14 post-infection can reduce MuHV-4 genital excretion from infected female mice until day 19 post-infection but is not sufficient to block it for the long term as seen at day 21 post-infection. Figure 2. View largeDownload slide Effect of a single treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Mice were infected intranasally with MuHV-4 (104 pfu). They were then injected with cidofovir (25 mg/kg, subcutaneously) at day 14 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 2. View largeDownload slide Effect of a single treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Mice were infected intranasally with MuHV-4 (104 pfu). They were then injected with cidofovir (25 mg/kg, subcutaneously) at day 14 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Once-daily cidofovir treatment of naive males prevents MuHV-4 replication and latency establishment While antiviral treatments could be used to reduce excretion and transmission from infected subjects, they could also prevent replication in naive ones. We therefore investigated the potency of a daily cidofovir treatment to block MuHV-4 replication and host colonization in naive males after sexual contact with excreting females. Briefly, we infected female BALB/c mice with the MuHV-4 WT Luc+ strain and followed them by in vivo imaging in order to detect MuHV-4 genital excretion. When the genital signal was observed (between days 17 and 21 depending on the females), excreting females were mated with naive males, treated or not with cidofovir from days 15 to 24 (day 0 was defined as the time of female intranasal infection). At day 24, males were separated from females and were followed by in vivo imaging to monitor viral replication (Figure 3a–c). Strikingly, no males displayed the genital signal after daily treatment with cidofovir (n = 0/15) while a transmission rate of 35% was observed in the control group (Figure 3b and c). Males of both groups were euthanized at day 41. In contrast with males from the PBS-treated group, none of the males from the cidofovir-treated group displayed seroconversion to MuHV-4 infection (Figure 3d) or latency establishment in the spleen (Figure 3e). Altogether, these results show that once-daily cidofovir administration to naive males (started 2 days before putting males in contact with genitally excreting females and maintained for the whole contact period with infected females) is sufficient to prevent replication and host colonization following sexual contact with MuHV-4-excreting females. Figure 3. View largeDownload slide Preventive daily treatment of males with cidofovir blocks MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not daily injection of cidofovir (25 mg/kg, subcutaneously) from day 15 to day 24 according to the experimental scheme. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal. Figure 3. View largeDownload slide Preventive daily treatment of males with cidofovir blocks MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not daily injection of cidofovir (25 mg/kg, subcutaneously) from day 15 to day 24 according to the experimental scheme. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal. A single preventive cidofovir treatment of naive males 2 to 6 days before sexual contact is not sufficient to prevent MuHV-4 sexual infection We then used a single preventive injection of cidofovir to naive males in order to test if it was sufficient to prevent viral replication and host colonization. Briefly, we repeated the same experimental design as described above except that males received a single injection of cidofovir at day 15 (day 0 was defined as the time of female intranasal infection) (Figure 4a). In vivo imaging did not highlight any significant difference between cidofovir- and PBS-treated groups (Figure 4b and c). Accordingly, similar rates of seroconversion (Figure 4d) or of latency establishment in the spleen (Figure 4e) were observed in both groups. Altogether, these results show that a single cidofovir administration to naive males 2 to 6 days before sexual contact with females excreting the virus in the genital tract is not sufficient to prevent viral replication and latency establishment in males. Figure 4. View largeDownload slide A single preventive treatment of males with cidofovir fails to block MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not a single injection of cidofovir (25 mg/kg, subcutaneously) at day 15 of the experiment. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). The broken line represents the mean value of mock-infected mice + 3 standard deviations. (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 4. View largeDownload slide A single preventive treatment of males with cidofovir fails to block MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not a single injection of cidofovir (25 mg/kg, subcutaneously) at day 15 of the experiment. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). The broken line represents the mean value of mock-infected mice + 3 standard deviations. (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Influence of a single post-exposure cidofovir treatment of naive males on their infection through the sexual route Finally, we tested if a single post-exposure treatment of exposed naive males could be sufficient to block systemic infection and latency establishment. Briefly, we repeated the same experimental scheme as the one described above, but instead of treating naive males 2 to 6 days before sexual contact with MuHV-4-excreting females, males received a single injection of cidofovir 24 h after contact with infected females (Figure 5a). Interestingly, only 1 of 32 (3%) treated males displayed an MuHV-4-associated genital signal, whereas 6 of 23 (26%) became infected in the control group (Figure 5b and c). Accordingly, seroconversion to MuHV-4 (Figure 5d) was also reduced in the cidofovir-treated group in comparison with the PBS-treated group. Although latency establishment (Figure 5e) tended also to be reduced in the cidofovir-treated group this was, however, not statistically different from the PBS-treated group (P = 0.056 by Fisher’s exact test). Altogether, these results suggest that, following sexual contact with MuHV-4-excreting females, a single post-exposure cidofovir administration to naive males reduces MuHV-4 replication and seroconversion against the virus. Figure 5. View largeDownload slide Post-exposure treatment of males with cidofovir can prevent establishment of MuHV-4 infection. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males. Males were treated 24 h post-contact with excreting females with a single injection of cidofovir (25 mg/kg, subcutaneously) or with PBS in the control group. Males were separated from females 48 h post-contact and were followed by in vivo imaging to detect virus replication. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 23 mice in PBS group, 32 mice in cidofovir group and 3 mice in the mock-infected group). (e) Viral genomes per spleen cell of males at euthanasia (n = 23 mice in the PBS group and 32 mice in the cidofovir group and 3 mice in the mock-infected group). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 5. View largeDownload slide Post-exposure treatment of males with cidofovir can prevent establishment of MuHV-4 infection. