Introduction
Epstein–Barr virus (EBV), a member of the γ-Herpesvirinae subfamily, is a ubiquitous human pathogen infecting >95% of the global adult population. Primary infection is often asymptomatic, but can also present with clinical infectious mononucleosis, which is characterized by fever, pharyngitis, and atypical lymphocytosis ( Kuri et al., 2020 ; Zanella et al., 2019 ). Beyond acute illness, EBV establishes lifelong persistence and is associated with a spectrum of malignancies, including Burkitt’s and Hodgkin’s lymphomas, nasopharyngeal carcinoma, gastric cancer, and post-transplant lymphoproliferative disorders, particularly in immunocompromised patients ( Chinna et al., 2023 ; Kutok and Wang, 2006 ). Other important disease associations of EBV are known, with EBV being necessary but not sufficient for the development of multiple sclerosis (MS) ( Lanz et al., 2022 ; Thomas et al., 2026 ).
Despite the significant clinical burden of EBV, no specific, direct-acting antiviral drug has been approved for the treatment of EBV infections to date. While several agents have demonstrated potent inhibition of EBV replication in vitro, they have shown limited efficacy in clinical trials ( Andrei et al., 2019 ; Chakravorty et al., 2022 ; Lv et al., 2024 ; Pagano et al., 2018 ). Therefore, the rigorous screening and development of novel antiviral compounds remain a critical priority, particularly in reducing the clinical impact on tumor incidence and potentially on MS ( Li et al., 2025 ). However, the evaluation of such agents is significantly hindered by the strict host tropism of human γ-herpesviruses, which limits the use of EBV in conventional small animal models. This challenge is further exacerbated by the limitation of in vitro cell culture systems permissive for efficient EBV productive replication, complicating the search for potent antivirals. Situations involving EBV infection and Kaposi sarcoma-associated herpesvirus (KSHV) infection are very similar to clinically problematic situations and urgently require the development of novel therapeutic options. Murine γ-herpesvirus 68 (MHV-68) has emerged as a robust surrogate model, that can be used to assess EBV or KSHV antivirals prior to human studies ( Briestenská and Briestenská, 2020 ; Simas and Efstathiou, 1998 ; Stewart, 1999 ). The MHV-68 is a naturally occurring rodent pathogen ( Blaškovič et al., 1980 ) that shares significant genetic colinearity and fundamental biological properties with human γ-herpesviruses ( Efstathiou et al., 1990 ; Mackett et al., 1997 ; Virgin et al., 1997 ). These shared features include the capacity to induce an infectious mononucleosis-like syndrome associated with splenomegaly ( Doherty et al., 1997 ; Flaño et al., 2002 ; Tripp et al., 1997 ; Usherwood et al., 1996 ), followed by the establishment of lifelong latency in B lymphocytes ( Sunil-Chandra et al., 1992b ; Weck et al., 1999 ). Furthermore, similar to its human counterparts, long-term MHV-68 infection is linked to the development of various lymphoproliferative diseases ( Sunil-Chandra et al., 1994 ).
The MHV-68 serves as an excellent tool, due to its high permissivity in model animals and cell culture, which allows for the direct assessment of antiviral effects in de novo lytic infection ( Cho et al., 2013 ; Mistríková and Briestenská, 2020 ). In contrast to human γ-herpesviruses, MHV-68 readily replicates in vivo in the lung epithelium of laboratory mice ( Blaškovič et al., 1984 ; Rajčáni et al., 1985 ; Sunil-Chandra et al., 1992a ) as well as in vitro in various cell lines derived from different animal species producing high titers of infectious virus ( Svobodová et al., 1982 ). Consequently, the use of recombinant reporter viruses to monitor the replication of various viruses, including MHV-68, has increased, providing a faster, more objective platform for antiviral screening ( Briestenska et al., 2019 ; Cho et al., 2013 ; Obergfäll et al., 2024 ). We used a recently generated recombinant MHV-68 that expresses firefly luciferase under the control of the viral lytic M3 promoter ( Hwang et al., 2008 ) to assess antiviral drug efficacy . The M3 promoter is highly responsive to the viral replication and transcription activator (Rta), making its activity a direct indicator of the lytic replication cycle ( Martinez-Guzman et al., 2003 ). This reporter virus was constructed using bacterial artificial chromosome (BAC)-based two-step allelic exchange ( Hwang et al., 2008 ; Smith and Enquist, 1999 ; Song et al., 2005 ; Wu et al., 2011 ), a method that ensures high genetic stability and allows for precise genomic modifications.
Crucially, multistep growth curve analysis previously demonstrated that the recombinant virus exhibits replication kinetics nearly identical to those of the wild-type MHV-68. Additionally, a strong correlation between luciferase activity and viral titers was confirmed by regression analysis, demonstrating that the luminescent signal accurately reflects viral lytic replication ( Briestenska et al., 2019 ; Obergfäll et al., 2025 , 2024 ). The present study deepens our understanding of MHV-68 infection in various cell types, as a model for assessing antiviral drug sensitivity, and identifying combinatorial drug synergies. Data are discussed in the context of γ-herpesvirus infections.
Materials and methods
Cell culture and virus
Primary human and murine fibroblasts (HFFs and MEFs; own repository), COS-7 and Vero 76 cells (ATCC, Manassas, VA, USA) were maintained at 37 °C, 5% CO 2 and 80% humidity. As cultivation medium, Eagle’s Minimal Essential Medium was used for HFFs (MEM 21,090,055, Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific) was used for COS-7, Vero 76 cells and MEFs, each supplemented with 10% fetal bovine serum (FBS, Capricorn, Ebsdorfergrund, Germany), 1 × GlutaMAX™ (Thermo Fisher Scientific, Waltham, MA, USA), and 10 μg/mL gentamicin (Thermo Fisher Scientific, Waltham, MA, USA). All cells were regularly monitored for the absence of mycoplasma contamination using a Lonza™ Mycoalert™ kit (Thermo Fisher Scientific,Waltham, MA, USA). The recombinant MHV-68 strain myodes was used in this study containing an expression cassette for firefly luciferase (Luc), which had been generated as described below ( Briestenska et al., 2019 ).
Antiviral compounds
The small molecule compounds BI-1347, CCT-251,921, MSC-2530,818 (all targeting the host kinase CDK8) brincidofovir (BCV), aciclovir (ACV), ganciclovir (GCV), and foscarnet (FOS) (all targeting the viral DNA polymerase) letermovir (LMV, targeting the viral terminase), and maribavir (MBV, targeting the viral protein kinase) were obtained from a commercial source (MedChemExpress, Monmouth Junction, NJ, USA). We further used the NEC inhibitors merbromin (MBM, Sigma Aldrich, St. Louis, MO, USA), GK2 and ibrutinib (IBR, both targeting the viral nuclear egress complex, MedChemExpress, Monmouth Junction, NJ, USA). These small molecules could be categorized in (i) approved drugs (ACV [against herpes simplex virus/HSV and varicella zoster virus/VZV, also applied as high-dose prophylaxis against HCMV], FOS [primarily HCMV and ACV-resistant HSV], GCV [primarily HCMV and HSV keratoconjunctivitis], LMV [HCMV], MBV [HCMV], IBR [cancer]), (ii) developmental/clinically relevant candidates (BCV, BI, MSC, CCT), and (iii) experimental compounds (GK2, MBM). Moreover, the new experimental reference inhibitor of EBV replication, tenofovir alafenamide, was added to the study (TAF, MedChemExpress, Monmouth Junction, NJ, USA). Stock solutions of all compounds were prepared at a 10 mM concentration in sterile DMSO (Sigma Aldrich, St. Louis, MO, USA), and aliquots were stored at −20 °C.
