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BACKGROUND AND AIMS. Epstein-Barr Virus (EBV)-associated Gastric Carcinoma (EBVaGC) is the most common EBV-associated cancer and accounts for ~10% of all gastric cancers (GC). Epstein-Barr virus Nuclear Antigen 1 (EBNA1), which is critical for the replication and maintenance of the EBV latent genome, is consistently expressed in all EBVaGC tumors. We previously developed small molecule inhibitors of EBNA1. In this study, we investigated the efficacy and selectivity of an EBNA1 inhibitor in cell-based and animal xenograft models of EBV-positive and EBV-negative gastric carcinoma. METHODS. We tested the potency of an EBNA1 inhibitor, VK-1727, in vitro and in xenograft studies, using EBV-positive (SNU-719 and YCCEL1) and EBV-negative (AGS and MKN74) GC cell lines. After treatment, we analyzed cell viability, proliferation, and RNA expression of EBV genes by RT-qPCR. RESULTS. Treatment with VK-1727 selectively inhibits cell cycle progression and proliferation in vitro. In animal studies, treatment with an EBNA1 inhibitor resulted in a significant dose-dependent decrease in tumor growth in EBVaGC xenograft models, but not in EBV-negative GC xenograft studies. Gene expression analysis revealed that short term treatment in cell culture tended towards viral gene activation, while long-term treatment in animal xenografts showed a significant decrease in viral gene expression. CONCLUSIONS. EBNA1 inhibitors are potent and selective inhibitors of cell growth in tissue culture and animal models of EBV-positive GC. Long-term treatment with EBNA1 inhibitors may lead to loss of EBV in mouse xenografts. These results suggest that pharmacological targeting of EBNA1 may be an effective strategy to treat patients with EBVaGC.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but its mechanism of protein stability is not completely understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2’ dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. We used mass spectrometry to identify EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460 to alanine or arginine abrogates EBNA1-driven DNA replication of oriP, while K461 mutations enhanced replication. These findings suggest that PLOD1 is a novel post-translational regulator of EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.ImportanceEBNA1 is essential for EBV latent infection and implicated in viral pathogenesis. We found that EBNA1 interacts with PLOD family of lysine hydroxylases and that this interaction is required for EBNA1 protein stability and function in viral persistence during viral latent infection. Identification of PLOD1 regulation of EBNA1 protein stability provide new opportunity to target EBNA1 for degradation in EBV associated disease.

Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus that contributes to the etiology of diverse human cancers and auto-immune diseases. EBV establishes a relatively benign, long-term latent infection in over 90 percent of the adult population. Yet, it also increases risk for certain cancers and auto-immune disorders depending on complex viral, host, and environmental factors that are only partly understood. EBV latent infection is found predominantly in memory B-cells, but the natural infection cycle and pathological aberrations enable EBV to infect numerous other cell types, including oral, nasopharyngeal, and gastric epithelia, B-, T-, and NK-lymphoid cells, myocytes, adipocytes, astrocytes, and neurons. EBV infected cells, free virus, and gene products can also be found in the CNS. In addition to the direct effects of EBV on infected cells and tissue, the effect of chronic EBV infection on the immune system is also thought to contribute to pathogenesis, especially auto-immune disease. Here, we review properties of EBV infection that may shed light on its potential pathogenic role in neurological disorders.