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males. Males were treated 24 h post-contact with excreting females with a single injection of cidofovir (25 mg/kg, subcutaneously) or with PBS in the control group. Males were separated from females 48 h post-contact and were followed by in vivo imaging to detect virus replication. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 23 mice in PBS group, 32 mice in cidofovir group and 3 mice in the mock-infected group). (e) Viral genomes per spleen cell of males at euthanasia (n = 23 mice in the PBS group and 32 mice in the cidofovir group and 3 mice in the mock-infected group). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Discussion γHVs are highly prevalent animal and human viruses that are associated with numerous pathological conditions. In immunocompromised people, EBV and KSHV cause cancers and other disorders such as Burkitt’s lymphoma, Kaposi’s sarcoma or Castleman disease.4,23 Moreover, primary infection of young adults with EBV causes infectious mononucleosis, which represents major health costs.24 EBV-induced infectious mononucleosis also increases the risk of developing multiple sclerosis to a similar degree as the strongest genetic risk factor.25 Preventing γHV infection in some defined populations is therefore a major public health challenge. Over the last 50 years, several antivirals have been developed to treat different infectious conditions.8 In the case of herpesviruses, viral DNA polymerase is a key target for the development of potent inhibitors. Thus, acyclic nucleoside phosphonates, such as cidofovir, are important broad-spectrum antiviral agents, which emerged as the most potent anti-γHV drugs.13 In this study, we tested the capacity of cidofovir to block γHV replication and host colonization in the context of sexual transmission.16 Briefly, we showed that once-daily treatment of either infected females or naive males is sufficient to significantly reduce viral excretion from infected females and viral replication and latency establishment in naive males (Figures 1 and 3). In particular, treatment of the uninfected males not only reduced initial viral replication in the male penis to levels undetectable by the IVIS system but also protected these mice from seroconversion to MuHV-4 and from latency establishment in the spleen (Figure 3). In contrast, cidofovir treatment of excreting females did not affect the levels of virus latency in the spleen but was sufficient to reduce virus excretion anyway (Figure 1). This approach could potentially be translated to human viruses as MuHV-4, EBV and KSHV display similar sensitivity to cidofovir even if measures of EC50 have not been performed in the same conditions.13 Moreover, a similar approach had already been shown to be successful in the case of sexual transmission of herpes simplex virus 2 (HSV-2) in human where once-daily valaciclovir treatment of the infected partner reduced the risk of HSV-2 transmission between heterosexual, monogamous couples discordant for HSV-2 infection.26,27 Such an approach could also apply to animals in specific conditions. For example, some γHVs, such as those associated with malignant catarrhal fever, cause recurrent problems in zoos where persistently infected and susceptible animals are housed in close contact.28,29 As these viruses are frequently transmitted upon parturition, treatment of persistently infected mothers around these periods could likely reduce virus excretion and help to protect neighbouring susceptible species. Of course, the sensitivity of these viruses to cidofovir will have to be determined. One of the major pitfalls of nucleoside analogues resides in their toxicity in the long term. We therefore tested the capacity of a single cidofovir injection (either to the infected or to the naive partner) before sexual contact to block MuHV-4 transmission (Figures 2 and 4). Neither of these two approaches were revealed as successful at the end of the experimental protocol while a reduction in genital excretion was observed until day 19 (P < 0.05 by χ2 test) in infected females treated with cidofovir (Figure 2). Therefore, although preventive cidofovir treatment has the potential to efficiently block γHV transmission, the establishment of protocols based on a single injection will be difficult and is likely directly linked with the in vivo t½ of the drug. Cidofovir has a long intracellular t½ of about 15 to 65 h and a single administration of 30 mg/kg (similar to the 25 mg/kg used in our protocols) protects mice from poxvirus infections 3 days later but not 5 days later.30 Similarly, we observed that a single cidofovir injection is sufficient to block excretion of MuHV-4 from infected females for 5 days (Figure 2; between days 14 and 19 post-infection). A single administration to naive males also delayed their infection (Figure 4). Therefore, usage of cidofovir as a preventive treatment to block sexual transmission of γHV will require a frequency of administration that allows maintenance of efficient concentration of the drug and its metabolites in cells but does not induce side effects such as nephrotoxicity. Alternatively, these objectives could be reached by the use of brincidofovir, a novel oral lipid-conjugated nucleotide analogue, which is converted into cidofovir in the target cells and has shown enhanced in vitro activity against some herpesviruses while displaying reduced toxicity in vivo.31 While preventive pre-exposure administration of antivirals is difficult to implement to block transmission, post-exposure treatment could display obvious advantages. Interestingly, we showed here that a single cidofovir administration to males 24 h post-sexual contact with MuHV-4-excreting females significantly reduced virus replication in the male genital area and seroconversion at euthanasia suggesting that a single cidofovir treatment could be efficient (Figure 5). However, reduction of viral genome copy numbers in the spleens was not statistically significant (Figure 5e) suggesting a reduced replication at the entry site but host colonization anyway. The feasibility of such an approach in the field could depend on the major mode of transmission of γHVs in those conditions. Endemic EBV and KSHV infections are maintained chiefly by carriers shedding virus into their saliva. In that context, single post-exposure administration is likely not feasible. However, numerous reports have shown that γHVs could be sexually transmitted in some circumstances.32 This was seen clearly for the KSHV transmission associated with HIV infection17 and may apply also to EBV.18,33 In such a context, post-exposure treatment could probably be an option. Altogether, we have shown that cidofovir could be used to efficiently block transmission of a γHV in conditions close to what happens in real life. Especially, it appeared that post-exposure treatment could be a very interesting approach in some epidemiological conditions regarding its efficacy and its ease of implementation. Acknowledgements We thank A. Vanderplasschen and L. Willems for helpful discussions and the technician and administrative team of the lab for very helpful assistance. Funding This work was supported by the VIR-IMPRINT ARC grant of the University of Liège and the BELVIR Interuniversity Attraction Pole (IAP). C. Z. is a research fellow of the ‘Fonds de la Recherche Scientifique - Fonds National Belge de la Recherche Scientifique’ (F.R.S. - FNRS). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Transparency declarations None to declare. Supplementary data Figures S1 to S3 are available as Supplementary data at JAC Online. References 1 Henle G , Henle W , Clifford P et al. Antibodies to Epstein-Barr virus in Burkitt's lymphoma and control groups . J Natl Cancer Inst 1969 ; 43 : 1147 – 57 . Google Scholar PubMed 2 Thorley-Lawson DA , Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas . N Engl J Med 2004 ; 350 : 1328 – 37 . Google Scholar CrossRef Search ADS PubMed 3 Verma SC , Robertson ES. Molecular biology and pathogenesis of Kaposi sarcoma-associated herpesvirus . FEMS Microbiol Lett 2003 ; 222 : 155 – 63 . Google Scholar CrossRef Search ADS PubMed 4 Cesarman E. Gammaherpesviruses and lymphoproliferative disorders . Annu Rev Pathol 2014 ; 9 : 349 – 72 . Google Scholar CrossRef Search ADS PubMed 5 Virgin HW , Wherry EJ , Ahmed R. Redefining chronic viral infection . Cell 2009 ; 138 : 30 – 50 . Google Scholar CrossRef Search ADS PubMed 6 Moutschen M , Léonard P , Sokal EM et al. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults . Vaccine 2007 ; 25 : 4697 – 705 . Google Scholar CrossRef Search ADS PubMed 7 Sokal EM , Hoppenbrouwers K , Vandermeulen C et al. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults . J Infect Dis 2007 ; 196 : 1749 – 53 . Google Scholar CrossRef Search ADS PubMed 8 De Clercq E , Li G. Approved antiviral drugs over the past 50 years . Clin Microbiol Rev 2016 ; 29 : 695 – 747 . Google Scholar CrossRef Search ADS PubMed 9 Friedrichs C , Neyts J , Gaspar G et al. Evaluation of antiviral activity against human herpesvirus 8 (HHV-8) and Epstein-Barr virus (EBV) by a quantitative real-time PCR assay . Antivir Res 2004 ; 62 : 121 – 3 . Google Scholar CrossRef Search ADS PubMed 10 Lin JC , De Clercq E , Pagano JS. Inhibitory effects of acyclic nucleoside phosphonate analogs, including (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, on Epstein-Barr virus replication . Antimicrob Agents Chemother 1991 ; 35 : 2440 – 3 . Google Scholar CrossRef Search ADS PubMed 11 Meerbach A , Holý A , Wutzler P et al. Inhibitory effects of novel nucleoside and nucleotide analogues on Epstein-Barr virus replication . Antivir Chem Chemother 1998 ; 9 : 275 – 82 . Google Scholar CrossRef Search ADS PubMed 12 Neyts J , De Clercq E. In vitro and in vivo inhibition of murine gamma herpesvirus 68 replication by selected antiviral agents . Antimicrob Agents Chemother 1998 ; 42 : 170 – 2 . Google Scholar PubMed 13 Coen N , Duraffour S , Naesens L et al. Evaluation of novel acyclic nucleoside phosphonates against human and animal gammaherpesviruses revealed an altered metabolism of cyclic prodrugs upon Epstein-Barr virus reactivation in P3HR-1 cells . J Virol 2013 ; 87 : 12422 – 32 . Google Scholar CrossRef Search ADS PubMed 14 Barton E , Mandal P , Speck SH. Pathogenesis and host control of gammaherpesviruses: lessons from the mouse . Annu Rev Immunol 2011 ; 29 : 351 – 97 . Google Scholar CrossRef Search ADS PubMed 15 Milho R , Smith CM , Marques S et al. In vivo imaging of murid herpesvirus-4 infection . J Gen Virol 2009 ; 90 : 21 – 32 . Google Scholar CrossRef Search ADS PubMed 16 François S , Vidick S , Sarlet M et al. Illumination of murine gammaherpesvirus-68 cycle reveals a sexual transmission route from females to males in laboratory mice . PLoS Pathog 2013 ; 9 : e1003292. Google Scholar CrossRef Search ADS PubMed 17 Schulz TF. Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8): epidemiology and pathogenesis . J Antimicrob Chemother 2000 ; 45 Suppl T3: 15 – 27 . Google Scholar CrossRef Search ADS PubMed 18 Dunmire SK , Grimm JM , Schmeling DO et al. The incubation period of primary Epstein-Barr virus infection: viral dynamics and immunologic events . PLoS Pathog 2015 ; 11 : e1005286. Google Scholar CrossRef Search ADS PubMed 19 Adler H , Messerle M , Wagner M et al. Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome . J Virol 2000 ; 74 : 6964 – 74 . Google Scholar CrossRef Search ADS PubMed 20 Adler H , Messerle M , Koszinowski UH. Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences . J Virol 2001 ; 75 : 5692 – 6 . Google Scholar CrossRef Search ADS PubMed 21 Gillet L , May JS , Stevenson PG. In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection . J Gen Virol 2009 ; 90 : 602 – 13 . Google Scholar CrossRef Search ADS PubMed 22 Latif MB , Machiels B , Xiao X et al. Deletion of murid herpesvirus 4 ORF63 affects the trafficking of incoming capsids toward the nucleus . J Virol 2015 ; 90 : 2455 – 72 . Google Scholar CrossRef Search ADS PubMed 23 Jha HC , Banerjee S , Robertson ES. The role of gammaherpesviruses in cancer pathogenesis . Pathogens 2016 ; 5 : 18. Google Scholar CrossRef Search ADS 24 Taylor GS , Long HM , Brooks JM et al. The immunology of Epstein-Barr virus-induced disease . Annu Rev Immunol 2015 ; 33 : 787 – 821 . Google Scholar CrossRef Search ADS PubMed 25 Geginat J , Paroni M , Pagani M et al. The enigmatic role of viruses in multiple sclerosis: molecular mimicry or disturbed immune surveillance? Trends Immunol 2017 ; 38 : 498 – 512 . Google Scholar CrossRef Search ADS PubMed 26 Corey L , Wald A , Patel R et al. Once-daily valacyclovir to reduce the risk of transmission of genital herpes . N Engl J Med 2004 ; 350 : 11 – 20 . Google Scholar CrossRef Search ADS PubMed 27 Crumpacker CS. Use of antiviral drugs to prevent herpesvirus transmission . N Engl J Med 2004 ; 350 : 67 – 8 . Google Scholar CrossRef Search ADS PubMed 28 Cooley AJ , Taus NS , Li H. Development of a management program for a mixed species wildlife park following an occurrence of malignant catarrhal fever . J Zoo Wildl Med 2008 ; 39 : 380 – 5 . Google Scholar CrossRef Search ADS PubMed 29 Frontoso R , Autorino GL , Friedrich KG et al. An acute multispecies episode of sheep-associated malignant catarrhal fever in captive wild animals in an Italian zoo . Transbound Emerg Dis 2016 ; 63 : 621 – 7 . Google Scholar CrossRef Search ADS PubMed 30 Quenelle DC , Collins DJ , Kern ER. Efficacy of multiple- or single-dose cidofovir against vaccinia and cowpox virus infections in mice . Antimicrob Agents Chemother 2003 ; 47 : 3275 – 80 . Google Scholar CrossRef Search ADS PubMed 31 Quenelle DC , Lampert B , Collins DJ et al. Efficacy of CMX001 against herpes simplex virus infections in mice and correlations with drug distribution studies . J Infect Dis 2010 ; 202 : 1492 – 9 . Google Scholar CrossRef Search ADS PubMed 32 Davison AJ. Evolution of sexually transmitted and sexually transmissible human herpesviruses . Ann N Y Acad Sci 2011 ; 1230 : E37 – 49 . Google Scholar CrossRef Search ADS PubMed 33 Higgins CD , Swerdlow AJ , Macsween KF et al. A study of risk factors for acquisition of Epstein-Barr virus and its subtypes . J Infect Dis 2007 ; 195 : 474 – 82 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Antiviral effect of the nucleoside analogue cidofovir in the context of sexual transmission of a gammaherpesvirus in mice

Journal of Antimicrobial Chemotherapy , Volume Advance Article (8) – May 16, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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0305-7453
