Nanotechnology Frontiers in γ-Herpesviruses Treatments.

Epstein-Barr Virus (EBV) and Kaposi's sarcoma associated-herpesvirus (KSHV) are γ-herpesviruses that belong to the Herpesviridae family. EBV infections are linked to the onset and progression of several diseases, such as Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), and lymphoproliferative malignancies arising in post-transplanted patients (PTDLs). KSHV, an etiologic agent of Kaposi's sarcoma (KS), displays primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Many therapeutics, such as bortezomib, CHOP cocktail medications, and natural compounds (e.g., quercetin or curcumin), are administrated to patients affected by γ-herpesvirus infections. These drugs induce apoptosis and autophagy, inhibiting the proliferative and cell cycle progression in these malignancies. In the last decade, many studies conducted by scientists and clinicians have indicated that nanotechnology and nanomedicine could improve the outcome of several treatments in γ-herpesvirus-associated diseases. Some drugs are entrapped in nanoparticles (NPs) expressed on the surface area of polyethylene glycol (PEG). These NPs move to specific tissues and exert their properties, releasing therapeutics in the cell target. To treat EBV- and KSHV-associated diseases, many studies have been performed in vivo and in vitro using virus-like particles (VPLs) engineered to maximize antigen and epitope presentations during immune response. NPs are designed to improve therapeutic delivery, avoiding dissolving the drugs in toxic solvents. They reduce the dose-limiting toxicity and reach specific tissue areas. Several attempts are ongoing to synthesize and produce EBV vaccines using nanosystems.

T Cell-Intrinsic Interferon Regulatory Factor 1 Expression Suppresses Differentiation of CD4+ T Cell Populations That Support Chronic Gammaherpesvirus Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with B cell lymphomas. To establish chronic infection, these viruses usurp B cell differentiation and drive a robust germinal center response to expand the latent viral reservoir and gain access to memory B cells. Germinal center B cells, while important for the establishment of latent infection, are also thought to be the target of viral transformation. The host and viral factors that impact the gammaherpesvirus-driven germinal center response are not clearly defined. We show that the global expression of the antiviral and tumor suppressor interferon regulatory factor 1 (IRF-1) selectively attenuates the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and restricts the expansion of the latent viral reservoir. In this study, we found that T cell-intrinsic IRF-1 expression recapitulates some aspects of the antiviral state imposed by IRF-1 during chronic MHV68 infection, including the attenuation of the germinal center response and viral latency in the spleen. We also discovered that global and T cell-intrinsic IRF-1 deficiency leads to an unhindered rise of interleukin-17A (IL-17A)-expressing and follicular helper T cell populations, two CD4+ T cell subsets that support chronic MHV68 infection. Thus, this study unveils a novel aspect of the antiviral activity of IRF-1 by demonstrating IRF-1-mediated suppression of specific CD4+ T cell subsets that support chronic gammaherpesvirus infection. IMPORTANCE Gammaherpesviruses infect over 95% of the adult population, last the lifetime of the host, and are associated with multiple cancers. These viruses usurp the germinal center response to establish lifelong infection in memory B cells. This manipulation of B cell differentiation by the virus is thought to contribute to lymphomagenesis, although exactly how the virus precipitates malignant transformation in vivo is unclear. IRF-1, a host transcription factor and a known tumor suppressor, restricts the MHV68-driven germinal center response in a B cell-extrinsic manner. We found that T cell-intrinsic IRF-1 expression attenuates the MHV68-driven germinal center response by restricting the CD4+ T follicular helper population. Furthermore, our study identified IRF-1 as a novel negative regulator of IL-17-driven immune responses, highlighting the multifaceted role of IRF-1 in gammaherpesvirus infection.

Proteomic approaches to investigate gammaherpesvirus biology and associated tumorigenesis.

The DNA viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are members of the gammaherpesvirus subfamily, a group of viruses whose infection is associated with multiple malignancies, including cancer. The primary host for these viruses is humans and, like all herpesviruses, infection with these pathogens is lifelong. Due to the persistence of gammaherpesvirus infection and the potential for cancer formation in infected individuals, there is a driving need to understand not only the biology of these viruses and how they remain undetected in host cells but also the mechanism(s) by which tumorigenesis occurs. One of the methods that has provided much insight into these processes is proteomics. Proteomics is the study of all the proteins that are encoded by a genome and allows for (i) identification of existing and novel proteins derived from a given genome, (ii) interrogation of protein-protein interactions within a system, and (iii) discovery of druggable targets for the treatment of malignancies. In this chapter, we explore how proteomics has contributed to our current understanding of gammaherpesvirus biology and their oncogenic processes, as well as the clinical applications of proteomics for the detection and treatment of gammaherpesvirus-associated cancers.