DAXX and ATRX are tumor suppressor proteins that form a histone H3.3 chaperone complex and are frequently mutated in cancers with the alternative lengthening of telomeres (ALT), such as pediatric glioblastoma. Rapid loss of function of either DAXX or ATRX are not by themselves sufficient to induce the ALT phenotype. However, cells lacking DAXX or ATRX can be readily selected for ALT-like features. Here, we show that DAXX and ATRX null glioblastoma cells with ALT-like features have defects in p53 chromatin binding and DNA damage response regulation. RNA-seq analysis of DAXX or ATRX null U87 glioblastoma cells with ALT-like features revealed that p53 pathway is among perturbed. ALT-selected DAXX and ATRX-null cells had aberrant response to DNA damaging agent etoposide. Both DAXX and ATRX-null ALT cells showed a loss of p53 binding at a subset of response elements. Complementation of DAXX null cells with a wild-type DAXX transgene rescued p53 binding and transcription, while the tumor associated mutation L130R that disrupts ATRX binding was incapable of rescuing p53 chromatin binding. We show that histone H3.3 binding is reduced in DAXX-null cells especially at subtelomeric p53 binding sites and telomere repeats. These findings indicate that DAXX and ATRX function to enable p53 chromatin binding through modulation of histone H3.3 binding, especially at sub-telomeric sites.

Epstein-Barr virus (EBV) establishes a lifelong latent infection that can be a causal agent for a diverse spectrum of cancers and autoimmune disease. A complex and dynamic viral lifecycle evades eradication by the host immune system and confounds antiviral therapeutic strategies. To date, there are no clinically approved vaccines or therapies that selectively target EBV as the underlying cause of EBV-associated disease. Here, we review the challenges and recent advances in the development of EBV-specific therapeutics for treatment of EBV-associated cancers.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.

Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS)., Author summary Trafficking of pathogenic B-cells to the CNS can drive CNS lymphoma and multiple sclerosis (MS). Here, we show that EBV+ B-cells can undergo an epigenetic switch conferring a neuroinvasive phenotype with upregulation of SPP1/Osteopontin, which when blocked reduces CNS penetrance.

Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers., Author summary EBV immortalization recapitulates aspects of natural B-cell development from germinal center reaction to memory B-cell differentiation. Here, we provide an integrated multi-omic approach to investigating the EBV-induced changes in B-cell transcriptome using RNA-seq, epigenome by ATAC-seq, and metabolome by mass spectrometry of polar metabolites. We identify purine metabolism as a major pathway that is significantly altered by EBV in each data set. We focus on EBNA1 and find that it binds directly to enhancer elements in a subset of genes, including key regulators of purine metabolism such as adenosine deaminase (ADA) and adenylate kinase 4 (AK4). EBV immortalized B-cells are strongly dependent on ADA for proliferation and inhibitors of ADA prevent B-cell immortalization by EBV. These findings identify purine metabolism as a major pathway targeted by EBV during primary infection and suggest that EBNA1 functions as a pioneering transcription factor that reprograms key regulatory genes in purine metabolism pathway.

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-β (TGF-β) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

Neonatal seizures disrupt normal synaptic maturation and often lead to later-life epilepsy and cognitive deficits. During early life, the brain exhibits heightened synaptic plasticity, in part due to a developmental overabundance of CaV1.2 L-type voltage gated calcium (Ca2+) channels (LT-VGCCs) and Ca2+-permeable AMPARs (CP-AMPARs) lacking GluA2 subunits. We hypothesized that early-life seizures overactivate these channels, in turn dysregulating Ca2+-dependent signaling pathways including that of methyl CPG binding protein 2 (MeCP2), a transcription factor implicated in the autism spectrum disorder (ASD) Rett Syndrome. Here, we show that in vivo hypoxia-induced seizures (HS) in postnatal day (P)10 rats acutely induced phosphorylation of the neuronal-specific target of activity-dependent MeCP2 phosphorylation, S421, as well as its upstream activator CaMKII T286. We next identified mechanisms by which activity-dependent Ca2+ influx induced MeCP2 phosphorylation using in vitro cortical and hippocampal neuronal cultures at embryonic day (E)18 + 10 days in vitro (DIV). In contrast to the prevalent role of NMDARs in the adult brain, we found that both CP-AMPARs and LT-VGCCs mediated MeCP2 S421 and CaMKII T286 phosphorylation induced by kainic acid (KA) or high potassium chloride (KCl) stimulation. Furthermore, in vivo post-seizure treatment with the broad-spectrum AMPAR antagonist NBQX, the CP-AMPAR blocker IEM-1460, or the LT-VGCC antagonist nimodipine blocked seizure-induced MeCP2 phosphorylation. Collectively, these results demonstrate that early-life seizures dysregulate critical activity-dependent developmental signaling pathways, in part via CP-AMPAR and LT-VGCC activation, providing novel age-specific therapeutic targets for convergent pathways underlying epilepsy and ASDs.

SummaryAutophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel bunyavirus that recently emerged in China. Infection with SFTSV is associated with case-fatality rates of up to 30%, and neither antivirals nor vaccines are available at present. Development of antiviral strategies requires the elucidation of virus-host cell interactions. Here, we analyzed host cell entry of SFTSV. Employing lentiviral and rhabdoviral vectors, we found that the Gn/Gc glycoproteins (Gn/Gc) of SFTSV mediate entry into a broad range of human and animal cell lines, as well as human macrophages and dendritic cells. The Gn/Gc proteins of La Crosse virus (LACV) and Rift Valley Fever Virus (RVFV), other members of the bunyavirus family, facilitated entry into an overlapping but not identical range of cell lines, suggesting that SFTSV, LACV, and RVFV might differ in their receptor requirements. Entry driven by SFTSV Gn/Gc was dependent on low pH but did not require the activity of the pH-dependent endosomal/lysosomal cysteine proteases cathepsins B and L. Instead, the activity of a cellular serine protease was required for infection driven by SFTSV and LACV Gn/Gc. Sera from convalescent SFTS patients inhibited SFTSV Gn/Gc-driven host cell entry in a dose-dependent fashion, demonstrating that the vector system employed is suitable to detect neutralizing antibodies. Finally, the C-type lectin DC-SIGN was found to serve as a receptor for SFTSV Gn/Gc-driven entry into cell lines and dendritic cells. Our results provide initial insights into cell tropism, receptor usage, and proteolytic activation of SFTSV and will aid in the understanding of viral spread and pathogenesis.

Multiple Sclerosis (MS) is the most common demyelinating disease of man with over 400,000 cases in the United States and over 2.5 million cases worldwide. There are over 64,000 citations in Pubmed dating back as far as 1887. Much has been learned over the past 129 years with a recent burst in therapeutic options (mostly anti-inflammatory) with newer medications in development that are neuroprotective and/or neuroreparative. However, with all these advancements the cause of MS remains elusive. There is a clear interplay of genetic, immunologic, and environmental factors that influences both the development and progression of this disorder. This chapter will give a brief overview of the history and pathogenesis of MS with attention to how host immune responses in genetically susceptible individuals contribute to the MS disease process. In addition, we will explore the role of infectious agents in MS as potential “triggers” of disease. Models of virus-induced demyelination will be discussed, with an emphasis on the recent interest in human herpesviruses and the role they may play in MS disease pathogenesis. Although we remain circumspect as to the role of any microbial pathogen in MS, we suggest that only through well-controlled serological, cellular immune, molecular, and animal studies we will be able to identify candidate agents. Ultimately, clinical interventional trials that either target a specific pathogen or class of pathogens will be required to make definitive links between the suspected agent and MS.

A myriad of factors favor the emergence and re-emergence of arthropod-borne viruses (arboviruses), including migration, climate change, intensified livestock production, an increasing volume of international trade and transportation, and changes to ecosystems (e.g., deforestation and loss of biodiversity). Consequently, arboviruses are distributed worldwide and represent over 30% of all emerging infectious diseases identified in the past decade. Although some arboviral infections go undetected or are associated with mild, flu-like symptoms, many are important human and veterinary pathogens causing serious illnesses such as arthritis, gastroenteritis, encephalitis and hemorrhagic fever and devastating economic loss as a consequence of lost productivity and high mortality rates among livestock. One of the most consistent molecular features of emerging arboviruses, in addition to their near exclusive use of RNA genomes, is the inclusion of viral, non-structural proteins that act as interferon antagonists. In this review, we describe these interferon antagonists and common strategies that arboviruses use to counter the host innate immune response. In addition, we discuss the complex interplay between host factors and viral determinants that are associated with virus emergence and re-emergence, and identify potential targets for vaccine and anti-viral therapies.