Construction and propagation of recombinant MHV-68-Luc
The MHV-68-Luc was engineered by inserting a firefly luciferase cassette, driven by the viral lytic M3 promoter, into the left end of the viral genome ( Hwang et al., 2008 ). The luciferase expression cassette was introduced into the viral genome using two-step allelic exchange as previously described ( Hwang et al., 2008 ; Smith and Enquist, 1999 ; Song et al., 2005 ; Wu et al., 2011 ). Briefly, this procedure employed Escherichia coli GS500 cells harboring the MHV-68 BAC and the GS111 strain carrying the pGS284 shuttle vector containing the luciferase construct. Specifically, the luciferase cassette was integrated into the BAC-cloned MHV-68 genome through RecA-mediated homologous recombination, forming a cointegrate. This was followed by a second recombination step to resolve the cointegrate and excise the vector backbone, leaving the reporter cassette within the viral genome. The integrity of the recombinant BAC was confirmed by PCR, restriction enzyme digestion, and sequencing. To reconstitute the infectious virus, the recombinant BAC DNA was cotransfected with a Cre recombinase-expressing plasmid into 293T cells, resulting in the excision of the BAC vector sequence. Following the observation of a viral cytopathic effect, the samples underwent three rounds of plaque purification. The resulting clones were screened by PCR to select for those that had successfully excised the bacterial vector sequences. The final recombinant virus was propagated in NIH3T3 cells and validated by sequencing to confirm the site-specific integration and integrity of the luciferase reporter ( Briestenska et al., 2019 ).
Neutral Red (NR) uptake cell viability assay
Uninfected cells (mock) were seeded at the same density used for the antiviral replication assays and treated with either the test compounds or a solvent control. The durations of compound treatment were adjusted to match the periods in the antiviral assays as follows: HFFs, 7 d; MEF, 7 d; Vero and COS-7 cells, 4 d. At the end of the treatment period, NR solution was added to a final concentration of 40 μg/mL, and cells were incubated for 2–4 h at 37 °C. The NR-containing supernatant was removed, and the cells were destained with a solution of 50% ethanol, 1% acetic acid, and 49% distilled water, before fluorescence was measured at 560/630 nm using the Victor X4 multilabel counter (PerkinElmer, Waltham, MA, USA).
Virus infection of cultured cells and quantitative readouts of MHV-68-Luc replication
The reporter virus MHV-68-Luc was allowed to multiply in Vero cells to produce a high-titer virus stock (2.91 × 10 7 PFU/mL, with viral plaque numbers determined in a 24-well setting at 4 days post-infection/d p.i.). A comparison of virus propagation showed that Vero cells (probably due to the lack of interferon restriction) supported productive MHV-68-Luc replication more efficiently than MEFs. Stock virus was then used to assess antiviral drugs in a 96-well plate setting, as previously described ( Obergfäll et al., 2025 , 2024 ). Briefly, 3 × 10⁴ cells were cultivated per 96-well plate and used for infection with MHV-68-Luc at an MOI of 0.001 (i.e., 0.001 PFU/cell), which corresponds to 75% of the maximum luciferase signal level at 4 days post-infection (d p.i.) for Vero cells and MEFs. For HFFs, the MOI was 0.2 (i.e., 0.2 PFU/cell), which corresponds to 75% of the maximum luciferase signal level at 7 d.p.i. MHV-68-Luc-infected cells were incubated for the cell-type-specific durations, lysed with 100 μl of lysis buffer per well, and applied to a reporter luminescence analysis with 50 μL of sample volume (0.1 M KH₂PO₄, 15 mM MgSO₄, 5 mM ATP, and 1 mM d -luciferin) in the Victor X4 multilabel counter. In the context of assessing antiviral drugs, the Luc reporter proved highly valuable in quantitatively evaluating the inhibition of viral replication by drugs. Control settings largely ruled out a falsely positive interpretation of anti-MHV-68-Luc drug activity based on a putative direct inhibitory effect against the M3 promoter driving the reporter. In particular, control panels showing blocking of extracellular virus release were helpful in indicating that the measured antiviral effects were not limited exclusively to intracellular reduction of the Luc reporter activity (see Fig. 1 and further data in Obergfäll et al., 2025 ).
Comparative quantitative readouts of HCMV and EBV replication
The quantitative readouts used for the measurement of HCMV replication ( Kicuntod et al., 2021 ; Obergfäll et al., 2024 ; Tillmanns et al., 2023 ; Wild et al., 2022 ), EBV replication ( Kögler et al., 2026 ; Yu et al., 2024 ), and antiviral drug assessment have been reported before. In brief, HCMV AD169-GFP (expressing the green fluorescent protein reporter) was used for the infection (multiplicity of 25% GFP-FU/7 d) of HFFs cultivated in a 96-well plate format. After 90 min of viral adsorption, the inocula were removed, media were refreshed, and cells were further cultivated under conditions of inhibitor treatment at 37 °C. At 7 d p.i., cells were fixed and subjected to an automated GFP fluorometry using the Perkin Elmer Victor X4 Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). For the assessment of EBV replication, the EBV-positive immortalized B cell line P3HR-1 was used as a producer culture, which was chemically induced for lytic EBV production (40 ng/mL of 12-O-tetradecanoylphorbol-13-acetate/TPA). Hereby, P3HR-1 cells were seeded in 96-well plates and were simultaneously induced with TPA (in the absence of a coinducer like sodium butyrate, to minimize cytotoxic effects) and treated with antiviral compounds for 10 d, before cell culture medium samples were subjected to quantitative assessment of EBV lytic productivity and release. Infectious media samples were used directly, without pelleting EBV virions, to perform proteinase K digestion for the specific extraction of EBV DNA, followed by the EBV/BGLF5-specific quantitative polymerase chain-reaction (qPCR) analysis. EBV qPCR was performed with 20 μl qPCR reaction mixture, containing 5 μl of either the EBV genomic test sample or the standard DNA solution. Additional components were 10 μL 2x TaqMan PCR Mastermix (Applied Biosystems, Waltham, MA, USA), 375 nM of each primer (5′-TGA CCT CTT GCA TGG CCT CT-3′ and 5′-CCT CTT TTC CAA GTC AGA ATT TGA C-3′) and 20 nM probe (5′-CCA TCT ACC CAT CCT ACA CTG CGC TTT ACA-3′), which was labelled with 6-carboxyfluorescein reporter dye and 6- carboxytetramethylrhodamine quencher dye. The thermal cycling conditions consisted of two initial steps of 2 min at 50 °C and 10 min at 95 °C followed by 40 amplification cycles (15 s at 95 °C, 1 min at 60 °C). Reactions were performed with an ABI Prism 7700 sequence detector (Applied Biosystems). For more information on the methods used in EBV-specific assessments, refer to the published protocols. ( Kögler et al., 2026 ; Obergfäll et al., 2025 ; Tillmanns et al., 2025 ; Yu et al., 2024 ).