Utilization of Host Cell Chromosome Conformation by Viral Pathogens: Knowing When to Hold and When to Fold.

Though viruses have their own genomes, many depend on the nuclear environment of their hosts for replication and survival. A substantial body of work has therefore been devoted to understanding how viral and eukaryotic genomes interact. Recent advances in chromosome conformation capture technologies have provided unprecedented opportunities to visualize how mammalian genomes are organized and, by extension, how packaging of nuclear DNA impacts cellular processes. Recent studies have indicated that some viruses, upon entry into host cell nuclei, produce factors that alter host chromatin topology, and thus, impact the 3D organization of the host genome. Additionally, a variety of distinct viruses utilize host genome architectural factors to advance various aspects of their life cycles. Indeed, human gammaherpesviruses, known for establishing long-term reservoirs of latent infection in B lymphocytes, utilize 3D principles of genome folding to package their DNA and establish latency in host cells. This manipulation of host epigenetic machinery by latent viral genomes is etiologically linked to the onset of B cell oncogenesis. Small DNA viruses, by contrast, are tethered to distinct cellular sites that support virus expression and replication. Here, we briefly review the recent findings on how viruses and host genomes spatially communicate, and how this impacts virus-induced pathology.

Update of the global distribution of human gammaherpesvirus 8 genotypes.

Human gammaherpesvirus 8 (HHV-8) consists of six major clades (A-F) based on the genetic sequence of the open reading frame (ORF)-K1. There are a few conflicting reports regarding the global distribution of the different HHV-8 genotypes. This study aimed to determine the global distribution of the different HHV-8 genotypes based on phylogenetic analysis of the ORF-K1 coding region using sequences published in the GenBank during 1997-2020 and construct a phylogenetic tree using the maximum likelihood algorithm with the GTR + I + G nucleotide substitution model. A total of 550 sequences from 38 countries/origins were analysed in this study. Genotypes A and C had similar global distributions and were prevalent in Africa and Europe. Genotype B was prevalent in Africa. Of the rare genotypes, genotype D was reported in East Asia and Oceania and genotype E in South America, while genotype F was prevalent in Africa. The highest genotypic diversity was reported in the American continent, with Brazil housing five HHV-8 genotypes (A, B, C, E, and F). In this study, we present update of the global distribution of HHV-8 genotypes, providing a basis for future epidemiological and evolutionary studies of HHV-8.

Herpesvirus Epigenetic Reprogramming and Oncogenesis.

Among all of the known biological carcinogens, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are two of the classical oncogenic herpesviruses known to induce the oncogenic phenotype. Many studies have revealed important functions related to epigenetic alterations of the EBV and KSHV genomes that mediate oncogenesis, but the detailed mechanisms are not fully understood. It is also challenging to fully describe the critical cellular events that drive oncogenesis as well as a comprehensive map of the molecular contributors. This review introduces the roles of epigenetic modifications of these viral genomes, including DNA methylation, histone modification, chromatin remodeling, and noncoding RNA expression, and elucidates potential strategies utilized for inducing oncogenesis by these human gammaherpesviruses.

Modeling oncogenic herpesvirus infections in humanized mice.

The creation of humanized mice generally involves the reconstitution of immunodeficient mice with human immune constituents. Different methodologies have been employed, and significant progress has been made towards the development of robustly humanized mouse models. Some of the techniques used include the injection of mature human immune cells, the injection of human hematopoietic stem cells (HSCs) capable of reconstituting radiation-depleted murine bone marrow, and the implantation of human fetal liver and thymus fragments under the kidney capsule to create a thymic organoid that can support thympoiesis. This review will serve as a brief introduction to the three most commonly utilized humanized mouse models for the study of gammaherpesvirus-driven pathogenesis, and highlight some of the critical discoveries these models have enabled.

Innate immunity and alpha/gammaherpesviruses: first impressions last a lifetime.

Innate immune system is considered the first line of defense during viral invasion, with the wealth of the literature demonstrating innate immune control of diverse viruses during acute infection. What is far less clear is the role of innate immune system during chronic virus infections. This short review focuses on alphaherpesviruses and gammaherpesviruses, two highly prevalent herpesvirus subfamilies that, following a brief, once in a lifetime period of acute lytic infection, establish life-long latent infection that is characterized by sporadic reactivation in an immunocompetent host. In spite of many similarities, these two viral families are characterized by distinct cellular tropism and pathogenesis. Here we focus on the published in vivo studies to review known interactions of these two viral subfamilies with the innate immunity of the intact host, both during acute and, particularly, chronic virus infection.

Gammaherpesvirus-infected germinal center cells express a distinct immunoglobulin repertoire.

The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1 In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation.