Viral infections of the CNS have been implicated in many debilitating illnesses including chronic neurodegenerative and neuroinflammatory disorders, neuropsychiatric conditions, acute encephalitis, and CNS malignancies. Although the effects of these manifold diseases are devastating, leading to death in some instances, the armamentarium of effective vaccines and therapeutics at our disposal to prevent and treat virus-mediated disorders of the CNS is astoundingly limited. It is, therefore, critical that we achieve a better understanding of the common mechanisms of neuroinflammation and neurodegeneration involved in these disorders so that more effective therapeutics can be developed. In this special issue of the Journal of Neuro-immune Pharmacology, we have assembled an extraordinary collection of contributions, including both comprehensive reviews and primary research articles, from leaders in the field of neurovirology. The manuscripts contained herein focus on a variety of CNS diseases caused by or associated with viruses from disparate families and highlight virus-induced mechanisms of neuropathogenesis supported by both clinical and basic research findings. Ultimately, the elucidation of pathways of virus-mediated neurotoxicity and the neuroinflammatory processes underlying these various conditions will lead to the development of treatment strategies for heterogenous disorders of the CNS associated with viral infection.

Arthropod-borne viruses (arboviruses) are of paramount concern as a group of pathogens at the forefront of emerging and re-emerging diseases. Although some arboviral infections are asymptomatic or present with a mild influenza-like illness, many are important human and veterinary pathogens causing serious illness ranging from rash and arthritis to encephalitis and hemorrhagic fever. Here, we discuss arboviruses from diverse families (Flaviviruses, Alphaviruses, and the Bunyaviridae) that are causative agents of encephalitis in humans. An understanding of the natural history of these infections as well as shared mechanisms of neuroinvasion and neurovirulence is critical to control the spread of these viruses and for the development of effective vaccines and treatment modalities.

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860–1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970–1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970–1350, a 22-amino-acid hydrophobic segment (1066–1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066–1087 as a fusion peptide.

The Bunyaviridae are a large group of viruses that infect a diversity of arthropod vectors and animal hosts. They have a worldwide distribution and can be the cause of human illness ranging from mild asymptomatic infection to hemorrhagic fever and fatal encephalitis. The growth of the human population, the expansion of agricultural and economic development, climatic changes, and the speed and frequency of global transportation all favor the emergence of bunyaviruses and other arthropod borne viruses. International monitoring of the Bunyaviridae and a greater understanding of their ecology and biology are needed to prepare for future outbreaks.

Increased golli MBP (golli) expression has been observed in the peripheral immune system of mice in the relapsing phase of EAE, raising the possibility that golli MBP expression in the periphery may contribute to relapses. Here we describe the generation of golli MBP-deficient mice and a comparison of the clinical course of EAE between heterozygous (golli+/−) and wild-type (golli+/+) mice. There was no difference between the two groups in incidence of disease, severity of the first episode of disease, or remission after the first episode. However, there was a significant reduction in relapses in golli+/− mice vs. controls, suggesting a role for golli proteins in the relapses in EAE.

Infection of the central nervous system by the type 1 human immunodeficiency virus (HIV-1) commonly results in a number of neurological impairments known, in their most severe form, as HIV-associated dementia (HAD). The persistence of HIV encephalitis (HIVE), the pathological correlate of HAD, in spite of highly active antiretroviral therapy (HAART) underscores the importance of continued research focused on the neurobiology of HIV. To elucidate direct and indirect mechanisms of HIV neuropathogenesis, current investigation is focused on neuroinvasion, HIV-1-mediated mechanisms of neuronal damage and apoptosis, and compartmentalized evolution of virus in the brain. The aim of this review is to provide a selective overview of the most recent research on the neurobiology of HIV, adding only a brief introduction regarding established principles.