Determination of drug interactions in combined antiviral treatment assays (Loewe additivity fixed-dose assay and Bliss independence checkerboard assay)
Drug–drug interactions were evaluated using two complementary approaches. In the Loewe additivity fixed-dose assay, an adapted HCMV luciferase-based replication system was applied to analyze serial dilution series of each compound alone and in combination, typically starting at a 5–10 × concentration of the virus-specific EC₅₀. Antiviral efficacy and combination indices (CI) were calculated using CompuSyn software (Version 1.0) according to ( Chou, 2005 ). A weighted CI (CI wt ) was determined using the formula 1 × CI₅₀ + 2 × CI₇₅ + 3 × CI₉₀ + 4 × CI₉₅)/10, and interactions were classified as synergistic (CI wt < 0.8), additive (0.8–1.2), or antagonistic (>1.2). To confirm the observed combinatorial drug effects, the Bliss independence checkerboard assay was performed in parallel, using an 8 × 8 drug concentration matrix in 96-well plates. Vero cells were infected with MHV-68-Luc at an MOI of 0.001 (corresponding to 75% of max. Luc activity at 4 d p.i.) or remained uninfected (mock). This MOI has been determined as the optimal level of infection that allows both, a most sensitive reporter-specific detection of virus replication (75% Luc activity) and quantitative inhibition of virus replication by antiviral drugs ( Obergfäll et al., 2025 ). Following a 90-minute virus adsorption period, the inocula were replaced with media containing various combinations of drugs, solvent control, or plain medium. All infections were conducted in biological triplicates. At 4 d p.i., the cells were lysed (100 µL/well), incubated for 15 min at room temperature, and then supplemented with 50 µL of luciferase buffer. Luminescence was subsequently measured using a Victor X4 microplate reader. The data were analyzed using MacSynergy II software ( Prichard et al., 1992 ), and were expressed as synergy volume plots within the 95% confidence interval.
Results and discussion
Establishment of the MHV-68 replication model, based on the quantitative luciferase reporter readout, in murine and non-murine host cell types
The cultured cell-based replication systems of γ-herpesviruses are often limited because they primarily establish latent infections, and may require an induction of viral lytic replication (e.g. with TPA or butyric acid in EBV-positive lymphoblastoid B cell systems) ( Gao et al., 2001 ; Gonnella et al., 2015 ). To address this limitation, an MHV-68-Luc model was developed in a 96-well format to quantify viral replication efficiency. The recombinant virus expresses luciferase, which enables quantitation of viral replication measured via luminescence. Previously, the reliability of this reporter system for antiviral drug assessment has been demonstrated in detail ( Obergfäll et al., 2025 ). The characterization included virus stock titration for the exact determination of the multiplicity of infection (MOI), the cell-based quantitation of luciferase reporter activity under drug treatment, and the illustration of a direct quantitative correlation with virus release. Further characterization of MHV-68-Luc has been reported before ( Briestenska et al., 2019 ; Obergfäll et al., 2025 ; Wu et al., 2011 ), including the infection of several permissive cells and PCR-based virus detection. Based on this optimization work, the following time points were considered most appropriate for the present study: 4 d p.i. for MEFs, COS-7, and Vero cells; and 7 d p.i. for HFFs. This system has now been successfully applied to Vero and COS-7 cells, HFFs and MEFs, demonstrating its broad applicability to different host cell types. In the present settings, cells were infected in the 96-well plate format, incubated in the presence of antiviral drugs, before viral reporter luciferase activity was measured 4 to 7 days (depending on the cell line) post-infection (d p.i.). Experimental replicates were performed in biological duplicates or triplicates (n = 2–3), to determine half-maximal effective concentrations for antiviral activity (EC₅₀ values), versus cytotoxicity (CC₅₀ values) assessed in mock-infected cells using the NR uptake assay in parallel. For this model system with MHV-68-Luc, it was considered important to demonstrate both, the drug-mediated reduction of virus-specific luciferase signals ( Fig. 1 A), and the reduction in production and release of progeny virus, which was demonstrated by the transfer of infectious culture supernatants ( Fig. 1 B). These data illustrate the validity of our established model for antiviral drug analysis.
Assessment of antiherpesviral reference drugs and mechanistically novel compounds against MHV-68
The MHV-68-Luc replication model was then applied to assess the antiviral efficacy of a selection of antiherpesviral reference drugs in different host cell environments. In a first step, Vero cells were used for the infection ( Fig. 2 ). To this end, clinically approved and developmental inhibitors of viral polymerases (i.e., ACV, BCV, FOS, GCV) terminases (i.e., LMV), or kinases (i.e., MBV) were analyzed for anti-MHC-68 activity ( Fig. 2 A, see also Table 1 ). While ACV, BCV, and GCV showed high activity at submicromolar concentrations (EC 50 of 0.3 ± 0.3 µM, 0.01 ± 0.003 µM, and 0.9 ± 0.7 µM, respectively), FOS, LMV, and MBV were relatively weakly active or inactive on the micromolar range (EC 50 values > 35 µM). Notably, the ACV values revealed slightly different shapes of the two curves. However, the mean EC 50 value of 0.3 ± 0.3 µM is consistent with our previously published data ( Obergfäll et al., 2025 ). Furthermore, we analyzed three related host-directed kinase inhibitors ( Fig. 2 B) that target the transcription-regulatory cyclin-dependent kinase 8 (CDK8). All three compounds were previously identified as exerting strong, broad anticytomegalovirus activity. ( Obergfäll et al., 2025 , 2024 ). In this setup of MHV-68-Luc replication, the three CDK8 inhibitors exerted a very pronounced antiviral effect, in the nanomolar range (EC 50 concentrations between 0.5 nM to 2.0 nM; Fig. 2 B). Moreover, our particular interest focused the recently identified inhibitors of the herpesviral nuclear egress complex (NEC) ( Tillmanns et al., 2024 ), which have the potential to exert a broad-spectrum inhibitory activity against this conserved target. Also, against MHV-68-Luc, the NEC inhibitors MBM, GK2, and IBR showed a strong antiviral activity at micromolar concentrations (EC 50 values between 1.9 µM and 7.3 µM; Fig. 2 C). Thus, the findings revealed an intriguing spectrum of anti-γ-herpesviral activities within this group of approved drugs (ACV, FOS, GCV, LMV, MBV and IBR, with the latter being approved for cancer therapy and all others being antiherpesviral drugs), as well as developmental/clinically relevant candidates (BCV, BI, MSC and CCT) and experimental compounds (GK2 and MBM). The experimental system using MHV-68-Luc (where all drugs were assessed purely experimentally) provides additional information on drug efficacy in cultured cell-based infections.