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

[Epstein-Barr virus (Herpesviridae: Gammaherpesvirinae: Lymphocryptovirus: Human gammaherpesvirus 4): replication strategies].

The Epstein-Barr virus (EBV), one of the most common in the human population, is capable of lifelong persistence in resting memory B-cells, in T-cells in case of type 2 EBV, and in some undifferentiated epithelial cells. In most people, EBV persistence is not accompanied by significant symptoms, but frequent virus activations are associated with the increased risks of severe diseases, such as chronic active Epstein-Barr virus infection, hemophagocytic lymphohistiocytosis, multiple sclerosis, systemic lupus erythematosus, gastric and nasopharyngeal carcinomas, and a variety of T- and B-cell lymphomas. Therefore, the molecular viral and host cell processes during asymptomatic or low-symptom EBV persistence are of great interest. This review describes the behavior of the viral DNA in an infected cell and the forms of its existence (linear, circular episome, chromosomally integrated forms), as well as methods of EBV genome copying. Two closely related cycles of viral reproduction are considered. Lytic activation is unfavorable for the survival of a particular viral genome in the cell, and may be a result of differentiation of a latently infected cell, or the arrival of stress signals due to adverse extracellular conditions. The EBV has a large number of adaptive mechanisms for limiting lytic reactivation and reducing hostility of host immune cells. Understanding the molecular aspects of EBV persistence will help in the future develop more effective targeted drugs for the treatment of both viral infection and associated diseases.

A Gammaherpesvirus MicroRNA Targets EWSR1 (Ewing Sarcoma Breakpoint Region 1) In Vivo To Promote Latent Infection of Germinal Center B Cells.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies, including B cell lymphomas. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong latent infection. To accomplish this, gammaherpesviruses employ an array of gene products, including microRNAs (miRNAs). Although numerous host mRNA targets of gammaherpesvirus miRNAs have been identified, the in vivo relevance of repression of these targets remains elusive due to species restriction. Murine gammaherpesvirus 68 (MHV68) provides a robust virus-host system to dissect the in vivo function of conserved gammaherpesvirus genetic elements. We identified here MHV68 mghv-miR-M1-7-5p as critical for in vivo infection and then validated host EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using novel, target-specific shRNA-expressing viruses, we determined that EWSR1 repression in vivo was essential for germinal center B cell infection. These findings provide the first in vivo demonstration of the biological significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.IMPORTANCE Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong infection. To accomplish this, gammaherpesviruses employ an array of gene products, including miRNAs, short noncoding RNAs that bind to and repress protein synthesis from specific target mRNAs. The in vivo relevance of repression of targets of gammaherpesvirus miRNAs remains highly elusive. Here, we identified a murine gammaherpesvirus miRNA as critical for in vivo infection and validated the host mRNA EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using a novel technology, we demonstrated that repression of EWSR1 was essential for in vivo infection of the critical B cell reservoir. These findings provide the first in vivo demonstration of the significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.

γ-herpesvirus latency attenuates Mycobacterium tuberculosis infection in mice.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a bacterial pathogen which is transmitted via aerosol and establishes a chronic lung infection. In naïve hosts, Mtb grows for several weeks without being restricted by IFNγ-producing T cells, which eventually accumulate and limit Mtb dissemination. In this study, we used a mouse model of Mtb/γ-herpesvirus (γHV) coinfection to test the hypothesis that latent γHV infection alters host resistance to Mtb. γHVs are DNA viruses which elicit a polyclonal T cell response and attenuate some acute bacterial pathogens in mice; whether γHVs modulate infection with Mtb is unknown. Here, mice harboring latent mouse gammaherpesvirus 68 (MHV68)-a γHV genetically and biologically related to human Epstein Barr virus (EBV)-were infected via aerosol with a low dose of virulent Mtb. Mtb burdens and IFNγ+ T cell frequencies in mice with latent MHV68 (MHV68POS mice) were subsequently measured and compared to control mice that did not harbor latent MHV68 (MHV68NEG mice). Relative to MHV68NEG controls, MHV68POS mice more effectively limited Mtb growth and dissemination, and had higher frequencies of CD4+IFNγ+ cells in lung-draining lymph nodes. Collectively, our results support a model wherein latent γHV confers moderate protection against subsequent Mtb infection.

The effect of maternal immunity on the equine gammaherpesvirus type 2 and 5 viral load and antibody response.