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the spinal cord in which HTLV-I Tax-specific cytotoxic T lymphocytes (CTL) have been suggested to be immunopathogenic. However, it is unknown whether the HTLV-I-specific CTL in the central nervous system differ from those in the periphery. We investigated functional T-cell receptor diversity in HTLV-I Tax11-19-specific CTL clones derived from peripheral blood and cerebrospinal fluid (CSF) of a HAM/TSP patient using analogue peptides of the viral antigen. CTL responses to the analogue peptides varied between T-cell clones, however, CTL clones from CSF showed limited recognition of the peptides when compared to those from peripheral blood. This suggests that CTL with highly focused specificity for HTLV-I Tax accumulate in the CSF and may contribute to the pathogenesis of HAM/TSP. Furthermore, this study provides a rationale for analogue peptide-based immunotherapeutic strategies focusing on the immunopathogenic T-cells in HTLV-I-associated neurologic disease.

In recent years, human herpesvirus 6 (HHV-6) has been investigated as a possible causative agent for MS. To determine if the detection of HHV-6 DNA in the serum of MS patients correlates with clinical parameters of MS disease progression, a total of 215 serum samples was obtained from 59 MS patients followed prospectively for a 5-month period. These samples were analyzed for the presence of HHV-6 DNA by nested PCR and compared in parallel to MS disease activity. HHV-6 DNA was amplified in 22% (4/18) of samples obtained during a period of clinical exacerbation. Significantly fewer (P = 0.008) sera, 5.6% (11/197), obtained from MS patients during clinical remission tested positive for the presence of HHV-6 DNA. This work demonstrates that the detection of serum HHV-6 DNA is significantly correlated with clinical exacerbations in MS. Moreover, the findings presented in this study have confirmed previous reports supporting an association between MS and HHV-6 and suggest a role for this human herpesvirus in the pathogenesis of MS.

63 Background: Worldwide, EBV is associated with ~10% of gastric cancer. Currently, no pharmaceutical-based therapies exist that selectively target EBV associated gastric cancers. EBV, in its latent oncogenic form, is dependent on the continuous expression of Epstein-Barr Nuclear Antigen 1 (EBNA1), a multifunctional dimeric protein critical for viral replication, genome maintenance and viral gene expression. Methods: The aim of this program is to advance the development of a New Chemical Entity (NCE) for latent infection of Epstein-Barr Virus (EBV) to treat EBV-associated gastric cancer. We have used structure-based drug design and medicinal chemistry methods to identify and develop a small molecule clinical candidate that selectively inhibits the DNA-binding activity of EBNA1. Results: The clinical candidate inhibits EBNA1 function with nanomolar potency in biochemical assays and low micromolecular activity in several cell-based assays. We demonstrate that our clinical candidate provides protection in 4 different xenograft models of EBV-driven tumor growth, including patient-derived xenografts. Furthermore, RNA analysis experiments confirm in vivo target engagement by the elimination of EBV in treated tumor tissue. The clinical candidate is selective, showing no activity in an EBV-negative xenograft experiments. The clinical candidate has met industry-accepted criteria for drug suitability, safety and toxicology including physicochemical properties, metabolic stability, selectivity in broad-based screens and bioavailability, and lack in vivoliabilities, including genotoxicity and 14-day toxicity studies. Conclusions: IND-enabling studies including safety pharmacology and toxicology and GMP manufacturing have begun with a projected First-In-Human clinical trial in Q4 2017. These data establish proof-of-concept for targeting EBNA1—a protein previously thought to be undruggable.

