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Persistent viral infections are widespread and represent significant public health burdens. Some viruses endure in a latent state by co-opting the host epigenetic machinery to manipulate viral gene expression. Small molecules targeting epigenetic pathways are now in the clinic for certain cancers and are considered as potential treatment strategies to reverse latency in HIV-infected individuals. In this review, we discuss how drugs interfering with one epigenetic pathway, protein acetylation, perturb latency of three families of pathogenic human viruses-retroviruses, herpesviruses, and papillomaviruses.

HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.

BackgroundExamination of a cohort of cats experimentally infected with feline immunodeficiency virus (FIV) for 5.75 years revealed detectable proviral DNA in peripheral blood mononuclear cells (PBMCs) harvested during the asymptomatic phase, undetectable plasma viral RNA (FIV gag), and rarely detectable cell-associated viral RNA. Despite apparent viral latency in peripheral CD4+ T cells, circulating CD4+ T cell numbers progressively declined in progressor animals. The aim of this study was to explore this dichotomy of peripheral blood viral latency in the face of progressive immunopathology. The viral replication status, cellular immunophenotypes, and histopathologic features were compared between popliteal lymph nodes (PLNs) and peripheral blood. Also, we identified and further characterized one of the FIV-infected cats identified as a long-term non-progressor (LTNP).ResultsPLN-derived leukocytes from FIV-infected cats during the chronic asymptomatic phase demonstrated active viral gag transcription and FIV protein translation as determined by real-time RT-PCR, Western blot and in situ immunohistochemistry, whereas viral RNA in blood leukocytes was either undetectable or intermittently detectable and viral protein was not detected. Active transcription of viral RNA was detectable in PLN-derived CD4+ and CD21+ leukocytes. Replication competent provirus was reactivated ex vivo from PLN-derived leukocytes from three of four FIV-infected cats. Progressor cats showed a persistent and dramatically decreased proportion and absolute count of CD4+ T cells in blood, and a decreased proportion of CD4+ T cells in PLNs. A single long-term non-progressor (LTNP) cat persistently demonstrated an absolute peripheral blood CD4+ T cell count indistinguishable from uninfected animals, a lower proviral load in unfractionated blood and PLN leukocytes, and very low amounts of viral RNA in the PLN.ConclusionCollectively our data indicates that PLNs harbor important reservoirs of ongoing viral replication during the asymptomatic phase of infection, in spite of undetectable viral activity in peripheral blood. A thorough understanding of tissue-based lentiviral reservoirs is fundamental to medical interventions to eliminate virus or prolong the asymptomatic phase of FIV infection.

IntroductionDistinct biological factors exist that affect the natural history of HIV and the host immune response between women and men. These differences must be addressed to permit the optimal design of effective HIV eradication strategies for much of the HIV-positive population.Methods and resultsHere, we review the literature on sex-based differences in HIV pathogenesis and natural history in tissues and anatomic compartments, HIV latency and transcriptional activity, and host immunity including the role of sex hormones. We then outline the potential effects of these differences on HIV persistence, and on the safety and efficacy of HIV eradication and curative interventions. Finally, we discuss the next steps necessary to elucidate these factors to achieve a cure for HIV, taking in account the complex ethical issues and the regulatory landscape in the hopes of stimulating further research and awareness in these areas.ConclusionsTargeted enrollment of women in clinical trials and careful sex-based analysis will be crucial to gain further insights into sex-based differences in HIV persistence and to design sex-specific approaches to HIV eradication, if required.

AimRecurrent herpetic stromal keratitis (rHSK), due to an immune response to reactivation of herpes simplex virus (HSV-1), can cause corneal blindness. The development of therapeutic interventions such as drugs and vaccines to decrease rHSK have been hampered by the lack of a small and reliable animal model in which rHSK occurs at a high frequency during HSV-1 latency. The aim of this study is to develop a rabbit model of rHSK in which stress from elevated temperatures increases the frequency of HSV-1 reactivations and rHSK.Materials and methodsRabbits latently infected with HSV-1 were subjected to elevated temperatures and the frequency of viral reactivations and rHSK were determined.ResultsIn an experiment in which rabbits latently infected with HSV-1 were subjected to ill-defined stress as a result of failure of the vivarium air conditioning system, reactivation of HSV-1 occurred at over twice the normal frequency. In addition, 60% of eyes developed severe rHSK compared to

Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells.

BackgroundIn vitro, disulfiram activated HIV transcription in a primary T-cell model of HIV latency and in a pilot clinical study increased plasma HIV RNA in individuals with adequate drug exposure. We assessed the effect of disulfiram on HIV transcription in a dose-escalation study.MethodsIn this prospective dose-escalation study, to optimise disulfiram exposure we included adults with HIV on suppressive antiretroviral therapy, with plasma HIV RNA of less than 50 copies per mL and a CD4 cell count greater than 350 cells per μL. Participants were allocated sequentially to one of three dosing groups (500 mg, 1000 mg, and 2000 mg) and received disulfiram daily for 3 days. Only the staff who did laboratory assays were masked to group assignment. The primary endpoint was change in cell-associated unspliced HIV RNA in CD4 cells. The primary analysis method was a negative binomial regression, with the number of copies as the outcome variable and the input total RNA or plasma volume as an exposure variable, which is equivalent to modelling copies or input. We used these models to estimate changes from before disulfiram to timepoints during and after disulfiram administration. This study is registered with ClinicalTrials.gov, number NCT01944371.FindingsOf 34 participants screened for eligibility at The Alfred Hospital (Melbourne, VIC, Australia), and San Francisco General Hospital (San Francisco, CA, USA), 30 people were enrolled between Sept 24, 2013, and March 31, 2014. The estimated fold increases in cell-associated unspliced HIV RNA from baseline were 1·7 (95% CI 1·3-2·2; p