Two types of gammaherpesviruses (γEHV) are known to infect horses, EHV-2 and EHV-5. Foals become infected early in life, probably via the upper respiratory tract, despite maternal antibodies. In this study, we analyzed samples from a herd of mares and their foals. The foals were followed from birth to 22 months of age and the dams during the first 6 months postpartum. Blood and nasal swab samples were taken regularly for evaluation of antibody responses, virus isolation and viral load by qPCR. EHV-2 was isolated on day 5, and EHV-5 on day 12, earlier than previously reported. γEHV specific antibodies were not detectable in serum of foals before colostrum intake but peaked a few days after colostrum. Overall, EHV-2 viral load peaked in nasal swab at three to four months of age, paralleled with decline in maternal antibodies, but EHV-5 viral load did not peak until month 12. Maternal antibodies had a notable effect on the viral load and induction of endogenous antibody production. Foals were grouped in two groups depending on the mare's γEHV specific total IgG levels in serum at birth, group-high and group-low. Group-high had higher levels of maternal γEHV specific total IgG and IgG4/7 for the first 3 months, but when the endogenous production had superseded maternal antibodies, group-low was higher. The maternal antibodies had an effect on the γEHV viral load. Group-low peaked in EHV-2 viral load one month earlier than group-high. These effects were more evident for EHV-5, as there were seven months between the viral load peaks for the groups. The study provides information on how maternal antibody transfer affects γEHV shedding and antibody production in offspring. It also extends our knowledge on the occurrence of EHV-2 and EHV-5 infection in foals during the first two years of life.

Novel feline viruses: Emerging significance of gammaherpesvirus and morbillivirus infections.

PRACTICAL RELEVANCE: New technologies capable of sequencing the genetic material in any given biological sample, combined with computer-based algorithms for sequence assembly and analysis, have revolutionised infectious disease research. The rate at which novel viruses are being discovered now exceeds our understanding of their clinical relevance. Novel viruses may contribute to diseases that are major causes of feline morbidity and mortality, including cancer and chronic kidney disease. The identification of new viral pathogens raises the prospect of not only improved patient outcomes through specific treatment but even disease prevention through viral control measures. CLINICAL CHALLENGES: It can be difficult to determine the role of a novel virus in disease development. Disease may be an occasional outcome, often years after infection. A high prevalence of infection in the general population can make disease associations harder to identify and almost impossible to rule out. Host cofactors such as immune dysfunction, genetic background or coinfections may be required for manifestation of disease, and one virus species may be linked to a range of pathological sequelae. Establishing causality relies on evaluating accumulating evidence from multiple investigations, which is often hard to access by practitioners. GLOBAL IMPORTANCE: The worldwide distribution of gammaherpesvirus and morbillivirus infections in domestic cats underlines the potential of these viruses to negatively impact feline health and welfare globally. EVIDENCE BASE: This review relies on grade la-III evidence.

Endosomal Toll-Like Receptors 7 and 9 Cooperate in Detection of Murine Gammaherpesvirus 68 Infection.

Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-α) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to levels in wild type (WT) cells but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7-/-Tlr9-/- double-knockout pDC, the IFN-α response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-α response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7-/-Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7-/-Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7-/-Tlr9-/- splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host.

Macaca arctoides gammaherpesvirus 1 (strain herpesvirus Macaca arctoides): virus sequence, phylogeny and characterisation of virus-transformed macaque and rabbit cell lines.

Herpesvirus Macaca arctoides (HVMA) has the propensity to transform macaque lymphocytes to lymphoblastoid cells (MAL-1). Inoculation of rabbits with cell-free virus-containing supernatant resulted in the development of malignant lymphomas and allowed isolation of immortalised HVMA-transformed rabbit lymphocytes (HTRL). In this study, the HVMA genome sequence (approx. 167 kbp), its organisation, and novel aspects of virus latency are presented. Ninety-one open reading frames were identified, of which 86 were non-repetitive. HVMA was identified as a Lymphocryptovirus closely related to Epstein-Barr virus, suggesting the designation as 'Macaca arctoides gammaherpesvirus 1' (MarcGHV-1). In situ lysis gel and Southern blot hybridisation experiments revealed that the MAL-1 cell line contains episomal and linear DNA, whereas episomal DNA is predominantly present in HTRL. Integration of viral DNA into macaque and rabbit host cell genomes was demonstrated by fluorescence in situ hybridisation on chromosomal preparations. Analysis of next-generation sequencing data confirmed this finding. Approximately 400 read pairs represent the overlap between macaque and MarcGHV-1 DNA. Both, MAL-1 cells and HTRL show characteristics of a polyclonal tumour with B- and T-lymphocyte markers. Based on analysis of viral gene expression and immunohistochemistry, the persistence of MarcGHV-1 in MAL-1 cells resemble the latency type III, whereas the expression pattern observed in HTRL was more comparable with latency type II. There was no evidence of the presence of STLV-1 proviral DNA in MAL-1 and HTRL. Due to the similarity to EBV-mediated cell transformation, MarcGHV-1 expands the available in vitro models by simian and rabbit cell lines.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Host Tumor Suppressor p18INK4c Functions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis.

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.