The Bunyaviridae is a large and diverse family of viruses whose members infect a wide range of arthropod vectors and animal or plant hosts. Bunyaviruses are distributed worldwide and their promiscuous use of an impressive variety of arthropod vectors amplifies the potential for bunyaviruses to increase their prevalence and geographic range given the right environmental or man-made conditions. Most bunyavirus infections are asymptomatic or present with a mild influenza-like illness. However, some members of the family are important human and veterinary pathogens that are the causative agents of devastating illnesses, including hemorrhagic fever, pulmonary disease, congenital abnormalities, and fatal encephalitis. In this chapter, we discuss the epidemiology, lifecycle, molecular biology, neuropathogenesis, and prevention and treatment strategies for emerging bunyaviruses that are predominantly associated with central nervous disease; these include members of the genera Phlebovirus (Rift Valley fever virus and Toscana virus) and Orthobunyavirus (La Crosse virus and Cache Valley virus).

Membrane cofactor protein (CD46) is a member of a family of glycoproteins that are regulators of complement and prevent activation of complement on autologous cells. Recently, CD46 has been identified as the cellular receptor for human herpesvirus Type 6 (HHV-6). Elevated levels of soluble CD46 have been described in several autoimmune disorders, and may be implicated in the pathogenesis of these diseases. As several reports have supported an association of HHV-6 and multiple sclerosis, it was of interest to compare levels of soluble CD46 in the sera of multiple sclerosis patients to that of healthy controls, other neurological disease controls, and other inflammatory disease controls. Using an immunoaffinity column comprised of immobilized monoclonal antibodies to CD46, serum levels of soluble CD46 were found to be significantly elevated in multiple sclerosis patients compared with healthy and other neurological disease controls. Moreover, multiple sclerosis patients who tested positive for HHV-6 DNA in serum had significantly elevated levels of soluble CD46 in their serum compared with those who were negative for HHV-6 DNA. A significant increase in soluble CD46 was also found in the serum of other inflammatory disease controls tested compared to healthy controls. Additionally, a significant correlation was demonstrated between levels of soluble CD46 in the serum and cerebrospinal fluid of multiple sclerosis patients. Collectively, these data suggest that elevated levels of soluble CD46 may contribute to the pathogenesis of inflammatory diseases, including multiple sclerosis.

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease associated with HTLV-I infection, in which chronically activated, HTLV-I-specific CD8+ CTL have been suggested to be immunopathogenic. In HLA-A2 HAM/TSP patients, CD8+ HTLV-I-specific CTLs recognize an immunodominant peptide of the HTLV-I Tax protein, Tax11–19. We examined the functional outcome on activation of both cloned peripheral blood and cerebrospinal spinal fluid-derived CTL and bulk PBMC from HAM/TSP patients by altered peptide ligands (APL) derived from HTLV-I Tax11–19. In CTL clones generated from PBMC and CSF of HLA-A2 HAM/TSP patients, an APL substituted at position 5 significantly decreased CTL responses when compared with the native peptide. Moreover, these ligands were also shown to inhibit CTL responses to the native peptide in bulk PBMC of HLA-A2 HAM/TSP patients. These data suggest that a modification of an antigenic peptide at the central position can manipulate the T cell responses in bulk PBMC from different individuals with an inflammatory disease. Additionally, these results have implications for the potential use of APL-based immunotherapy in this T cell-mediated CNS disease.