Suberoylanilide hydroxamic acid (SAHA) has been assessed in clinical trials as part of a "shock and kill" strategy to cure HIV-infected patients. While it was effective at inducing expression of HIV RNA ("shock"), treatment with SAHA did not result in a reduction of reservoir size ("kill"). We therefore utilized a combined analysis of effects of SAHA on the host transcriptome and proteome to dissect its mechanisms of action that may explain its limited success in "shock and kill" strategies. CD4+ T cells from HIV seronegative donors were treated with 1μM SAHA or its solvent dimethyl sulfoxide (DMSO) for 24h. Protein expression and post-translational modifications were measured with iTRAQ proteomics using ultra high-precision two-dimensional liquid chromatography-tandem mass spectrometry. Gene expression was assessed by Illumina microarrays. Using limma package in the R computing environment, we identified 185 proteins, 18 phosphorylated forms, 4 acetylated forms and 2982 genes, whose expression was modulated by SAHA. A protein interaction network integrating these 4 data types identified the HIV transcriptional repressor HMGA1 to be upregulated by SAHA at the transcript, protein and acetylated protein levels. Further functional category assessment of proteins and genes modulated by SAHA identified gene ontology terms related to NFκB signaling, protein folding and autophagy, which are all relevant to HIV reactivation. In summary, SAHA modulated numerous host cell transcripts, proteins and post-translational modifications of proteins, which would be expected to have very mixed effects on the induction of HIV-specific transcription and protein function. Proteome profiling highlighted a number of potential counter-regulatory effects of SAHA with respect to viral induction, which transcriptome profiling alone would not have identified. These observations could lead to a more informed selection and design of other HDACi with a more refined targeting profile, and prioritization of latency reversing agents of other classes to be used in combination with SAHA to achieve more potent induction of HIV expression.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis.

Infection of cells with DNA viruses triggers innate immune responses mediated by DNA sensors. cGMP-AMP synthase (cGAS) is a key DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which binds to and activates stimulator of interferon genes (STING), leading to IFN production and an antiviral response. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. We report that KSHV infection activates the cGAS-STING pathway, and that cGAS and STING also play an important role in regulating KSHV reactivation from latency. We screened KSHV proteins for their ability to inhibit this pathway and identified six viral proteins that block IFN-β activation through this pathway. This study is the first report identifying multiple viral proteins encoded by a human DNA virus that inhibit the cGAS-STING DNA sensing pathway. One such protein, viral interferon regulatory factor 1 (vIRF1), targets STING by preventing it from interacting with TANK binding kinase 1 (TBK1), thereby inhibiting STING's phosphorylation and concomitant activation, resulting in an inhibition of the DNA sensing pathway. Our data provide a unique mechanism for the negative regulation of STING-mediated DNA sensing. Moreover, the depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. The vIRF1-expressing cells also inhibited IFN-β production following infection with DNA pathogens. Collectively, our results demonstrate that gammaherpesviruses encode inhibitors that block cGAS-STING-mediated antiviral immunity, and that modulation of this pathway is important for viral transmission and the lifelong persistence of herpesviruses in the human population.

BackgroundQuantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.MethodsWe developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.FindingsIn suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.InterpretationsOur results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.Research in contextIn this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Infection with varicella zoster virus (VZV) causes varicella (chickenpox), which can be severe in immunocompromised individuals, infants and adults. Primary infection is followed by latency in ganglionic neurons. During this period, no virus particles are produced and no obvious neuronal damage occurs. Reactivation of the virus leads to virus replication, which causes zoster (shingles) in tissues innervated by the involved neurons, inflammation and cell death - a process that can lead to persistent radicular pain (postherpetic neuralgia). The pathogenesis of postherpetic neuralgia is unknown and it is difficult to treat. Furthermore, other zoster complications can develop, including myelitis, cranial nerve palsies, meningitis, stroke (vasculopathy), retinitis, and gastroenterological infections such as ulcers, pancreatitis and hepatitis. VZV is the only human herpesvirus for which highly effective vaccines are available. After varicella or vaccination, both wild-type and vaccine-type VZV establish latency, and long-term immunity to varicella develops. However, immunity does not protect against reactivation. Thus, two vaccines are used: one to prevent varicella and one to prevent zoster. In this Primer we discuss the pathogenesis, diagnosis, treatment, and prevention of VZV infections, with an emphasis on the molecular events that regulate these diseases. For an illustrated summary of this Primer, visit: http://go.nature.com/14xVI1.