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10.1093/jac/dky161
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Abstract

Abstract Objectives To investigate the efficacy of cidofovir to block gammaherpesvirus replication in the context of sexual transmission. Methods A luciferase-expressing strain of murid herpesvirus 4 (MuHV-4) was used to monitor genital virus excretion from infected female BALB/c mice and sexual transmission to naive males. The efficiency of cidofovir to block genital excretion from infected females or replication and host colonization of naive males after sexual contact was tested by treating infected females (either once daily or at a single timepoint), naive males before exposure (either once daily or at a single timepoint) or males 24 h post-exposure. Results We showed that daily treatment of infected females can reduce MuHV-4 genital shedding by 75%. Similarly, daily preventive treatment of naive males was sufficient to block viral replication and latency establishment in males. In contrast, a single administration of cidofovir to infected females at day 14 post-infection or to naive males 2 to 6 days before contact with MuHV-4-excreting females was not sufficient to significantly reduce viral shedding from females or infection of males, respectively. Interestingly, a single administration of cidofovir to males 24 h after contact with MuHV-4-infected females excreting the virus in the genital tract significantly reduced virus replication in males and seroconversion. Conclusions Altogether, our results show that cidofovir can significantly reduce gammaherpesvirus replication, excretion and colonization of the naive partner in the context of sexual transmission. Such treatments could therefore be recommended in some specific conditions where gammaherpesvirus infections could be deleterious. Introduction Gammaherpesviruses (γHVs) are important pathogens that are ubiquitous in both human and animal populations. Thus, Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) infect, respectively, up to 90% and 30% of humans worldwide and are associated with several human malignancies such as Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinoma, Kaposi’s sarcoma and post-transplant lymphoproliferative disease.1–3 Efficient control of these infections is therefore of major interest, particularly in immunocompromised people.4 The very high infection prevalence of these infections is due to efficient transmission from carriers to new hosts5 and reflects the fact that these viruses have evolved to coexist with immune responses. Indeed, γHVs establish persistent, productive infections, with virus carriers both making antiviral immune responses that protect against disease and excreting infectious virions. While interrupting transmission is the sine qua non of infection control, strategies based on vaccination have therefore been mainly unsuccessful to reach this goal. For example, vaccination with the EBV gp350, to block virion binding to B cells, failed to reduce infection rates while protecting against infectious mononucleosis.6,7 Other approaches to block transmission are therefore needed. Antiviral drugs are another way to fight viral infections.8 No antiviral drugs are currently licensed for treatment of KSHV or EBV infections. However, several antiherpesvirus drugs have been shown to block these infections in vitro, especially those targeting the viral DNA polymerase or viral DNA replication, such as aciclovir, ganciclovir, foscarnet and cidofovir.8–12 Cidofovir, also called HPMPC {for (S)-1-[3-hydroxy-2-(phosphomethoxy)propyl]cytosine}, is a nucleotide analogue that needs to be phosphorylated by cellular kinases to its diphosphate form to become biologically active and block viral DNA polymerase.13 When evaluated in an in vivo model, cidofovir proved to be very efficacious in protecting mice from γHV-induced disease whereas aciclovir, ganciclovir and foscarnet had little or no effect.12 While cidofovir and related molecules are promising compounds for future clinical development to fight γHV infection, their use in the context of viral transmission has never been investigated. Analysing EBV and KSHV transmission has proved difficult as these viruses have no well-established in vivo infection models. Related animal viruses, such as murid herpesvirus 4 (MuHV-4), provide another way to address such questions.14 Indeed, using luciferase imaging of MuHV-4 infection,15 we recently observed genital MuHV-4 excretion following intranasal inoculation of female mice and transmission to naive males after sexual contact.16 Interestingly, that way of transmission also occurs for the KSHV transmission associated with HIV infection17 and may also apply to EBV.18 In this study, we therefore want to investigate whether cidofovir could block γHV replication in conditions of sexual transmission close to natural settings. Materials and methods Ethics statement and animals Experiments, maintenance and care of mice complied with the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The Committee on the Ethics of Animal Experiments of the University of Liège, Belgium approved the protocol (Permit Number: 1502). All efforts were made to limit animal suffering. BALB/c mice were purchased from ENVIGO Laboratories and were housed under conventional conditions in the Department of Infectious Diseases, University of Liège, Liège, Belgium. Female mice were used at the age of 8 weeks and their weight was 20 ± 1.5 g. Mice were infected intranasally with 104 pfu of MuHV-4 diluted in 30 μL of sterile PBS, under general anaesthesia with isoflurane. Animals were randomly allocated to experimental groups. Antiviral compound Mice were injected subcutaneously at different timepoints according to experimental design with 25 mg/kg/day cidofovir (Gilead Sciences) diluted in PBS. Viral strain All viruses were derived from a MuHV-4 bacterial artificial chromosome (BAC).19 We used a strain expressing luciferase under the control of the M3 promoter that has been described previously (WT Luc+ strain).15 The loxP-flanked BAC/eGFP cassette was removed by subsequent growths of the virus in NIH-3T3-CRE cells until eGFP+ cells were no longer visible.20 Virus stocks were grown in BHK-21 cells cultured in DMEM (Gibco) supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% FCS. Cell culture medium was cleared of cell debris by low-speed centrifugation (1000 g, 30 min). Viruses were then concentrated by high-speed centrifugation (100 000 g, 90 min) and titrated by plaque assay on BHK-21 cells as described elsewhere.21 In vivo imaging Mice were anaesthetized, injected intraperitoneally with luciferin (60 mg/kg) and imaged 10 min later with an IVIS Spectrum (Perkin Elmer). For quantitative comparisons, Living Image software (Perkin Elmer) was used to obtain the average radiance [photons/s/cm2/steradian (p/s/cm2/sr)] over each region of interest. For the reliable comparison of data, an average background measured in the right abdominal region was removed from the measured intensities. Animals were considered as positive if the genital signal was superior to the mean value of 10 uninfected mice + 3 standard deviations (represented by a broken line in the figures). Quantification of anti-MuHV-4-specific antibodies by ELISA MuHV-4 virions pelleted as described above were disrupted by addition to the viral preparation of Triton X-100 to a concentration of 0.1% (v/v). Nunc Maxisorp ELISA plates (Nalgene Nunc) were coated for 18 h at 4°C with Triton X-100-disrupted MuHV-4 virions (106 pfu equivalents/well), blocked in PBS/0.1% Tween 20/3% BSA, and incubated with mouse sera (diluted 1/200 in PBS/0.1% Tween 20). Bound antibodies were detected with alkaline-phosphatase-conjugated goat anti-mouse immunoglobulin (Ig) polyclonal antibody (Sigma–Aldrich). Washing was performed with PBS/0.1% Tween 20. p-Nitrophenylphosphate (Sigma–Aldrich) was used as substrate for colorimetry and absorbance was read at 405 nm using a Benchmark ELISA plate reader (Thermo). Sera were considered as positive when absorbance was superior to the mean value of mock-infected mice + 3 standard deviations (represented by a broken line in the figures). Viral genome quantification MuHV-4 genomic coordinates 40 264–44 385 were amplified as described (ORF25 gene, forward primer 5′-ATGGTATAGCCGCCTTTGTG-3′, reverse primer 5′-ACAAGTGGATGAAGGGTTGC-3′).22 The PCR products were quantified by hybridization with a TaqMan probe (genomic coordinates 43 088–43 117, 5′ 6-FAM-TTCATAAGTTTTATGCTGATCCAGTGGTTG-BHQ1 3′) and converted to genome copies by comparison with a standard curve of cloned plasmid template serially diluted in control mouse spleen DNA and amplified in parallel (iCycler, Bio-Rad). Cellular DNA was quantified by amplifying part of the mouse interstitial retinoid binding protein (IRBP) gene (forward primer 5′-ATCCCTATGTCATCTCCTACYTG-3′, reverse primer 5′-CCRCTGCCTTCCCATGTYTG-3′). The PCR products were quantified with Sybr green (Invitrogen), the copy number was calculated by comparison with a standard curve of cloned mouse IRBP template amplified in parallel. Amplified products were distinguished from paired primers by melting curve analysis and the correct sizes of the products confirmed by electrophoresis and staining with ethidium bromide. Statistical analyses Fisher’s exact test or unpaired, two-sided Student’s t-test was used for comparisons between two sets of data. If multiple groups of data were compared simultaneously, an ANOVA was used and Bonferroni post-tests were used to compare groups. A P value <0.05 was used for statistical significance. Results Daily cidofovir treatment of infected females leads to reduction of MuHV-4 genital excretion In order to test the effect of cidofovir treatment on MuHV-4 transmission, we firstly investigated if cidofovir could reduce MuHV-4 genital excretion from infected female mice. We infected 8-week-old BALB/c female mice with a MuHV-4 strain expressing luciferase under the control of the M3 lytic promoter (WT Luc strain).15 Indeed, using this strain, we previously showed that, following intranasal infection, a luciferase signal is observed in the genital tract of ∼80% of infected female mice, that this signal is associated with viral excretion and that this excretion occurs in most of the cases between days 17 and 21 post-infection.16 Moreover, we showed that excretion of infectious virus in the female genital tract allows sexual transmission to naive males (Figure S1, available as Supplementary data at JAC Online).16 Therefore, cidofovir was given on a daily basis from day 15 post-infection until day 22 post-infection (n = 20) (Figure 1a) while control mice received PBS. Mice were then followed by daily in vivo imaging between days 17 and 22 post-infection in order to detect the MuHV-4 genital signal. While ∼90% of untreated females showed MuHV-4 excretion during this period as expected, only 20% of females excreted the virus after treatment with cidofovir (Figure 1b and c and Figure S2). In contrast, cidofovir treatment did not reduce seroconversion to MuHV-4 (Figure 1d) or the level of latent genomes in spleens (Figure 1e). Altogether, these results show that once-daily cidofovir administration reduces MuHV-4 genital excretion from MuHV-4-infected female mice without reducing latency establishment. Figure 1. View largeDownload slide Effect of once-daily treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu). They were then injected daily with cidofovir (25 mg/kg, subcutaneously) from day 15 to day 22 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 10 randomly subsampled mice from groups of 20 mice, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). ***P < 0.001 and *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 1. View largeDownload slide Effect of once-daily treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu). They were then injected daily with cidofovir (25 mg/kg, subcutaneously) from day 15 to day 22 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 10 randomly subsampled mice from groups of 20 mice, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). ***P < 0.001 and *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. A single cidofovir treatment of infected females does not reduce MuHV-4 genital excretion Daily cidofovir treatment is usually used in mice.12 However, in humans a weekly administration is sufficient and allows the reduction of associated adverse effects. Therefore, in order to address the effect of a single cidofovir treatment, we repeated the same experiment by replacing the daily injections by a single injection of cidofovir performed at day 14 post-infection as genital excretion usually starts from day 17 (Figure 2a).16 At 19 days post-infection, only 2 females of 20 (10%) showed genital excretion after treatment while 8 females of 20 (40%) had displayed the genital signal in the control group (P < 0.05 by χ2 test; Figure 2b). However, at 21 days post-infection, there was no difference between the two groups (P > 0.05 by χ2 test) (Figure 2b and c and Figure S3). Again, cidofovir treatment did not reduce seroconversion to MuHV-4 (Figure 2d) or the level of latent genomes in spleens (Figure 2e). Altogether, these results show that a single cidofovir administration at day 14 post-infection can reduce MuHV-4 genital excretion from infected female mice until day 19 post-infection but is not sufficient to block it for the long term as seen at day 21 post-infection. Figure 2. View largeDownload slide Effect of a single treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Mice were infected intranasally with MuHV-4 (104 pfu). They were then injected with cidofovir (25 mg/kg, subcutaneously) at day 14 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 2. View largeDownload slide Effect of a single treatment with cidofovir on MuHV-4 genital excretion by female mice. (a) Experimental scheme. Mice were infected intranasally with MuHV-4 (104 pfu). They were then injected with cidofovir (25 mg/kg, subcutaneously) at day 14 post-infection and were imaged by IVIS from day 17 to day 22 post-infection. (b) Kaplan–Meier plot of time of occurrence of the genital signal among MuHV-4-infected mice treated with PBS or cidofovir (n = 20 mice per group). (c) Maximal intensity of the genital luciferase signal among groups (n = 20 mice per group). (d) Anti-MuHV-4 serology at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell at euthanasia (n = 20 