Table 1
Antiviral EC 50 [µM], cytotoxicity CC 50 [µM], and SI values, collected for anti-MHV-68 activities in Vero cells, MEFs, and HFFs a .
a For virus system, cell types, and antiviral readouts see Materials and methods; n.d., not determined (for all cases in which no EC 50 could be determined, SI was set as n.d.).
| EC 50 | CC 50 | SI | ||
| Vero | ||||
| Approved and developmental antivirals | ACV | 0.3 ± 0.3 | > 100 | > 333.3 |
| BCV | 0.01 ± 0.003 | > 10 | > 1000 | |
| FOS | 36.4 ± 10.8 | >1000 | > 27.5 | |
| GCV | 0.9 ± 0.7 | > 100 | > 111.1 | |
| LMV | 44.0 ± 3.2 | > 100 | 2.2 | |
| MBV | 88.9 ± 13.6 | > 1000 | > 11.2 | |
| Antiviral CDK8 inhibitors | BI | 0.0005 ± 0.00,003 | > 10 | > 20,000 |
| MSC | 0.002 ± 0.001 | > 100 | > 50,000 | |
| CCT | 0.002 ± 0.07 | > 100 | > 10,000 | |
| Antiviral NEC inhibitors | MBM | 7.3 ± 0.5 | 12.9 | 3.5 |
| GK2 | 5.4 ± 2.2 | 58.9 ± 0.01 | 10.9 | |
| IBR | 1.9 ± 1.0 | 47.7 ± 1.0 | 25.1 | |
| MEF | ||||
| Approved and developmental antivirals | ACV | 0.69 ± 0.26µM | > 100 | > 144.9 |
| BCV | 0.14 ± 0.07 | > 10 | > 71.4 | |
| FOS | 41.5 ± 17.5 | > 1000 | 24.0 | |
| GCV | 9.4 ± 6.3 | > 100 | 10.6 | |
| LMV | > 50 | 66.9 ± 8.6 | n.d. | |
| MBV | 59.6 ± 2.2 | 179.6 ± 10.2 | 3.0 | |
| Antiviral CDK8 inhibitors | BI | 0.006 ± 0.002 | > 10 | > 1666 |
| MSC | 0.01 ± 0.009 | > 1000 | > 10,000 | |
| CCT | 0.016 ± 0.015 | > 100 | > 6250 | |
| Antiviral NEC inhibitors | MBM | 22.2 ± 5.5 | 59.5 ± 9.2 | 2.7 |
| GK2 | 29.6 ± 0.8 | 59.3 ± 0.2 | 2.0 | |
| IBR | 13.1 ± 1.2 | 12.2 ± 2.0 | 0.9 | |
| HFF | ||||
| Approved and developmental antivirals | ACV | > 5 | > 100 | n.d. |
| BCV | > 0.5 | > 10 | n.d. | |
| FOS | > 500 | > 1000 | n.d. | |
| GCV | > 50 | > 100 | n.d. | |
| LMV | > 50 | 81.6 ± 18.6 | n.d. | |
| MBV | 268.7 ± 55.5 | 163.5 ± 9.2 | n.d. | |
| Antiviral CDK8 inhibitors | BI | 0.04 ± 0.02 | > 10 | > 250.0 |
| MSC | 1.8 ± 1.8 | > 100 | > 55.6 | |
| CCT | 0.48 ± 0.33 | > 100 | > 208.3 | |
| Antiviral NEC inhibitors | MBM | 48.9 ± 22,4 | 52.4 ± 7.3 | 1.1 |
| GK2 | > 50 | 42.7 ± 2.2 | n.d. | |
| IBR | 5.5 ± 5.0 | 28.7 ± 3.0 | 5.3 |
The results obtained from MHV-68-Luc-infected simian Vero cells were then compared with the data derived from murine and human primary fibroblasts (MEFs, MOI 0.001, corresponding to 75% of max. Luc activity at 4 d p.i.; HFFs, MOI 0.2, corresponding to 90% of max Luc. activity at 7 d p.i. due to low permissiveness). It is noteworthy that the cellular environment of these murine and human fibroblasts appeared to be less applicable for the assessment of MHV-68-specific antiviral drugs ( Fig. 3 , Fig. 4 ). A comparative evaluation is given with cell type-specific EC 50 , CC 50 , and SI values of antiviral drugs ( Table 1 ). As shown in Table 1 for Vero cells, the agents demonstrating strongest anti-MHV-68-Luc activity, in the low micromolar or submicromolar range, were ACV, BCV, GCV, BI, MSC, CCT, and IBR (upper panel). These drug activities were found much less defined in MEFs (middle) and particularly in HFFs (lower). This observation may be explained by the restricted replication efficiency of MHV-68 in these primary cells (probably due to interferon restriction that is missing in Vero cells), which may likewise translate into a diminished detection sensitivity of antivirals. In essence, a part of the results obtained with MHV-68-Luc, described in the present study as well as in an earlier report (i.e. there compared to a panel of other herpesviruses and non-herpesviruses; ( Obergfäll et al., 2025 ), was found fully reproducible, thus supporting our approach.
Comparative antiviral drug properties spanning three different herpesviruses
In our study, the MHV-68 was applied as a well-characterized γ-herpesvirus that represents a model for human Epstein-Barr virus (EBV). Thus, concerning the sensitivity of MHV-68 to antivirals, a comparative analysis was performed together with EBV and also with human cytomegalovirus (HCMV), since a general focus on HCMV had been given in our recent characterization of novel antiherpesviral drugs ( Table 2 ). This comparison included antivirals described in detail for MHV-68 by Section 3.2 . Interestingly, as a central result, the profiles of antiviral drug sensitivities were not directly assigned to the distinction between the γ-herpesviruses, MHV-68 and EBV, and the β-herpesvirus HCMV. Actually, MHV-68 and EBV showed limited parallels for the five drugs analyzed, such as the SI values for MBV and CCT ranged above 10 ( Table 2 ). TAF could be verified as a reference compound that inhibits EBV replication (EC 50 0.5 ± 0.2 µM), as identified by others before ( Drosu et al., 2020 ). Of note, we detected similar drug sensitivities of HCMV and MHV-68 for CCT, GCV, and BCV, both in SI values and the range of EC 50 . The EC 50 values comprised 0.002 ± 0.07, 0.9 ± 0.7, and 0.01 ± 0.003 µM for MHV-68, versus 0.004 ± 0.002, 0.9 ± 0.2, and 0.002 ± 0.001 µM for HCMV, respectively. Also remarkable was the finding that MBV showed micromolar-submicromolar activity against both EBV and HCMV (4.1 ± 2.1 versus 0.2 ± 0.02 µM, respectively), albeit our current studies demonstrated that the EBV kinase BGLF4, in difference to HCMV pUL97, does not show enzymatic sensitivity to MBV ( Kögler et al., 2026 ). For MBV, other researchers have earlier suggested an anti-EBV inhibitory mechanism, which may be directed to other viral proteins, but independent of any MBV-mediated kinase inhibition ( Gershburg and Pagano, 2002 ). Combined, these results illustrate both, certain points of identical, but in other parts strongly different drug sensitivity panels of the three herpesviruses.