The partial successes of the Phase 2 gB-based vaccine trials for HCMV highlight the very real likelihood that vaccine-mediated induction of antibodies that neutralize the fusion pathway of fibroblast infection is not sufficient as a singular strategy to confer protective efficacy against primary HCMV infection. Alternative strategies that serve as adjuncts to gB-based vaccines are likely required to target different aspects of the complex lifecycle of HCMV infection. There has been considerable recent interest in targeting the gH/gL/UL128/UL130/UL131 pentamer complex (gH/gL-PC) to neutralize the endocytic pathway of HCMV infection of epithelial and endothelial cells. Since both cell types are critical during primary mucosal infection, intrahost spread, and shedding of HCMV in an infected host, the gH/gL-PC represents a high-value target for vaccination to interrupt the HCMV lifecycle. The natural history of HCMV is exceedingly complex and incompletely resolved, and the protective efficacy generated by gH/gL-PC remains to be validated in clinical trials. Yet, there are salient aspects of its lifecycle that offer clues about how other novel vaccine strategies can be targeted to especially susceptible parts of the viral proteome to significantly disrupt HCMV's ability to infect susceptible hosts. In particular, the protracted evolution of Herpesvirales has endowed HCMV with two remarkable properties of its natural history: (1) lifelong persistence within immune hosts that develop extraordinarily large antiviral immune responses and (2) the ability to reinfect those with prior immunity. The latter phenotype strongly implies that, if HCMV can overcome prior immunity to initiate a new infection, it is likely irrelevant whether prior immunity derives from prior infection or prior vaccination. Both phenotypes are unified by the extensive devotion of the HCMV coding repertoire (~50%) to viral proteins that modulate host cell signaling, trafficking, activation, antigen presentation, and resistance to apoptosis. Collectively, these viral proteins are the likely reason for the high barrier to success for the 4-decade effort to design an HCMV vaccine, and they represent the viral proteins that make HCMV be the virus that it is. James Hanshaw wrote in 1971 that, based on a 15-year retrospective of congenital HCMV cases, "… any thoughtful program designed at prevention or treatment deserves consideration". Drawing upon natural history data from the nonhuman primate model of HCMV persistence and pathogenesis, a "thoughtful program" is put forth that HCMV immune-modulating proteins should be considered as vaccine candidates.

Many important aspects of human retroviral infections cannot be fully evaluated using only in vitro systems or unmodified animal models. An alternative approach involves the use of humanized mice, which consist of immunodeficient mice that have been transplanted with human cells and/or tissues. Certain humanized mouse models can support robust infection with human retroviruses including different strains of human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV). These models have provided wide-ranging insights into retroviral biology, including detailed information on primary infection, in vivo replication and pathogenesis, latent/persistent reservoir formation, and novel therapeutic interventions. Here we describe the humanized mouse models that are most commonly utilized to study retroviral infections, and outline some of the important discoveries that these models have produced during several decades of intensive research.

The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation.

ObjectiveThe eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir.DesignWe investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals.MethodsWe measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-long terminal repeat (2-LTR) circle HIV-1 DNA and immunophenotypes of CD4 T cells in 72 HIV-1-infected individuals on suppressive ART (23 individuals initiated ART less than 1 year post-infection, and 49 individuals initiated ART greater than 1 year post-infection). Correlations were analysed using nonparametric tests.ResultsThe enhanced expression of a few select host restriction factors, p21, schlafen 11 and PAF1, was strongly associated with reduced CD4 T-cell associated HIV RNA during ART (P

HIV can enter a state of latency that allows it to persist for decades in antiretroviral drug-treated patients. In a recent Nature paper, Deng et al. (2015) show that this latent reservoir also contains a large number of cytotoxic T lymphocyte escape mutants, presenting another challenge to HIV cure efforts.

Biological circuits can be controlled by two general schemes: environmental sensing or autonomous programs. For viruses such as HIV, the prevailing hypothesis is that latent infection is controlled by cellular state (i.e., environment), with latency simply an epiphenomenon of infected cells transitioning from an activated to resting state. However, we find that HIV expression persists despite the activated-to-resting cellular transition. Mathematical modeling indicates that HIV's Tat positive-feedback circuitry enables this persistence and strongly controls latency. To overcome the inherent crosstalk between viral circuitry and cellular activation and to directly test this hypothesis, we synthetically decouple viral dependence on cellular environment from viral transcription. These circuits enable control of viral transcription without cellular activation and show that Tat feedback is sufficient to regulate latency independent of cellular activation. Overall, synthetic reconstruction demonstrates that a largely autonomous, viral-encoded program underlies HIV latency—potentially explaining why cell-targeted latency-reversing agents exhibit incomplete penetrance.