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Once-daily cidofovir treatment of naive males prevents MuHV-4 replication and latency establishment While antiviral treatments could be used to reduce excretion and transmission from infected subjects, they could also prevent replication in naive ones. We therefore investigated the potency of a daily cidofovir treatment to block MuHV-4 replication and host colonization in naive males after sexual contact with excreting females. Briefly, we infected female BALB/c mice with the MuHV-4 WT Luc+ strain and followed them by in vivo imaging in order to detect MuHV-4 genital excretion. When the genital signal was observed (between days 17 and 21 depending on the females), excreting females were mated with naive males, treated or not with cidofovir from days 15 to 24 (day 0 was defined as the time of female intranasal infection). At day 24, males were separated from females and were followed by in vivo imaging to monitor viral replication (Figure 3a–c). Strikingly, no males displayed the genital signal after daily treatment with cidofovir (n = 0/15) while a transmission rate of 35% was observed in the control group (Figure 3b and c). Males of both groups were euthanized at day 41. In contrast with males from the PBS-treated group, none of the males from the cidofovir-treated group displayed seroconversion to MuHV-4 infection (Figure 3d) or latency establishment in the spleen (Figure 3e). Altogether, these results show that once-daily cidofovir administration to naive males (started 2 days before putting males in contact with genitally excreting females and maintained for the whole contact period with infected females) is sufficient to prevent replication and host colonization following sexual contact with MuHV-4-excreting females. Figure 3. View largeDownload slide Preventive daily treatment of males with cidofovir blocks MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not daily injection of cidofovir (25 mg/kg, subcutaneously) from day 15 to day 24 according to the experimental scheme. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal. Figure 3. View largeDownload slide Preventive daily treatment of males with cidofovir blocks MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not daily injection of cidofovir (25 mg/kg, subcutaneously) from day 15 to day 24 according to the experimental scheme. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal. A single preventive cidofovir treatment of naive males 2 to 6 days before sexual contact is not sufficient to prevent MuHV-4 sexual infection We then used a single preventive injection of cidofovir to naive males in order to test if it was sufficient to prevent viral replication and host colonization. Briefly, we repeated the same experimental design as described above except that males received a single injection of cidofovir at day 15 (day 0 was defined as the time of female intranasal infection) (Figure 4a). In vivo imaging did not highlight any significant difference between cidofovir- and PBS-treated groups (Figure 4b and c). Accordingly, similar rates of seroconversion (Figure 4d) or of latency establishment in the spleen (Figure 4e) were observed in both groups. Altogether, these results show that a single cidofovir administration to naive males 2 to 6 days before sexual contact with females excreting the virus in the genital tract is not sufficient to prevent viral replication and latency establishment in males. Figure 4. View largeDownload slide A single preventive treatment of males with cidofovir fails to block MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not a single injection of cidofovir (25 mg/kg, subcutaneously) at day 15 of the experiment. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). The broken line represents the mean value of mock-infected mice + 3 standard deviations. (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 4. View largeDownload slide A single preventive treatment of males with cidofovir fails to block MuHV-4 sexual transmission. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males, which received or not a single injection of cidofovir (25 mg/kg, subcutaneously) at day 15 of the experiment. Males were followed by IVIS from day 24 to day 31. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 15 mice per group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 15 mice per group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 5 mice). The broken line represents the mean value of mock-infected mice + 3 standard deviations. (e) Viral genomes per spleen cell of males at euthanasia (n = 15 mice per group, except the mock-infected group, which was made of 3 mice). Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Influence of a single post-exposure cidofovir treatment of naive males on their infection through the sexual route Finally, we tested if a single post-exposure treatment of exposed naive males could be sufficient to block systemic infection and latency establishment. Briefly, we repeated the same experimental scheme as the one described above, but instead of treating naive males 2 to 6 days before sexual contact with MuHV-4-excreting females, males received a single injection of cidofovir 24 h after contact with infected females (Figure 5a). Interestingly, only 1 of 32 (3%) treated males displayed an MuHV-4-associated genital signal, whereas 6 of 23 (26%) became infected in the control group (Figure 5b and c). Accordingly, seroconversion to MuHV-4 (Figure 5d) was also reduced in the cidofovir-treated group in comparison with the PBS-treated group. Although latency establishment (Figure 5e) tended also to be reduced in the cidofovir-treated group this was, however, not statistically different from the PBS-treated group (P = 0.056 by Fisher’s exact test). Altogether, these results suggest that, following sexual contact with MuHV-4-excreting females, a single post-exposure cidofovir administration to naive males reduces MuHV-4 replication and seroconversion against the virus. Figure 5. View largeDownload slide Post-exposure treatment of males with cidofovir can prevent establishment of MuHV-4 infection. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males. Males were treated 24 h post-contact with excreting females with a single injection of cidofovir (25 mg/kg, subcutaneously) or with PBS in the control group. Males were separated from females 48 h post-contact and were followed by in vivo imaging to detect virus replication. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 23 mice in PBS group, 32 mice in cidofovir group and 3 mice in the mock-infected group). (e) Viral genomes per spleen cell of males at euthanasia (n = 23 mice in the PBS group and 32 mice in the cidofovir group and 3 mice in the mock-infected group). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Figure 5. View largeDownload slide Post-exposure treatment of males with cidofovir can prevent establishment of MuHV-4 infection. (a) Experimental scheme. Female mice were infected intranasally with MuHV-4 (104 pfu) and were imaged from day 17 to day 21 post-infection. When the genital signal occurred, females were mated with naive males. Males were treated 24 h post-contact with excreting females with a single injection of cidofovir (25 mg/kg, subcutaneously) or with PBS in the control group. Males were separated from females 48 h post-contact and were followed by in vivo imaging to detect virus replication. (b) Kaplan–Meier plot of time of occurrence of the genital signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (c) Maximal intensity of the genital luciferase signal among males treated with PBS or cidofovir (n = 23 mice in the PBS group and 32 mice in the cidofovir group). (d) Anti-MuHV-4 serology in males at euthanasia (n = 23 mice in PBS group, 32 mice in cidofovir group and 3 mice in the mock-infected group). (e) Viral genomes per spleen cell of males at euthanasia (n = 23 mice in the PBS group and 32 mice in the cidofovir group and 3 mice in the mock-infected group). *P < 0.05. Avg, average; CDV, cidofovir; d, day; in, intranasal; ns, not significant. Discussion γHVs are highly prevalent animal and human viruses that are associated with numerous pathological conditions. In immunocompromised people, EBV and KSHV cause cancers and other disorders such as Burkitt’s lymphoma, Kaposi’s sarcoma or Castleman disease.4,23 Moreover, primary infection of young adults with EBV causes infectious mononucleosis, which represents major health costs.24 EBV-induced infectious mononucleosis also increases the risk of developing multiple sclerosis to a similar degree as the strongest genetic risk factor.25 Preventing γHV infection in some defined populations is therefore a major public health challenge. Over the last 50 years, several antivirals have been developed to treat different infectious conditions.8 In the case of herpesviruses, viral DNA polymerase is a key target for the development of potent inhibitors. Thus, acyclic nucleoside phosphonates, such as cidofovir, are important broad-spectrum antiviral agents, which emerged as the most potent anti-γHV drugs.13 In this study, we tested the capacity of cidofovir to block γHV replication and host colonization in the context of sexual transmission.16 Briefly, we showed that once-daily treatment of either infected females or naive males is sufficient to significantly reduce viral excretion from infected females and viral replication and latency establishment in naive males (Figures 1 and 3). In particular, treatment of the uninfected males not only reduced initial viral replication in the male penis to levels undetectable by the IVIS system but also protected these mice from seroconversion to MuHV-4 and from latency establishment in the spleen (Figure 3). In contrast, cidofovir treatment of excreting females did not affect the levels of virus latency in the spleen but was sufficient to reduce virus excretion anyway (Figure 1). This approach could potentially be translated to human viruses as MuHV-4, EBV and KSHV display similar sensitivity to cidofovir even if measures of EC50 have not been performed in the same conditions.13 Moreover, a similar approach had already been shown to be successful in the case of sexual transmission of herpes simplex virus 2 (HSV-2) in human where once-daily valaciclovir treatment of the infected partner reduced the risk of HSV-2 transmission between heterosexual, monogamous couples discordant for HSV-2 infection.26,27 Such an approach could also apply to animals in specific conditions. For example, some γHVs, such as those associated with malignant catarrhal fever, cause recurrent problems in zoos where persistently infected and susceptible animals are housed in close contact.28,29 As these viruses are frequently transmitted upon parturition, treatment of persistently infected mothers around these periods could likely reduce virus excretion and help to protect neighbouring susceptible species. Of course, the sensitivity of these viruses to cidofovir will have to be determined. One of the major pitfalls of nucleoside analogues resides in their toxicity in the long term. We therefore tested the capacity of a single cidofovir injection (either to the infected or to the naive partner) before sexual contact to block MuHV-4 transmission (Figures 2 and 4). Neither of these two approaches were revealed as successful at the end of the experimental protocol while a reduction in genital excretion was observed until day 19 (P < 0.05 by χ2 test) in infected females treated with cidofovir (Figure 2). Therefore, although preventive cidofovir treatment has the potential to efficiently block γHV transmission, the establishment of protocols based on a single injection will be difficult and is likely directly linked with the in vivo t½ of the drug. Cidofovir has a long intracellular t½ of about 15 to 65 h and a single administration of 30 mg/kg (similar to the 25 mg/kg used in our protocols) protects mice from poxvirus infections 3 days later but not 5 days later.30 Similarly, we observed that a single cidofovir injection is sufficient to block excretion of MuHV-4 from infected females for 5 days (Figure 2; between days 14 and 19 post-infection). A single administration to naive males also delayed their infection (Figure 4). Therefore, usage of cidofovir as a preventive treatment to block sexual transmission of γHV will require a frequency of administration that allows maintenance of efficient concentration of the drug and its metabolites in cells but does not induce side effects such as nephrotoxicity. Alternatively, these objectives could be reached by the use of brincidofovir, a novel oral lipid-conjugated nucleotide analogue, which is converted into cidofovir in the target cells and has shown enhanced in vitro activity against some herpesviruses while displaying reduced toxicity in vivo.31 While preventive pre-exposure administration of antivirals is difficult to implement to block transmission, post-exposure treatment could display obvious advantages. Interestingly, we showed here that a single cidofovir administration to males 24 h post-sexual contact with MuHV-4-excreting females significantly reduced virus replication in the male genital area and seroconversion at euthanasia suggesting that a single cidofovir treatment could be efficient (Figure 5). However, reduction of viral genome copy numbers in the spleens was not statistically significant (Figure 5e) suggesting a reduced replication at the entry site but host colonization anyway. The feasibility of such an approach in the field could depend on the major mode of transmission of γHVs in those conditions. Endemic EBV and KSHV infections are maintained chiefly by carriers shedding virus into their saliva. In that context, single post-exposure administration is likely not feasible. However, numerous reports have shown that γHVs could be sexually transmitted in some circumstances.32 This was seen clearly for the KSHV transmission associated with HIV infection17 and may apply also to EBV.18,33 In such a context, post-exposure treatment could probably be an option. Altogether, we have shown that cidofovir could be used to efficiently block transmission of a γHV in conditions close to what happens in real life. Especially, it appeared that post-exposure treatment could be a very interesting approach in some epidemiological conditions regarding its efficacy and its ease of implementation. Acknowledgements We thank A. Vanderplasschen and L. Willems for helpful discussions and the technician and administrative team of the lab for very helpful assistance. Funding This work was supported by the VIR-IMPRINT ARC grant of the University of Liège and the BELVIR Interuniversity Attraction Pole (IAP). C. Z. is a research fellow of the ‘Fonds de la Recherche Scientifique - Fonds National Belge de la Recherche Scientifique’ (F.R.S. - FNRS). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Transparency declarations None to declare. Supplementary data Figures S1 to S3 are available as Supplementary data at JAC Online. References 1 Henle G , Henle W , Clifford P et al. Antibodies to Epstein-Barr virus in Burkitt's lymphoma and control groups . J Natl Cancer Inst 1969 ; 43 : 1147 – 57 . Google Scholar PubMed 2 Thorley-Lawson DA , Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas . N Engl J Med 2004 ; 350 : 1328 – 37 . Google Scholar CrossRef Search ADS PubMed 3 Verma SC , Robertson ES. Molecular biology and pathogenesis of Kaposi sarcoma-associated herpesvirus . FEMS Microbiol Lett 2003 ; 222 : 155 – 63 . Google Scholar CrossRef Search ADS PubMed 4 Cesarman E. Gammaherpesviruses and lymphoproliferative disorders . Annu Rev Pathol 2014 ; 9 : 349 – 72 . Google Scholar CrossRef Search ADS PubMed 5 Virgin HW , Wherry EJ , Ahmed R. Redefining chronic viral infection . Cell 2009 ; 138 : 30 – 50 . Google Scholar CrossRef Search ADS PubMed 6 Moutschen M , Léonard P , Sokal EM et al. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults . Vaccine 2007 ; 25 : 4697 – 705 . Google Scholar CrossRef Search ADS PubMed 7 Sokal EM , Hoppenbrouwers K , Vandermeulen C et al. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults . J Infect Dis 2007 ; 196 : 1749 – 53 . Google Scholar CrossRef Search ADS PubMed 8 De Clercq E , Li G. Approved antiviral drugs over the past 50 years . Clin Microbiol Rev 2016 ; 29 : 695 – 747 . Google Scholar CrossRef Search ADS PubMed 9 Friedrichs C , Neyts J , Gaspar G et al. Evaluation of antiviral activity against human herpesvirus 8 (HHV-8) and Epstein-Barr virus (EBV) by a quantitative real-time PCR assay . Antivir Res 2004 ; 62 : 121 – 3 . Google Scholar CrossRef Search ADS PubMed 10 Lin JC , De Clercq E , Pagano JS. Inhibitory effects of acyclic nucleoside phosphonate analogs, including (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, on Epstein-Barr virus replication . Antimicrob Agents Chemother 1991 ; 35 : 2440 – 3 . Google Scholar CrossRef Search ADS PubMed 11 Meerbach A , Holý A , Wutzler P et al. Inhibitory effects of novel nucleoside and nucleotide analogues on Epstein-Barr virus replication . Antivir Chem Chemother 1998 ; 9 : 275 – 82 . Google Scholar CrossRef Search ADS PubMed 12 Neyts J , De Clercq E. In vitro and in vivo inhibition of murine gamma herpesvirus 68 replication by selected antiviral agents . Antimicrob Agents Chemother 1998 ; 42 : 170 – 2 . Google Scholar PubMed 13 Coen N , Duraffour S , Naesens L et al. Evaluation of novel acyclic nucleoside phosphonates against human and animal gammaherpesviruses revealed an altered metabolism of cyclic prodrugs upon Epstein-Barr virus reactivation in P3HR-1 cells . J Virol 2013 ; 87 : 12422 – 32 . Google Scholar CrossRef Search ADS PubMed 14 Barton E , Mandal P , Speck SH. Pathogenesis and host control of gammaherpesviruses: lessons from the mouse . Annu Rev Immunol 2011 ; 29 : 351 – 97 . Google Scholar CrossRef Search ADS PubMed 15 Milho R , Smith CM , Marques S et al. In vivo imaging of murid herpesvirus-4 infection . J Gen Virol 2009 ; 90 : 21 – 32 . Google Scholar CrossRef Search ADS PubMed 16 François S , Vidick S , Sarlet M et al. Illumination of murine gammaherpesvirus-68 cycle reveals a sexual transmission route from females to males in laboratory mice . PLoS Pathog 2013 ; 9 : e1003292. Google Scholar CrossRef Search ADS PubMed 17 Schulz TF. Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8): epidemiology and pathogenesis . J Antimicrob Chemother 2000 ; 45 Suppl T3: 15 – 27 . Google Scholar CrossRef Search ADS PubMed 18 Dunmire SK , Grimm JM , Schmeling DO et al. The incubation period of primary Epstein-Barr virus infection: viral dynamics and immunologic events . PLoS Pathog 2015 ; 11 : e1005286. Google Scholar CrossRef Search ADS PubMed 19 Adler H , Messerle M , Wagner M et al. Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome . J Virol 2000 ; 74 : 6964 – 74 . Google Scholar CrossRef Search ADS PubMed 20 Adler H , Messerle M , Koszinowski UH. Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences . J Virol 2001 ; 75 : 5692 – 6 . Google Scholar CrossRef Search ADS PubMed 21 Gillet L , May JS , Stevenson PG. In vivo importance of heparan sulfate-binding glycoproteins for murid herpesvirus-4 infection . J Gen Virol 2009 ; 90 : 602 – 13 . Google Scholar CrossRef Search ADS PubMed 22 Latif MB , Machiels B , Xiao X et al. Deletion of murid herpesvirus 4 ORF63 affects the trafficking of incoming capsids toward the nucleus . J Virol 2015 ; 90 : 2455 – 72 . Google Scholar CrossRef Search ADS PubMed 23 Jha HC , Banerjee S , Robertson ES. The role of gammaherpesviruses in cancer pathogenesis . Pathogens 2016 ; 5 : 18. Google Scholar CrossRef Search ADS 24 Taylor GS , Long HM , Brooks JM et al. The immunology of Epstein-Barr virus-induced disease . Annu Rev Immunol 2015 ; 33 : 787 – 821 . Google Scholar CrossRef Search ADS PubMed 25 Geginat J , Paroni M , Pagani M et al. The enigmatic role of viruses in multiple sclerosis: molecular mimicry or disturbed immune surveillance? Trends Immunol 2017 ; 38 : 498 – 512 . Google Scholar CrossRef Search ADS PubMed 26 Corey L , Wald A , Patel R et al. Once-daily valacyclovir to reduce the risk of transmission of genital herpes . N Engl J Med 2004 ; 350 : 11 – 20 . Google Scholar CrossRef Search ADS PubMed 27 Crumpacker CS. Use of antiviral drugs to prevent herpesvirus transmission . N Engl J Med 2004 ; 350 : 67 – 8 . Google Scholar CrossRef Search ADS PubMed 28 Cooley AJ , Taus NS , Li H. Development of a management program for a mixed species wildlife park following an occurrence of malignant catarrhal fever . J Zoo Wildl Med 2008 ; 39 : 380 – 5 . Google Scholar CrossRef Search ADS PubMed 29 Frontoso R , Autorino GL , Friedrich KG et al. An acute multispecies episode of sheep-associated malignant catarrhal fever in captive wild animals in an Italian zoo . Transbound Emerg Dis 2016 ; 63 : 621 – 7 . Google Scholar CrossRef Search ADS PubMed 30 Quenelle DC , Collins DJ , Kern ER. Efficacy of multiple- or single-dose cidofovir against vaccinia and cowpox virus infections in mice . Antimicrob Agents Chemother 2003 ; 47 : 3275 – 80 . Google Scholar CrossRef Search ADS PubMed 31 Quenelle DC , Lampert B , Collins DJ et al. Efficacy of CMX001 against herpes simplex virus infections in mice and correlations with drug distribution studies . J Infect Dis 2010 ; 202 : 1492 – 9 . Google Scholar CrossRef Search ADS PubMed 32 Davison AJ. Evolution of sexually transmitted and sexually transmissible human herpesviruses . Ann N Y Acad Sci 2011 ; 1230 : E37 – 49 . Google Scholar CrossRef Search ADS PubMed 33 Higgins CD , Swerdlow AJ , Macsween KF et al. A study of risk factors for acquisition of Epstein-Barr virus and its subtypes . J Infect Dis 2007 ; 195 : 474 – 82 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 16, 2018

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