Table 2
Comparing MHV-68, EBV and HCMV in their sensitivity to selected antiviral drugs [µM] a .
a For virus systems and antiviral readouts see Materials and methods; TAF, tenofovir alafenamide; n.d., not determined.
| EC 50 | CC 50 | SI | |
| MHV-68-Luc (Vero) | |||
| MBV | 88.9 ± 13.6 | > 1000 | > 11.2 |
| CCT | 0.002 ± 0.07 | > 100 | > 10,000 |
| GCV | 0.9 ± 0.7 | > 100 | > 111.1 |
| BCV | 0.01 ± 0.003 | > 10 | > 1000 |
| TAF | n.d. | > 100 | n.d. |
| EBV qPCR (P3HR-1) | |||
| MBV | 4.1 ± 2.1 | > 100 | > 24 |
| CCT | 2.5 ± 1.0 | > 100 | > 40 |
| GCV | > 50 | > 50 | n.d. |
| BCV | > 50 | > 50 | n.d. |
| TAF | 0.5 ± 0.2 | > 100 | >196 |
| HCMV AD169-GFP (HFF) | |||
| MBV | 0.2 ± 0.02 | 163.5 ± 9.2 | > 820 |
| CCT | 0.004 ± 0.002 | > 100 | > 25,500 |
| GCV | 0.9 ± 0.2 | > 100 | > 111 |
| BCV | 0.002 ± 0.001 | > 10 | > 5000 |
| TAF | n.d. | > 100 | n.d. |
Analysis of anti-MHV-68 cotreatments to define synergistic drug combinations
Finally, we addressed the question of putative synergistic effects detectable for cotreatment with antiviral drug combinations. In earlier studies, we have focused on antiviral drug synergies in the case of HCMV ( Wild et al., 2022 , 2023 , 2021 ), and we now used these experienced tools of detecting drug–drug interaction for MHV-68-Luc in Vero cells. Both well-established assay systems, of Loewe additivity fixed-dose and Bliss independence checkerboard assays, were performed for distinct combinatorial drug treatments in parallel ( Fig. 5 ). The three compounds used in our analysis, i.e. IBR, MBV, and BCV, had shown results of intermediate, low, and high anti-MHV-68 activity, respectively (compare Fig. 2 and Table 1 ). Notably, the combinations of both IBR + MBV and IBR + BCV revealed statistically relevant synergistic interactions according to the Loewe additivity system ( Fig. 5 A-B, right panels). The respective CI wt values comprised 0.28 ± 0.16 and 0.57 ± 0.51 for these two drug combinations ( Table 3 ). Likewise, using the Bliss independence system, the synergistic effects could be confirmed for IBR + MBV and IBR + BCV ( Fig. 5 A-B, left panels). When comparing additional replicates of the Bliss independence checkerboard assay, however, it should be noted that the combination IBR + MBV indicated some intermediate position ( Table 4 ). The interpretation of synergistic (3 replicates) indicated equality to the interpretation of additive (3 replicates). This result pointed to the combination phenotype of marginally synergistic with a tendency to additive. As far as the Loewe additivity fixed-dose assay was concerned, the interpretation was constant in stating a synergistic effect for IBR + MBV. At this stage of investigations, however, it cannot be concluded whether similar synergistic interactions might occur with other herpesviruses. Combined, these results strongly argue for an enhanced combinatorial antiviral efficacy, for both cotreatments of IBR + MBV and IBR + BCV, in the MHV-68-Luc infection model.
Table 3
Results and interpretation of Loewe additivity fixed-dose drug interaction assays.
a Individual experimental replicates were performed in quadruplicate measurements. b The CI wt value was calculated as (0.1 × CI 50 +0.2 × CI 75 +0.3 × CI 90 +0.4 × CI 95 ). c Interpretation of CI wt values according to the following classification: <0.1–0.3, strongly synergistic; 0.3–0.7, synergistic; 0.7–0.85, moderately synergistic; 0.85–0.9, slightly synergistic; 0.90–1.10, additive; 1.10–1.20, slightly antagonistic; 1.20–1.45, moderately antagonistic; 1.45–3.3, antagonistic; 3.3–>10, strongly antagonistic.
| Compound combination | Replicates a | CI wt b | Interaction type c |
| IBR + MBV | 2 | 0.28 ± 0.16 | strongly synergistic |
| IBR + BCV | 2 | 0.57 ± 0.51 | synergistic |
Table 4
Results and interpretation of Bliss independence checkerboard assays.
a The 95% confidence intervals refer to three biological replicates per assay. b Interpretation of total values (sum) of synergy volumes according to the following classification: below −100, strongly antagonistic; −100 to +50, additive; +50 to +100, moderately synergistic; above +100 strongly synergistic.
| Compound combination | Replicates | 95% confidence interval synergy volume (µM 2 %) a | Interaction type b | ||
| Positive | Negative | Sum | |||
| IBR + BCV | 3 | 1054.02 | 0.00 | 1054.02 | strongly synergistic |
| 3 | 632.10 | 0.00 | 632.10 | ||
| IBR + MBV | 3 | 472.43 | 0.03 | 472.40 | synergistic |
| 3 | 90.27 | 17.63 | 72.64 | ||
| 3 | 1.21 | 33.45 | 32.24 | additive | |
| 3 | 62.92 | 31.43 | 31.49 |
Conclusions
Infections with human γ-herpesviruses, in particular with Epstein-Barr virus (EBV), represent ubiquitous, worldwide clinical problems. Existing experimental systems of EBV infections are time- and labor-consuming. Here, we used murine γ-herpesvirus infection; of murine, human, and simian cells, based on the MHV-68-Luc replication reporter system. The main results were the establishment of a new γ-herpesviral lytic system in cell culture, with a quantitative readout, applying the luciferase reporter for antiviral drug assessments. The reporter system proved to be very flexible and easy in its application. It provided a high reliability in the determination of viral replication and antiviral drug efficacy in quantitative terms. Of note, the viral reporter is driven by the M3 promoter (see Sections 2.3 and 1.), and for none of the analyzed antiviral drugs, any indication of direct activity against the M3 promoter/Luc expression has been observed. This theoretical option seems to be highly improbable, due to the fact that infected-cell Luc reporter profiles correlated well with MHV-68-Luc progeny virus production and release. ( Fig. 1 A-B). It should be noted that secondary drug effects could slightly interfere with MHV-68-Luc reporter expression. However, two factors argue against rating antiviral compounds as false positives. First, the measured reduction in virus production and release ( Obergfäll et al., 2025 ) contradicts such a rating. Second, microscopic monitoring of viral cytopathic effects (CPE) clearly demonstrated a reduction in all cases of antiviral drug activity, as determined by Luc reporter analysis. These results suggest a correlation between reduced viral CPE formation and antiviral drug effects. Consequently, the antiviral system described in this study is considered significant in assessing antiviral drug activity and combinatorial drug synergies. To confirm the validity of the data and its interpretation in future assessments, implementing a luciferase reporter assay could be considered. This would involve cotransfecting cells with an M3 promoter reporter plasmid responsive to the viral transactivator Rta and an MHV-68 Rta expression plasmid. This setup could confirm the specific effects of antiviral drugs on MHV-68-Luc replication (expected to be nonreactive against the M3 promoter or Rta activity) and exclude assay-based side effects. In summary, the present study emphasized the helpful applicability of MHV-68-Luc as a model of productive γ-herpesvirus infection, irrespective of options in technical optimization.