HIV latency is the chief obstacle to eradicating HIV but is widely believed to be an evolutionary accident providing no lentiviral fitness advantage. However, findings of latency being "hardwired" into HIV's gene-regulatory circuitry appear inconsistent with latency being an evolutionary accident, given HIV's rapid mutation rate. Here, we propose that latency is an evolutionary "bet-hedging" strategy whose frequency has been optimized to maximize lentiviral transmission by reducing viral extinction during mucosal infections. The model quantitatively fits the available patient data, matches observations of high-frequency latency establishment in cell culture and primates, and generates two counterintuitive but testable predictions. The first prediction is that conventional CD8-depletion experiments in SIV-infected macaques increase latent cells more than viremia. The second prediction is that strains engineered to have higher replicative fitness—via reduced latency—will exhibit lower infectivity in animal-model mucosal inoculations. Therapeutically, the theory predicts treatment approaches that may substantially enhance "activate-and-kill" HIV-cure strategies.

Histone deacetylase inhibitors (HDACi) are being evaluated in a "shock-and-kill" therapeutic approach to reverse human immunodeficiency virus type-1 (HIV) latency from CD4(+) T cells. Using this approach, HDACi have induced HIV RNA synthesis in latently infected cells from some patients. The hope is that the increase in viral production will lead to killing of the infected cell either by the virus itself or by the patient's immune system, a "sterilizing cure." Although administered within the context of combination antiretroviral therapy, the infection of bystander cells remains a concern. In this study, we investigated the effect of HDACi (belinostat, givinostat, panobinostat, romidepsin, and vorinostat) on the productive infection of macrophages. We demonstrate that the HDACi tested do not alter the initial susceptibility of macrophages to HIV infection. However, we demonstrate that HDACi decrease HIV release from macrophages in a dose-dependent manner (belinostat < givinostat < vorinostat < panobinostat < romidepsin) via degradation of intracellular HIV through the canonical autophagy pathway. This mechanism involves unc-51-like autophagy-activating kinase 1 (ULK1) and the inhibition of the mammalian target of rapamycin and requires the formation of autophagosomes and their maturation into autolysosomes in the absence of increased cell death. These data provide further evidence in support of a role for autophagy in the control of HIV infection and suggest that careful consideration of off-target effects will be essential if HDACi are to be a component of a multipronged approach to eliminate latently infected cells.

Highly active antiretroviral therapy (HAART) is very effective in suppressing HIV-1 replication and restoring immune functions in HIV-infected individuals. However, it fails to eradicate the latent viral reservoirs and fully resolve chronic inflammation in HIV infection. The "shock-and-kill" strategy was recently proposed to induce latent HIV expression in the presence of HAART. Recent studies have shown that the protein kinase C (PKC) agonists are highly potent in inducing latent HIV expression from the viral reservoirs in vitro and ex vivo and in protecting primary CD4(+) T cells from HIV infection through down-modulation of their HIV coreceptor expression. The PKC agonists are excellent candidates for advancing to clinical HIV eradication strategies. This article will present a critical review of the structure and function of known PKC agonists, their mechanisms for the reactivation of latent HIV expression, and the potential of these compounds for advancing clinical HIV eradication strategies.

Short interspersed nuclear elements (SINEs) are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68) infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS)-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit.

Background: Behcet's Disease (BD) is one of the chronic inflammatory diseases with unknown etiology. The relationship between infectious agents and BD has not been clearly identified yet. Human cytomegalovirus (HCMV) causes a primary infection in the first years of life and remains latent in various tissues and cells. This study aimed to find out the frequency of HCMV among BD patients. Methods: In this cross-sectional study, serum samples from 103 BD patients were analyzed for HCMV by molecular and serological tests. The results were analyzed by IBM SPSS version 20. Results: The mean age of BD patients was 38.67±8.82, and 53% (54/103) were male. The mean duration of BD was 18.42±6.12 years. HCMV IgG antibody was found in 96% and 94% of males and females, respectively, and the IgM antibody was positive only in one male patient. HCMV DNA was not detected in the samples. Conclusion: Our result showed, there is no evidence of Cytomegalovirus infection among Behcet's patients. Like other studies IgG antibodies against CMV are prominent among the general population.and Behcet patients. While, IgM against HCMV was positive only in one patients. But, no CMV-DNA detected among 103 Behcet patients. However, the absence of the virus genome in a positive IgM person can be due to the Short-term DNAemia or due to rheumatoid factor interference. In addition, there is a time lag between primary infection and IgM antibody production (IgM level can remain undetectable because of delayed seroconversion owing to immunosuppressive agents). IgM antibodies can also persist for a long time after infection in some healthy individuals.

BackgroundHIV-1 can persist for the duration of a patient's life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle.ResultsWe developed a novel molecular clock-based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past.ConclusionsThese results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis.