Several reports have suggested an association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) based on immunohistochemical demonstration of HHV-6 antigens in inflammatory lesions, detection of increased HHV-6 specific serum antibody titers, and amplification of HHV-6 DNA from sera and cerebrospinal fluid of MS patients but not in controls. Characterization of the cellular immune response of MS patients to HHV-6 may further clarify the role of HHV-6 in MS and provide insight into the pathogenesis of this immune-mediated disease. We have compared lymphoproliferative responses to HHV-6A (U1102)-, HHV-6B (Z29)-, and HHV-7 (H7SB)-infected cell lysates in healthy controls and patients with MS. Most healthy controls (71%) proliferated to HHV-6B lysate, and fewer (33%) responded to the HHV-6A lysate. In contrast, 67% of MS patients had a lymphoproliferative response to HHV-6A, which is a significant increase in comparison with healthy controls. A similar frequency of lymphoproliferative response (78%) to HHV-6B was demonstrated in MS patients. Lymphoproliferation to HHV-7 lysate was demonstrated in 23% of healthy controls and 28% of MS patients. These results indicate that the lymphoproliferative response to the HHV-6A variant, which was recently reported to have greater neurotropism, is increased in MS patients.

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3′- gag /5′- prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0, 2, 6, 10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I-infected, activated CD4+ lymphocytes.

Thrombin-induced platelet microbicidal protein (tPMP) exerts potent in vitro microbicidal activity against pathogens commonly found in the bloodstream, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Localized platelet release of tPMP may be important in defense against infections involving the vascular endothelium caused by tPMP-susceptible organisms. In contrast, pathogens capable of surviving in the presence of tPMP could then exploit the platelet as an adhesive surface for attachment to damaged endothelium. To examine these hypotheses, we derived a tPMP-resistant (tPMP(r)) C. albicans strain from its tPMP-sensitive (tPMP(s)) parental strains were equivalent in vitro as assessed by genotyping (electrophoretic karyotype and restriction endonuclease analysis of genomic DNA), biotyping, germination, platelet aggregation, adherence to vascular endothelial cells, and growth characteristics. In addition, the tPMP(r) phenotype was stable following multiple in vitro and in vivo passages. We then investigated the in vivo relevance of tPMP susceptibility on endovascular infection using a rabbit model of endocarditis and hematogenous dissemination. Rabbits with transaortic catheters (n = 15 in each group) were challenged with either the tPMP(s) or tPMP(r) C. albicans strain. All rabbits developed C. albicans-induced endocarditis, as determined by the presence of infected vegetations. In rabbits challenged with tPMP(s) strain (P < 0.001). These results were seen in the absence of differences in either initial adherence of strains to cardiac valves or vegetation weights. Furthermore, although these C. albicans strains induced equivalent rates and extent of hematogenous renal infection, only the tPMP(r) strain disseminated hematogenously to the spleen (15 of 15 rabbits) versus 0 of 15 [tpmp(s) strain]; P < 0.0001). Thus, tPMP(r) C. albicans caused more-severe endocarditis and produced greater metastatic sequelae than the tPMP(s) counterpart.

La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.

La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066–1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066–1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative to LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.