As part of our long-term research efforts, we have continuously worked to make drug inhibitory efficacies comparable. This has been challenging due to the various herpesviruses used in this study and related studies requiring different in vitro conditions, including the selection of appropriate cell lines, readout systems, and experimental protocols. To this end, we employed appropriate readouts (e.g., Luc, qPCR, GFP, etc.). These protocols were successfully established in previous publications ( Kicuntod et al., 2021 ; Kögler et al., 2026 ; Obergfäll et al., 2025 , 2024 ; Tillmanns et al., 2023 ; Wild et al., 2022 , 2021 ; Yu et al., 2024 ). It should be noted that these assay systems had to be standardized, including the use of internal controls and reference inhibitors (e.g., GCV, CDV, BCV, TFA, FOS, ACV, and LMV for MHV-68, EBV, and other herpesviruses; see ( Obergfäll et al., 2025 ); for HCMV and other herpesviruses, see ( Marschall et al., 2012 ; Wild et al., 2021 ; Yu et al., 2024 ). This was done to ensure reliability and high reproducibility. These controls especially helped assure reliable outcomes in terms of comparable EC₅₀, CC₅₀, and SI values, proving their applicability as assay-independent parameters. The present study assessed both conventional antiherpesviral drugs and mechanistically novel compounds that could be analyzed in a comparative setting. The antiviral analysis identified drug combination treatments that exhibited statistically significant synergies.
In more general terms, these questions have been continuously relevant for animal and human herpesviruses, such as EBV and HCMV, as well as their murine relatives MHV-68 and MCMV. The potential targets for use of antivirals in EBV infection, i.e. acute severe types of infections, have not clearly been defined and so far, and have failed to provide substantial clinical benefits. Moreover, the treatment of EBV-associated epithelial cancers (NPC, gastric carcinoma), adjunctive treatment of lymphomas (Burkitt’s lymphoma, PTLD, rare NK or T cell lymphomas), or potential use in treating MS remain challenging. The WHO International Agency for Research on Cancer (IARC) classifies EBV as a group 1 human carcinogen, consistent with their finding sufficient evidence for EBV induced carcinogenesis. Also, EBV is classified as a requisite environmental risk factor or primary trigger for the development of MS, with therapeutic trials ongoing.
There is an urgent need for antivirals that can target the early phases of pre-tumoral infection, especially since these tumors are often treatment-resistant. On the one hand, the current situation of inadequate antitumor agent efficacy faces the importance of Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) in driving tumorigenesis. In this context, antivirals could provide a potentially life-saving adjunct mode of therapy by reducing tumor induction and progression, as well as post-therapy morbidity. According to the novel modes of action shown in this study, this would be mostly beneficial, including drug–drug synergies with potentially fewer side effects due to their targeting of crucial virus-supportive processes. Redirecting such antivirals to other EBV-driven diseases under investigation, such as MS and other autoimmune disorders, could provide additional utility for novel antiviral therapeutic options. In this context, using MHV-68 model systems allows us to obtain complex phenotypic information. This is particularly relevant for combination therapies involving novel agents that affect viral or cellular processes, including direct-acting antivirals already licensed for herpesvirus treatment.
Funding
The project was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) DFG 401821119–Research Training Group GRK2504.1–2/A1; DFG individual grant (MA 1289/21–1); and Volkswagen-Stiftung (AZ-9B783-M.M./S.B.T.).
CRediT authorship contribution statement
Christina Wangen: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Debora Obergfäll: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Data curation. Katarína Briestenská: Writing – review & editing, Writing – original draft, Resources, Methodology. Jela Mistríková: Writing – original draft, Supervision, Resources, Methodology. William D. Rawlinson: Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Formal analysis. Manfred Marschall: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Formal analysis, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have declared that no competing interests exist.
Acknowledgments
The authors are grateful to all members of the M.M. research group for very valuable scientific contributions and for continuous discussion, and M.M. greatly appreciates the excellent technical assistance by Regina Müller and Sabrina Wagner (Virology, FAU/UKER, Erlangen). The authors like to thank Prof. Ren Sun and Prof. Ting-Ting Wu (University of California, Los Angeles, USA) for kindly providing the essential molecular tools and methodology for recombinant virus construction. We are also grateful to Dr. Anna Kostrábová (Dept. Microbiol. and Virol., Faculty Nat. Sci., Comenius Univ. Bratislava, SK) for her guidance and expertise in the virus construction process.
Contributor Information
Christina Wangen, Email: christina.wangen@uk-erlangen.de.
Debora Obergfäll, Email: debora.obergfaell@fau.de.
Katarína Briestenská, Email: katarina.briestenska@savba.sk.
Jela Mistríková, Email: jela.mistrikova@savba.sk.
William D. Rawlinson, Email: w.rawlinson@unsw.edu.au.
Manfred Marschall, Email: manfred.marschall@fau.de.
Data availability
Data will be made available on request.
References
Andrei G., Trompet E., Snoeck R. Novel therapeutics for Epstein(-)Barr Virus. Molecules. 2019;24(5):997. doi: 10.3390/molecules24050997.
Blaškovič D., Stancekova M., Svobodová J., Mistríková J. Isolation of five strains of herpesviruses from two species of free living small rodents. Acta Virol. 1980;24(6):468.
Blaškovič D., Stanekova D., Rajčáni J. Experimental pathogenesis of murine herpesvirus in newborn mice. Acta Virol. 1984;28(3):225–231.
Briestenská J., Briestenská K. Murid herpesvirus 4 (MuHV-4, prototype strain MHV-68) as an important model in global research of human oncogenic gammaherpesviruses. Acta. Virol. 2020;64(2):167–176. doi: 10.4149/av_2020_206.
Briestenska K., Samsulova V., Polakova M., Mistrikova J. Recombinant luciferase-expressing murine gammaherpesvirus 68 as a tool for rapid antiviral screening. Acta. Virol. 2019;63(4):439–449. doi: 10.4149/av_2019_411.
Chakravorty S., Afzali B., Kazemian M. EBV-associated diseases: current therapeutics and emerging technologies. Front. Immunol. 2022;13 doi: 10.3389/fimmu.2022.1059133.
Chen H., Lye M.F., Gorgulla C., Ficarro S.B., Cuny G.D., Scott D.A., Wu F., Rothlauf P.W., Wang X., Fernandez R., Pesola J.M., Draga S., Marto J.A., Hogle J.M., Arthanari H., Coen D.M. A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex. PLoS. Pathog. 2023;19(11) doi: 10.1371/journal.ppat.1011781.
Chinna P., Bratl K., Lambarey H., Blumenthal M.J., Schafer G. The impact of Co-infections for Human gammaherpesvirus infection and associated pathologies. Int. J. Mol. Sci. 2023;24(17) doi: 10.3390/ijms241713066.
Cho H.J., Jeong S.G., Park J.E., Han J.A., Kang H.R., Lee D., Song M.J. Antiviral activity of angelicin against gammaherpesviruses. Antiviral Res. 2013;100(1):75–83. doi: 10.1016/j.antiviral.2013.07.009.
Chou, T.M.N., 2005. CompuSyn for Drug Combinations: PC Software and User’s Guide: A Computer Program for Quantitation of Synergism and Antagonism in Drug Combinations, and the Determination of IC50 and ED50 and LD50 Values. Paramus, NJ.
Doherty P.C., Tripp R.A., Hamilton-Easton A.M., Cardin R.D., Woodland D.L., Blackman M.A. Tuning into immunological dissonance: an experimental model for infectious mononucleosis. Curr. Opin. Immunol. 1997;9(4):477–483. doi: 10.1016/s0952-7915(97)80098-2.
Drosu N.C., Edelman E.R., Housman D.E. Tenofovir prodrugs potently inhibit Epstein-Barr virus lytic DNA replication by targeting the viral DNA polymerase. Proc. Natl. Acad. Sci. USA. 2020;117(22):12368–12374. doi: 10.1073/pnas.2002392117.