Feline immunodeficiency virus (FIV) is a naturally-occurring, large animal model of lentiviral-induced immunodeficiency syndrome, and has been used as a model of HIV pathogenesis and therapeutic interventions. HIV reservoirs in the form of latent virus remain the primary roadblock to viral eradication and cure, and FIV has been previously established an animal model of lentiviral latency. The goal of this study was to determine whether administration of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) to aviremic, chronically FIV-infected cats would induce latent viral reactivation in vivo. A proof-of-concept experiment in a Transwell co-culture system demonstrated the ability of SAHA to reactivate latent virus which was replication competent and able to infect naïve cells. Oral SAHA (250mg/m(2)) was administered with food to four asymptomatic, experimentally FIV-infected cats and one uninfected control cat, and a limited pharmacokinetic and pharmacodynamic analysis was performed. A statistically significant increase in cell-associated FIV RNA was detected in the cat with the greatest serum SAHA exposure, and cell-free viral RNA was detected at one time point in the three cats that achieved the highest levels of SAHA in serum. Interestingly, there was a significant decrease in viral DNA burden at 2h post drug administration in the same three cats. Though the sample size is small and the drug response was modest, this study provides evidence that in vivo treatment of FIV-infected cats with the HDACi SAHA can induce viral transcriptional reactivation, which may be dependent upon the concentration of SAHA achieved in blood. Importantly, alternative putative antilatency therapy drugs, and multimodal drug combinations, could be studied in this in vivo system. The FIV/cat model provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus in vivo.

Halper-Stromberg et al. use a humanized mouse model to demonstrate that broadly neutralizing antibodies, when administered with a combination of HIV latency activators, can reduce persistent HIV reservoirs, as measured by plasma virus rebound. Their results support the use of broadly neutralizing antibodies in HIV-reservoir-purging strategies.

ObjectiveAlthough HAART effectively suppresses viral replication, it fails to eradicate latent viral reservoirs. The 'shock and kill' strategy involves the activation of HIV from latent reservoirs and targeting them for eradication. Our goal was to develop new approaches for activating HIV from latent reservoirs.DesignWe investigated capacity of Ingenol B (IngB), a newly modified derivative of Ingenol ester that was originally isolated from a Brazilian plant in Amazon, for its capacity and mechanisms of HIV reactivation.MethodsReactivation of HIV-1 by IngB was evaluated in J-Lat A1 cell culture model of HIV latency as well as in purified primary CD4 T cells from long-term HAART-treated virologically-suppressed HIV-infected individuals. The underlining molecular mechanisms of viral reactivation were investigated using flow cytometry, RT-qPCR and chromatin immunoprecipitation.ResultsIngB is highly effective in reactivating HIV in J-Lat A1 cells with relatively low cellular toxicity. It is also able to reactivate latent HIV in purified CD4 T cells from HAART-treated HIV-positive individuals ex vivo. Our data show that IngB may reactivate HIV expression by both activating protein kinase C (PKC)δ-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and directly inducing NF-κB protein expression. Importantly, IngB has a synergistic effect with JQ1, a BET bromodomain inhibitor, in latent HIV reactivation.ConclusionsIngB is a new promising compound to activate latent HIV reservoirs. Our data suggest that formulating novel derivatives from Ingenol esters may be an innovative approach to develop new lead compounds to reactivate latent HIV.

HypothesisPretreatment with antiherpetic medications and steroids decreases likelihood of development of delayed facial paralysis (DFP) after otologic surgery.BackgroundHeat-induced reactivation of herpes simplex virus type 1 (HSV1) in geniculate ganglion neurons (GGNs) is thought to cause of DFP after otologic surgery. Antiherpetic medications and dexamethasone are used to treat DFP. Pretreatment with these medications has been proposed to prevent development of DFP.MethodsRat GGN cultures were latently infected with HSV1 expressing a lytic protein-GFP chimera. Cultures were divided into pretreatment groups receiving acyclovir (ACV), acyclovir-plus-dexamethasone (ACV + DEX), dexamethasone alone (DEX), or untreated media (control). After pretreatment, all cultures were heated 43°C for 2 hours. Cultures were monitored daily for reactivation with fluorescent microscopy. Viral titers were determined from culture media.ResultsHeating cultures to 43°C for 2 hours leads to HSV1 reactivation and production of infectious virus particles (59 ± 6.8%); heating cultures to 41°C showed a more variable frequency of reactivation (60 ± 40%), compared with baseline rates of 14.4 ± 5%. Cultures pretreated with ACV showed lower reactivation rates (ACV = 3.7%, ACV + DEX = 1.04%) compared with 44% for DEX alone. Viral titers were lowest for cultures treated with ACV or ACV + DEX.ConclusionGGN cultures harboring latent HSV1 infection reactivate when exposed to increased temperatures that can occur during otologic surgery. Pretreatment with ACV before heat provides prophylaxis against heat-induced HSV reactivation, whereas DEX alone is associated with higher viral reactivation rates. This study provides evidence supporting the use of prophylactic antivirals for otologic surgeries associated with high rates of DFP.

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity.

