Is murine gammaherpesvirus-68 (MHV-68) a suitable immunotoxicological model for examining immunomodulatory drug-associated viral recrudescence?

Figures & data

Figure 1. Overview of gammaherpesvirus pathogenesis. Primary gammaherpesvirus infection typically occurs in the respiratory tract and results in an initial lytic infection that is generally cleared within a few weeks via a robust CTL response in immune-competent individuals. The host is unable to clear all virally infected cells, and the virus down-regulates lytic genes and enters latency. Latency persists for the life of the infected host. Viral re-activation can periodically occur (shown by the bidirectional arrow) during immunosuppression that may be associated with lymphomagenesis in some patients.

Figure 2. Gammaherpesvirus genomic organization and gene expression. MHV-68, EBV, and KSHV genomes are shown as indicated. Solid boxes refer to areas of similar genome conservation amongst gammaherpesviruses. 5′ and 3′ terminal repeats (TR) are not shown for simplicity. Arrows refer to direction of transcription. Unique genes or genes that play a known role in viral pathogenesis/recrudescence are labeled in the table and are discussed within the text.

Table 1. Mechanisms of gammaherpesvirus immune control.

Cell type/effector	Impact on pathogenesis
CD8^+ T-lymphocytes	Primary mechanism to control acute lytic infection; shuts down lytic replication and down-regulates latency and re-activation
CD4^+ T-lymphocytes	Collaborate with CD8^+ T-lymphocytes to mediate lytic and latent infection; assist in long-term maintenance of CD8^+ T-lymphocytes response; other roles in immunity unclear
Interferon	IFN-α/β and -γ expression can induce an antiviral state and reduce lytic and latent replication as well as reduce re-activation
Antibody	Development of neutralizing antibody against virions occurs within a few weeks of infection and helps reduce re-activation and viral production
Apoptosis	Programmed cell-mediated death of infected cells which reduces lytic and latent replication and re-activation; decreases persistent viral replication

Table 2. Activating factors involved with gammaherpesvirus recrudescence.

Factors	Description and potential inducers
RAS/RAF	Phorbol esters, BCR cross-linkers have been shown to increase viral re-activation
Toll-like Receptors (TLRs)	Stimulators of TLR 3, 4, 5, and 9 result in increased viral reactivation
NF-κB	Inhibition of NF-κB results in increased viral replication High NF-κB expression promotes proliferation and survival of latently infected cells
Epigenetics	Histone deacetylase inhibitors increase viral re-activation and have been shown to reactivate latently infected MHV-68 cell lines
DNA Damage (DDR)	Unclear mechanism; may play a role in viral replication Chemicals such as etoposide, camptothecin, cisplatin, and staurosporine induce DDR and increase MHV-68 lytic gene expression in vitro

Table 3. Overview of MHV-68 model.

	Experimental summary	Description/comments
Characterize infection model and define molecular tools	1. Determine route of infection 2. Characterize infection 3. Characterize lytic and latent infection 4. Establish assays to detect virus	1. Intranasal (IN) vs Intraperitoneal (IP) 2. Characterize infection progression in WT and immnuosuppressed mice 3. Determine time-course of lytic infection and latency establishment 4. Determine most robust methods for viral detection (e.g. qPCR, viral titration, IHC, RNA ISH)
Validate model with known immunomodulants	1. Select immunomodulatory compound(s) likely to induce viral recrudescence 2. Determine dosing regimen	1. Choose compound(s) with well-known clinical history that are likely to induce recrudescence (e.g. CsA) as positive control for the assay 2. Establish dosing regimen and timelines of dosing after latency establishment to show proof of concept
Test model with novel immunomodulants	1. Treat animals with immunosuppressive compounds after establishment of latency to assay recrudescence 2. Determine utility of model using novel immunomodulatory agents	1. Viral recrudescence after treatment with immunomodulatory compounds provides an indication that the compound being tested has potential for association with neoplasia 2. MHV-68 model potentially provides a strong tool with which to predict how an immunomodulant might impact human disease 3. Timeline for recrudescence after immunosuppressive treatment may vary with agent used 4. Novel compounds may not induce viral reactivation but may still indicate increased risk of neoplasia via non-viral mechanisms 5. Novel compounds that induce viral reactivation may not necessarily be associated with increased risk of neoplasia 6. Need for validation of model via alternative assays

Table 4. Utility of MHV-68 model for risk assessment of immunomodulatory compounds.

	Advantages	Disadvantages	Similarities to EBV/KSHV
Acute infection	1. Infection can be modeled in vivo 2. Infection can be modeled in vitro in various cell lines 3. Infects multiple cell types (macrophages, dendritic cells, and B-cells)	1. Route of transmission unclear 2. Only few cell types established become latently or persistently infected in vitro	1. Acute infection symptoms similar to EBV (pathogenic features of lung and lymphoid tissue) 2. CTL response to control acute infection 3. Acute infection usually resolved but virus never cleared 4. KSHV acute infection largely undefined
Latency	1. Established cell lines (A20, S11) to study latency in vitro 2. Virus can be used in vivo with default latency pathway 3. Immunosuppression can induce viral reactivation (lymphomagenesis)	1. Lymphomagenesis not as robust as EBV in vivo 2. Lymphomagenesis can take long periods to develop in immunocompetent or immunosuppressed animals	1. Latency primarily occurs in B-cells for all of these viruses 2. MHV-68 genes involved in latency are most homologous to KSHV
Pathology of PTLD	1. Use of immunosuppressive drugs can model virus reactivation and/or lymphomagenesis 2. Mouse models can be used to study a variety of viral reactivation mechanisms	1. MHV-68 may not fully represent PTLD progression 2. In vivo experiments (CsA) take a long time to cause LPD	1. Immunosuppression results in viral reactivation for all these viruses 2. Latency usually occurs in immunocompetent mice and/or humans
Lymphomagenesis	1. Established small animal model that is highly tractable 2. Potential for in vitro as well as in vivo modeling with latent cell lines and high titer viral stocks	1. Lymphomagenesis not as robust as EBV in vivo 2. Lymphomagenesis can take long periods of time to develop in in vivo models	1. MHV-68 disease progression similar to EBV 2. MHV-68 disease progression may have similarities to KSHV, but KSHV pathogenesis is not well-understood