Efstathiou S., Ho Y.M., Hall S., Styles C.J., Scott S.D., Gompels U.A. Murine herpesvirus 68 is genetically related to the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri. J. Gen. Virol. 1990;71(Pt 6):1365–1372. doi: 10.1099/0022-1317-71-6-1365.
Flaño E., Woodland D.L., Blackman M.A. A mouse model for infectious mononucleosis. Immunol. Res. 2002;25(3):201–217. doi: 10.1385/IR:25:3:201.
Gao X., Ikuta K., Tajima M., Sairenji T. 12-O-tetradecanoylphorbol-13-acetate induces Epstein-Barr virus reactivation via NF-kappaB and AP-1 as regulated by protein kinase C and mitogen-activated protein kinase. Virology. 2001;286(1):91–99. doi: 10.1006/viro.2001.0965.
Gershburg E., Pagano J.S. Phosphorylation of the Epstein-Barr virus (EBV) DNA polymerase processivity factor EA-D by the EBV-encoded protein kinase and effects of the l-riboside benzimidazole 1263W94. J. Virol. 2002;76(3):998–1003. doi: 10.1128/JVI.76.3.998-1003.2002.
Gonnella R., Granato M., Farina A., Santarelli R., Faggioni A., Cirone M. PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction. Biochim. Biophys. Acta. 2015;1853(7):1586–1595. doi: 10.1016/j.bbamcr.2015.03.011.
Hwang S., Wu T.T., Tong L.M., Kim K.S., Martinez-Guzman D., Colantonio A.D., Uittenbogaart C.H., Sun R. Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo. J. Virol. 2008;82(24):12498–12509. doi: 10.1128/JVI.01152-08.
Kicuntod J., Alkhashrom S., Häge S., Diewald B., Müller R., Hahn F., Lischka P., Sticht H., Eichler J., Marschall M. Properties of oligomeric interaction of the Cytomegalovirus core nuclear egress complex (NEC) and its sensitivity to an NEC inhibitory small molecule. Viruses. 2021;13(3):462. doi: 10.3390/v13030462.
Kögler M., Wangen C., Hammerschmitt A., Obergfäll D., Hahn F., Marschall M. Identification of a new phosphorylated host interactor of the Epstein-Barr Virus (EBV) kinase BGLF4 suggests key points for EBV-specific antiviral drug targeting. Int. J. Mol. Sci. 2026;27(6) doi: 10.3390/ijms27062627.
Kuri A., Jacobs B.M., Vickaryous N., Pakpoor J., Middeldorp J., Giovannoni G., Dobson R. Epidemiology of Epstein-Barr virus infection and infectious mononucleosis in the United Kingdom. BMC. Public Health. 2020;20(1):912. doi: 10.1186/s12889-020-09049-x.
Kutok J.L., Wang F. Spectrum of Epstein-Barr virus-associated diseases. Annu Rev. Pathol. 2006;1:375–404. doi: 10.1146/annurev.pathol.1.110304.100209.
Lanz T.V., Brewer R.C., Ho P.P., Moon J.S., Jude K.M., Fernandez D., Fernandes R.A., Gomez A.M., Nadj G.S., Bartley C.M., Schubert R.D., Hawes I.A., Vazquez S.E., Iyer M., Zuchero J.B., Teegen B., Dunn J.E., Lock C.B., Kipp L.B., Cotham V.C., Ueberheide B.M., Aftab B.T., Anderson M.S., DeRisi J.L., Wilson M.R., Bashford-Rogers R.J.M., Platten M., Garcia K.C., Steinman L., Robinson W.H. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature. 2022;603(7900):321–327. doi: 10.1038/s41586-022-04432-7.
Li V., McKay F.C., Tscharke D.C., Smith C., Khanna R., Lechner-Scott J., Rawlinson W.D., Lloyd A.R., Taylor B.V., Morahan J.M., Steinman L., Giovannoni G., Bar-Or A., Levy M., Drosu N., Potter A., Caswell N., Smith L., Brady E.C., Frost B., Hodgkinson S., Hardy T.A., Broadley S.A., Australian Anti E.B.V.D.F.M.S.W.G. Repurposing licensed drugs with activity against Epstein-Barr virus for treatment of multiple sclerosis: a systematic approach. CNS. Drugs. 2025;39(3):305–320. doi: 10.1007/s40263-024-01153-5.
Lv M., Ding Y., Zhang Y., Liu S. Targeting EBV-encoded products: implications for drug development in EBV-associated diseases. Rev. Med. Virol. 2024;34(1) doi: 10.1002/rmv.2487.
Mackett M., Stewart J.P., de V.P.S., Chee M., Efstathiou S., Nash A.A., Arrand J.R. Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68. J. Gen. Virol. 1997;78(Pt 6):1425–1433. doi: 10.1099/0022-1317-78-6-1425.
Marschall M., Stamminger T., Urban A., Wildum S., Ruebsamen-Schaeff H., Zimmermann H., Lischka P. In vitro evaluation of the activities of the novel anticytomegalovirus compound AIC246 (letermovir) against herpesviruses and other human pathogenic viruses. Antimicrob. Agents. Chemother. 2012;56(2):1135–1137. doi: 10.1128/AAC.05908-11.
Martinez-Guzman D., Rickabaugh T., Wu T.T., Brown H., Cole S., Song M.J., Tong L., Sun R. Transcription program of murine gammaherpesvirus 68. J. Virol. 2003;77(19):10488–10503. doi: 10.1128/JVI.77.19.10488-10503.2003.
Mistríková J., Briestenská K. Murid herpesvirus 4 (MuHV-4, prototype strain MHV-68) as an important model in global research of human oncogenic gammaherpesviruses. Acta. Virol. 2020;64(2):167–176. doi: 10.4149/av_2020_206.
Obergfäll D., Hahn F., Kicuntod J., Wangen C., Kogler M., Wagner S., Kaufer B., Marschall M. New assay systems to characterize the broad-spectrum antiherpesviral and non-herpesviral activity of cyclin-dependent kinase (CDK) 8 inhibitors. Pharmaceuticals. (Basel) 2025;18(10) doi: 10.3390/ph18101560.
Obergfäll D., Wild M., Sommerer M., Barillas Dahm M., Kicuntod J., Tillmanns J., Kögler M., Lösing J., Dhotre K., Müller R., Wangen C., Wagner S., Phan Q.V., Wiebusch L., Briestenska K., Mistrikova J., Kerr-Jones L., Stanton R.J., Voigt S., Hahn F., Marschall M. Cyclin-dependent kinase 8 represents a positive regulator of Cytomegalovirus replication and a novel host target for antiviral strategies. Pharmaceutics. 2024;16(9) doi: 10.3390/pharmaceutics16091238.
Pagano J.S., Whitehurst C.B., Andrei G. Antiviral drugs for EBV. Cancers. (Basel) 2018;10(6):197. doi: 10.3390/cancers10060197.
Prichard M., Aseltine K., Shipman C., Jr . University of Michigan; Ann Arbor, MI: 1992. MacSynergy II User’s Manual.
Rajčáni J., Blaškovič D., Svobodová J., Ciampor F., Huckova D., Stanekova D. Pathogenesis of acute and persistent murine herpesvirus infection in mice. Acta Virol. 1985;29(1):51–60.