BackgroundTranscriptionally silent human immunodeficiency virus type 1 (HIV-1) DNA persists in resting memory CD4(+) T cells despite antiretroviral therapy. In a primary cell model, the antialcoholism drug disulfiram has been shown to induce HIV-1 transcription in latently infected resting memory CD4(+) T cells at concentrations achieved in vivo.MethodsWe conducted a single-arm pilot study to evaluate whether 500 mg of disulfiram administered daily for 14 days to HIV-1-infected individuals on stable suppressive antiretroviral therapy would result in reversal of HIV-1 latency with a concomitant transient increase in residual viremia or depletion of the latent reservoir in resting memory CD4(+) T cells.ResultsDisulfiram was safe and well tolerated. There was a high level of subject-to-subject variability in plasma disulfiram levels. The latent reservoir did not change significantly (1.16-fold change; 95% confidence interval [CI], .70- to 1.92-fold; P = .56). During disulfiram administration, residual viremia did not change significantly compared to baseline (1.53-fold; 95% CI, .88- to 2.69-fold; P = .13), although residual viremia was estimated to increase by 1.88-fold compared to baseline during the postdosing period (95% CI, 1.03- to 3.43-fold; P = .04). In a post hoc analysis, a rapid and transient increase in viremia was noted in a subset of individuals (n = 6) with immediate postdose sampling (HIV-1 RNA increase, 2.96-fold; 95% CI, 1.29- to 6.81-fold; P = .01).ConclusionsAdministration of disulfiram to patients on antiretroviral therapy does not reduce the size of the latent reservoir. A possible dose-related effect on residual viremia supports future studies assessing the impact of higher doses on HIV-1 production. Disulfiram affects relevant signaling pathways and can be safely administered, supporting future studies of this drug.

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α-/- (lack functional CD8 T cells and CD8α+ DCs), CD8β-/- (have functional CD8α+ T cells and CD8α+ DCs), and β2m-/- (lack functional CD8 T cells but have CD8α+ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+ DCs) versus WT C3H (have functional CD8α T cells and CD8α+ DCs) mice. We also determined whether the phenotype of CD8α-/- and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+ T cells or bone marrow (BM) derived CD8α+ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.

Despite significant advances in our understanding of HIV, a cure has not been realized for the more than 34 million infected with this virus. HIV is incurable because infected individuals harbor cells where the HIV provirus is integrated into the host's DNA but is not actively replicating and thus is not inhibited by antiviral drugs. Similarly, these latent viruses are not detected by the immune system. In this Review, we discuss HIV-1 latency and the mechanisms that allow this pathogenic retrovirus to hide and persist by exploiting the cellular vehicles of immunological memory.

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in different neuronal subtypes (A5+ and KH10+) in murine trigeminal ganglia, results which correlate with restricted productive infection in these neurons in vitro. HSV-2 latency-associated transcript (LAT) contains a cis-acting regulatory element near the transcription start site that promotes productive infection in A5+ neurons and a second element in exon 1 that inhibits productive infection in KH10+ neurons. HSV-1 contains no such regulatory sequences, demonstrating different mechanisms for regulating productive HSV infection in neurons.

Antiretroviral therapy for HIV infection needs lifelong access and strict adherence to regimens that are both expensive and associated with toxic effects. A curative intervention will be needed to fully stop the epidemic. The failure to eradicate HIV infection during long-term antiretroviral therapy shows the intrinsic stability of the viral genome in latently infected CD4T cells and other cells, and possibly a sustained low-level viral replication. Heterogeneity in latently infected cell populations and homoeostatic proliferation of infected cells might affect the dynamics of virus production and persistence. Despite potent antiretroviral therapy, chronic immune activation, inflammation, and immune dysfunction persist, and are likely to have important effects on the size and distribution of the viral reservoir. The inability of the immune system to recognise cells harbouring latent virus and to eliminate cells actively producing virus is the biggest challenge to finding a cure. We look at new approaches to unravelling the complex virus-host interactions that lead to persistent infection and latency, and discuss the rationale for combination of novel treatment strategies with available antiretroviral treatment options to cure HIV.

HIV latency is a major barrier to viral eradication from infected individuals. Lusic et al. (2013) show that HIV latency is established by spatially positioning the proviral chromatin in close proximity to promyelocytic leukemia (PML) nuclear bodies, a reversible process that recruits the methyltransferase enzyme G9a to the latent viral promoter.

The Herpesviridae include the Epstein-Barr Virus (EBV) and the Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are oncogenic gamma-herpesviruses. These viruses manipulate host cellular mechanisms, including through ubiquitin-mediated pathways, to promote viral replication and oncogenesis. Ubiquitin, a regulatory protein which tags substrates for degradation or alters their function, is manipulated by both EBV and KSHV to facilitate viral persistence and cancer development. EBV infects approximately 90% of the global population and is implicated in malignancies including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma. EBV latency proteins, notably LMP1 and EBNA3C, use ubiquitin-mediated mechanisms to inhibit apoptosis, promote cell proliferation, and interfere with DNA repair, contributing to tumorigenesis. EBV's lytic proteins, including BZLF1 and BPLF1, further disrupt cellular processes to favor oncogenesis. Similarly, KSHV, a causative agent of Kaposi's Sarcoma and lymphoproliferative disorders, has a latency-associated nuclear antigen (LANA) and other latency proteins that manipulate ubiquitin pathways to degrade tumor suppressors, stabilize oncogenic proteins, and evade immune responses. KSHV's lytic cycle proteins, such as RTA and Orf64, also use ubiquitin-mediated strategies to impair immune functions and promote oncogenesis. This review explores the ubiquitin-mediated interactions of EBV and KSHV proteins, elucidating their roles in viral oncogenesis. Understanding these mechanisms offers insights into the similarities between the viruses, as well as provoking thought about potential therapeutic targets for herpesvirus-associated cancers.

Background: A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB), but we do not know how TB impacts the HIV reservoir., Methods: Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Haïtien d'Étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince, Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring., Results: In the identification cohort, we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR, 183-1173 vs 117; IQR, 24-279 intact provirus per million CD4, respectively; P =0.0001). This difference also was seen in the validation cohort (n=31), (median 102; IQR, 0-737 vs 0; IQR, 0-24.5 intact provirus per million CD4, P =0.03) for TB vs no-TB history groups, respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524, P = 0.0025) and interleukin-2 (r=0.622, P =0.0002)., Conclusions: People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells, even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas., Competing Interests: The authors report no competing financial interests., (Copyright © 2024 Pathogens and Immunity.)

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Viruses normally reprogram the host cell metabolic pathways as well as metabolic sensors to facilitate their persistence. The serine-threonine liver kinase B1 (LKB1) is a master upstream kinase of 5'-AMP-activated protein kinase (AMPK) that senses the energy status and therefore regulates the intracellular metabolic homeostasis. Previous studies showed that AMPK restricts Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in endothelial cells during primary infection and promotes primary effusion lymphoma (PEL) cell survival. However, the role of LKB1 in KSHV lytic reactivation and KSHV-associated malignancies is unclear. In this study, we found that LKB1 is phosphorylated or activated in KSHV-positive PEL cells. Mechanistically, KSHV-encoded vCyclin mediated LKB1 activation in PEL cells, as vCyclin knockout ablated, while vCyclin overexpression enhanced LKB1 activation. Furthermore, knockdown of LKB1 inactivated AMPK and induced KSHV reactivation, as indicated by the increased expression of viral lytic genes and the increased virions in supernatants. Accordingly, AMPK inhibition by functional knockdown or a pharmacologic inhibitor, Compound C, promoted KSHV reactivation in PEL cells. Furthermore, inhibition of either LKB1 or AMPKα1 efficiently induced cell death by apoptosis of PEL cells both in vitro and in vivo . Together, these results identify LKB1 as a vulnerable target for PEL, which could be potentially exploited for treating other virus-associated diseases.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human cancers, such as primary effusion lymphoma (PEL). Here, we showed that serine-threonine liver kinase B1 (LKB1), upstream of 5 ' AMP-activated protein kinase (AMPK), is activated by KSHV-encoded vCyclin and maintains KSHV latency in PEL cells. Inhibition of either LKB1 or AMPK enhances KSHV lytic replication from latency, which at least partially accounts for PEL cell death by apoptosis. Compound C, a potent AMPK inhibitor, induced KSHV reactivation and efficiently inhibited PEL progression in vivo . Thus, our work revealed that LKB1 is a potential therapeutic target for KSHV-associated cancers., Competing Interests: The authors declare no conflict of interest.

Equid alphaherpesvirus 1 (EHV-1) has been linked to the emergence of neurological disorders, with the horse racing industry experiencing significant impacts from outbreaks of equine herpesvirus myeloencephalopathy (EHM). Building robust immune memory before pathogen exposure enables rapid recognition and elimination, preventing infection. This is crucial for effectively managing EHV-1. Removing neuropathogenic factors and immune evasion genes to develop live attenuated vaccines appears to be a successful strategy for EHV-1 vaccines. We created mutant viruses without ORF38 and ORF37/38 and validated their neuropathogenicity and immunogenicity in hamsters. The ∆ORF38 strain caused brain tissue damage at high doses, whereas the ∆ORF37/38 strain did not. Dexamethasone was used to confirm latent herpesvirus infection and reactivation. Dexamethasone injection increased viral DNA load in the brains of hamsters infected with the parental and ∆ORF38 strains, but not in those infected with the ∆ORF37/38 strain. Immunizing hamsters intranasally with the ∆ORF37/38 strain as a live vaccine produced a stronger immune response compared to the ∆ORF38 strain at the same dose. The hamsters demonstrated effective protection against a lethal challenge with the parental strain. This suggests that the deletion of ORF37/38 may effectively inhibit latent viral infection, reduce the neuropathogenicity of EHV-1, and induce a protective immune response.

Research is underway to develop a vaccine to prevent and cure infection from herpes simplex virus (HSV). It emphasizes the critical need for immunization to address public health issues and the shortcomings of existing treatment options. Furthermore, studies on the HSV vaccine advance the field of immunology and vaccine creation, which may help in the battle against other viral illnesses. The current lack of such a vaccine is, in part, due to herpes viral latency in sensory ganglions. Current vaccines rely on tissue-resident memory CD8 + T cells, which are known to provide protection against subsequent HSV reinfection and reactivation without correlating with other immune subsets. For that reason, there is no effective vaccine that can provide protection against latent or recurrent herpes infection. This review focuses on conventional methods for evaluating the efficacy of a herpes vaccine using differential CD8 + T cells and important unaccounted immune aspects for designing an effective vaccine against herpes.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi's sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production.