Simas J.P., Efstathiou S. Murine gammaherpesvirus 68: a model for the study of gammaherpesvirus pathogenesis. Trends. Microbiol. 1998;6(7):276–282. doi: 10.1016/s0966-842x(98)01306-7.
Smith G.A., Enquist L.W. Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus. J. Virol. 1999;73(8):6405–6414. doi: 10.1128/jvi.73.8.6405-6414.1999.
Song M.J., Hwang S., Wong W.H., Wu T.T., Lee S., Liao H.I., Sun R. Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis. Proc. Natl. Acad. Sci. USA. 2005;102(10):3805–3810. doi: 10.1073/pnas.0404521102.
Stewart J. Of mice and men: murine gammaherpesvirus 68 as a model. Epstein Barr Virus Report. 1999;6:31. -31.
Sunil-Chandra N.P., Arno J., Fazakerley J., Nash A.A. Lymphoproliferative disease in mice infected with murine gammaherpesvirus 68. Am. J. Pathol. 1994;145(4):818–826.
Sunil-Chandra N.P., Efstathiou S., Arno J., Nash A.A. Virological and pathological features of mice infected with murine gamma-herpesvirus 68. J. Gen. Virol. 1992;73(Pt 9):2347–2356. doi: 10.1099/0022-1317-73-9-2347.
Sunil-Chandra N.P., Efstathiou S., Nash A.A. Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo. J. Gen. Virol. 1992;73(Pt 12):3275–3279. doi: 10.1099/0022-1317-73-12-3275.
Svobodová J., Blaskovic D., Mistríková J. Growth characteristics of herpesviruses isolated from free living small rodents. Acta. Virol. 1982;26(4):256–263.
Thomas O.G., Rykaczewska U., Galesic M., van der Burgt R.T.M., Hallen N., Ferro F., Bronge M., Marti Z., Li Y., Rique A.H., Lin J., Krstic A., Gromadzka A., Szonder A.L., Sorini C., Reina-Campos M., Sun T., Rubio Rodriguez-Kirby L.A., Dumral O., Berglund R., Kakhki M.P., Adzemovic M.Z., Zeitelhofer M., Akpinar B., Tengvall K., Nilsson O.B., Holmgren E., Cucuzza C.S., Hogelin K.A., Gafvelin G., Fink K., Castelo-Branco G., Needhamsen M., Khademi M., Piehl F., Graslund T., Alfredsson L., Lund H., Uhlen P., Kockum I., Martin R., Jagodic M., Gronlund H., Guerreiro-Cacais A.O., Olsson T. Anoctamin-2-specific T cells link Epstein-Barr virus to multiple sclerosis. Cell. 2026;189(2):585–602.e38. doi: 10.1016/j.cell.2025.12.032. e538.
Tillmanns J., Battisti V., Kicuntod J., Hahn F., Obergfäll D., Geiger P., Wagner S., Buschmann H., Lesch B., Lischka P., Sticht H., Langer T., Marschall M. The conserved core nuclear egress complex (NEC) as an antiherpesviral drug target: pharmacophore-based identification of NEC-specific inhibitors. Antiviral Res. 2025;239 doi: 10.1016/j.antiviral.2025.106168.
Tillmanns J., Häge S., Borst E.M., Wardin J., Eickhoff J., Klebl B., Wagner S., Wangen C., Hahn F., Socher E., Marschall M. Assessment of covalently binding warhead compounds in the validation of the Cytomegalovirus nuclear egress complex as an antiviral target. Cells. 2023;12(8):1162. doi: 10.3390/cells12081162.
Tillmanns J., Kicuntod J., Lösing J., Marschall M. Getting better'-is it a feasible strategy of broad pan-antiherpesviral drug targeting by using the nuclear egress-directed mechanism? Int. J. Mol. Sci. 2024;25(5):2823. doi: 10.3390/ijms25052823.
Tripp R.A., Hamilton-Easton A.M., Cardin R.D., Nguyen P., Behm F.G., Woodland D.L., Doherty P.C., Blackman M.A. Pathogenesis of an infectious mononucleosis-like disease induced by a murine gamma-herpesvirus: role for a viral superantigen? J. Exp. Med. 1997;185(9):1641–1650. doi: 10.1084/jem.185.9.1641.
Usherwood E.J., Ross A.J., Allen D.J., Nash A.A. Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells. J. Gen. Virol. 1996;77(Pt 4):627–630. doi: 10.1099/0022-1317-77-4-627.
Virgin H.W.T., Latreille P., Wamsley P., Hallsworth K., Weck K.E., Dal Canto A.J., Speck S.H. Complete sequence and genomic analysis of murine gammaherpesvirus 68. J. Virol. 1997;71(8):5894–5904. doi: 10.1128/jvi.71.8.5894-5904.1997.
Weck K.E., Kim S.S., Virgin H.I., Speck S.H. B cells regulate murine gammaherpesvirus 68 latency. J. Virol. 1999;73(6):4651–4661. doi: 10.1128/jvi.73.6.4651-4661.1999.
Wild M., Hahn F., Bruckner N., Schütz M., Wangen C., Wagner S., Sommerer M., Strobl S., Marschall M. Cyclin-dependent kinases (CDKs) and the Human Cytomegalovirus-encoded CDK ortholog pUL97 represent highly attractive targets for synergistic drug combinations. Int. J. Mol. Sci. 2022;23(5):2493. doi: 10.3390/ijms23052493.
Wild M., Karner D., Eickhoff J., Wagner S., Kicuntod J., Chang W., Barry P., Jonjic S., Lenac Rovis T., Marschall M. Combined treatment with host-directed and anticytomegaloviral kinase inhibitors: mechanisms, synergisms and drug resistance barriers. Pharmaceutics. 2023;15(12):2680. doi: 10.3390/pharmaceutics15122680.
Wild M., Kicuntod J., Seyler L., Wangen C., Bertzbach L.D., Conradie A.M., Kaufer B.B., Wagner S., Michel D., Eickhoff J., Tsogoeva S.B., Bauerle T., Hahn F., Marschall M. Combinatorial drug treatments reveal promising anticytomegaloviral profiles for clinically relevant pharmaceutical kinase inhibitors (PKIs) Int. J. Mol. Sci. 2021;22(2):575. doi: 10.3390/ijms22020575.
Wu T.T., Liao H.I., Tong L., Leang R.S., Smith G., Sun R. Construction and characterization of an infectious murine gammaherpesivrus-68 bacterial artificial chromosome. J. Biomed. Biotechnol. 2011;2011 doi: 10.1155/2011/926258.
Yu D., Wagner S., Schütz M., Jeon Y., Seo M., Kim J., Bruckner N., Kicuntod J., Tillmanns J., Wangen C., Hahn F., Kaufer B.B., Neipel F., Eickhoff J., Klebl B., Nam K., Marschall M. An antiherpesviral host-directed strategy based on CDK7 covalently binding drugs: target-selective, picomolar-dose, cross-virus reactivity. Pharmaceutics. 2024;16(2):158. doi: 10.3390/pharmaceutics16020158.
Zanella L., Riquelme I., Buchegger K., Abanto M., Ili C., Brebi P. A reliable Epstein-Barr Virus classification based on phylogenomic and population analyses. Sci. Rep. 2019;9(1):9829. doi: 10.1038/s41598-019-45986-3.
Associated Data
Data Availability Statement
Data will be made available on request.