Gammaherpesviruses are ubiquitous, lifelong pathogens associated with multiple cancers that infect over 95% of the adult population. Increases in viral reactivation, due to stress and other unknown factors impacting the immune response, frequently precedes lymphomagenesis. One potential stressor that could promote viral reactivation and increase viral latency would be the myriad of infections from bacterial and viral pathogens that we experience throughout our lives. Using murine gammaherpesvirus 68 (MHV68), a mouse model of gammaherpesvirus infection, we examined the impact of bacterial challenge on gammaherpesvirus infection. We challenged MHV68 infected mice during the establishment of latency with nontypeable Haemophilus influenzae (NTHi) to determine the impact of bacterial infection on viral reactivation and latency. Mice infected with MHV68 and then challenged with NTHi, saw increases in viral reactivation and viral latency. These data support the hypothesis that bacterial challenge can promote gammaherpesvirus reactivation and latency establishment, with possible consequences for viral lymphomagenesis., Competing Interests: 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

HIV/AIDS cannot be cured because of the persistence of the viral reservoir. Because of the complexity of the cellular composition and structure of the human organs, HIV reservoirs of anatomical site are also complex. Recently, although a variety of molecules have been reported to be involved in the establishment and maintenance of the viral reservoirs, or as marker of latent cells, the research mainly focuses on blood and lymph nodes. Now, the characteristics of the viral reservoir in tissue are not yet fully understood. In this study, various tissues were collected from SIVmac239-infected monkeys, and the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in them were compared with character of the anatomical viral reservoir under early treatment. The results showed that short-term combination antiretroviral therapy (cART) starting from 3 days after infection could significantly inhibit viremia and reduce the size of the anatomical viral reservoir, but it could not eradicate de novo infections and ongoing replication of virus. Moreover, the effects of early cART on the level of total SIV DNA, SIV 2-LTR DNA, and cell-associated virus RNA in different tissues were different, which changed the size distribution of viral reservoir in anatomical site. Finally, the contribution of nonlymphoid tissues, especially liver and lung, to the viral reservoir increased after treatment, while the contribution of intestinal lymphoid to the viral reservoir significantly reduced. These results suggested that early treatment effectively decreased the size of viral reservoir, and that the effects of cART on the tissue viral reservoir varied greatly by tissue type. The results implied that persistent existence of virus in nonlymphoid tissues after short-term treatment suggested that the role of nonlymphoid tissues cannot be ignored in development strategies for AIDS therapy.

Human alphaherpesvirus 1 (HSV-1) establishes life-long latency in sensory neurons in trigeminal ganglia (TG), brainstem neurons, and other CNS neurons. Two important segments of the brainstem were examined in this study: principal sensory nucleus of the spinal trigeminal tract (Pr5) because it receives direct afferent inputs from TG, and locus coeruleus (LC) because it is indirectly connected to Pr5 and LC sends axonal projections to cortical structures, which may facilitate viral spread from brainstem to the brain. The only viral gene abundantly expressed during latency is the latency associated transcript (LAT). Previous studies revealed 8-week old female C57Bl/6 mice infected with a LAT null mutant (dLAT2903) versus wild-type (wt) HSV-1 exhibit higher levels of senescence markers and inflammation in LC of females. New studies revealed 1-year old mice latently infected with wt HSV-1 or dLAT2903 contained differences in neuroinflammation and senescence in Pr5 and LC versus young mice. In summary, these studies confirm HSV-1 promotes neuro-inflammation in the brainstem, which may accelerate neurodegenerative disease., Competing Interests: 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., (Copyright © 2024 Oklahoma State University, College of Veterinary Medicine. Published by Elsevier B.V. All rights reserved.)

CD80 is the best-known costimulatory molecule for effective T cell functions. Many different reports have summarized the role of CD80 in HSV-1 and its functions in maintaining adaptive immunity, which is the main player in causing herpes stromal keratitis (HSK). To determine the effects of absence or overexpression of CD80 in HSV-1 infection, we infected CD80 -/- and WT mice with a recombinant HSV-1 expressing murine CD80 (HSV-CD80) in place of the latency associated transcript (LAT). Parental dLAT2903 virus lacking LAT was used as a control. After infection, critical components of infection like virus replication, eye disease, early cellular infiltrates into the corneas and trigeminal ganglia (TG), latency-reactivation in the infected mice were determined. Our findings reveal that the absence of CD80 in the CD80 -/- mice infected with both viruses did not affect the viral titers in the mice eyes or eye disease, but it played a significant role in critical components of HSV-induced immunopathology. The WT mice infected with dLAT2903 virus had significantly higher levels of latency compared with the CD80 -/- mice infected with dLAT2903 virus, while levels of latency as determined by gB DNA expression were similar between the WT and CD80 -/- mice infected with HSV-CD80 virus. In contrast to the differences in the levels of latency between the infected groups, the absence of CD80 expression in the CD80 -/- mice or its overexpression by HSV-CD80 virus did not have any effect on the time of reactivation. Furthermore, the absence of CD80 expression contributed to more inflammation in the CD80 -/- -infected mice. Overall, this study suggests that in the absence of CD80, inflammation increases, latency is reduced, but reactivation is not affected. Altogether, our study suggests that reduced latency correlated with reduced levels of inflammatory molecules and blocking or reducing expression of CD80 could be used to mitigate the immune responses, therefore controlling HSV-induced infection.