Human immunodeficiency virus type 1 (HIV-1) infection affects more that 32 million people worldwide (2007) and is associated with central nervous system (CNS) dysfunction in at least 30% of infected individuals, despite treatment with highly active antiretroviral therapy (HAART). The spectrum of cognitive disorders ranges from mild impairment of higher cortical functions to dementia (HIV associated dementia, HAD) and HIV-associated neurocognitive disorders (HAND). The use of HAART has lessened the severity but not the prevalence of HAND, and improving neurological outcomes will require more effective antiviral drug effects in the CNS and adjunctive therapies directed toward mechanisms and pathways of HIV-induced neurodegeneration. Identifying such targets requires understanding the events preceding irreversible neuronal damage in the CNS: HIV entry, replication in cellular reservoirs, neuroimmune activation, production of neurotoxins (HIV proteins and cellular factors), and neuronal repair responses. Studies of HIV infected individuals and simian immunodeficiency virus (SIV)–infected macaques, and numerous in vitro model systems of HIV neurodegeneration have suggested a central role for chemokines and their receptors in modulating these pathogenic processes during the course of CNS infection. This chapter addresses selected aspects of our current understanding of HIV neuropathogenesis, based on in vivo and in vitro studies, which are modulated directly and indirectly through chemokine/chemokine receptor interactions. Some of these associations are firmly established through published studies, and others are more speculative. Without doubt, many of the steps in HIV neuropathogenesis apply to other neurodegenerative diseases, and common approaches for neuroprotection will most likely emerge.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) gp160s obtained from the brain are often genetically distinct from those isolated from other organs, suggesting the presence of brain-specific selective pressures or founder effects that result in the compartmentalization of viral quasi-species. Whereas HIV has also been found to compartmentalize within different regions of the brain, the extent of brain-regional compartmentalization of SIV in rhesus macaques has not been characterized. Furthermore, much is still unknown about whether phenotypic differences exist in envelopes from different brain regions. To address these questions, env DNA sequences were amplified from four SIVmac239-infected macaques and subjected to phylogenetic and phenetic analysis. The authors demonstrated that sequences from different areas of the brain form distinct clades, and that the long-term progressing macaques demonstrated a greater degree of regional compartmentalization compared to the rapidly progressing macaques. In addition, regional compartmentalization occurred regardless of the presence of giant-cell encephalitis. Nucleotide substitution rates at synonymous and nonsynonymous sites (ds:dn rates) indicated that positive selection varied among envelopes from different brain regions. In one macaque, envelopes from some but not all brain regions acquired changes in a conserved CD4-binding motif GGGDPE at amino acids 382 to 387. Furthermore, gp160s with the mutation G383E were able to mediate cell-to-cell fusion in a CD4-independent manner and were more susceptible to fusion inhibition by pooled plasma from infected macaques. Reversion of this mutation by site-directed mutagenesis resulted in reduction of CD4-independence and resistance to fusion inhibition in cell fusion assays. These studies demonstrate that SIV evolution within the brain results in a heterogeneous viral population with different phenotypes among different regions.

Publisher Summary This chapter focuses on evidence suggesting an association of human herpesvirus-6 (HHV-6) with four major disorders of the central nervous system (CNS): childhood febrile seizures, mesial temporal-lobe epilepsy (MTLE), multiple sclerosis (MS), and progressive multifocal leukoencephalopathy (PML). Detection of HHV-6 genome in the brain per se does not necessarily indicate productive infection, and establishing a primary pathogenic role for HHV-6 in CNSdisorders is challenging. Nevertheless, HHV-6 has been associated with aseptic meningoencephalitis, encephalitis, transverse myelitis, Guilain-Barre´ syndrome, and cerebellar astrocytoma. Although the HHV-6A variant has increased neurotropism and is associated with viral persistence and reactivation in the CNS, both HHV-6 variants are clearly neurotropic. HHV-6A has been isolated from the CNS of acquired immuno deficiency syndrome (AIDS) patients with areas of demyelination and associated with MS. MS is the most prevalent demyelinating disease of the CNS. The variability of the diseases suggests that many factors, including genetic and immune components, are involved in the spectrum of clinical syndromes that are defined as MS. The HHV-6B variant is a causative agent of exanthem subitum and accounts for the majority of symptomatic HHV-6 infections in infants.

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.

The pathogenesis and etiology of Multiple Sclerosis (MS) are yet to be well defined. Epidemiologic evidence suggests that MS is a multi-factorial disease which develops as a result of host genetics, immune response, and environment. Several lines of evidence, including the documentation of microbes, which induce a variety of demyelinating diseases in both humans and animals, suggest that a virus may comprise the environmental component in the etiology of this disorder. While many viruses have been proposed as etiologic agents in MS, none of these viruses has been firmly associated with disease pathogenesis. Additionally, mechanism(s) by which virus-host interactions may lead to demyelination are not fully understood. It is suggested that multiple agents may induce a virus-specific or a cross-reactive autoimmune process resulting in clinical disease in a subset of genetically susceptible individuals. The involvement of multiple infectious agents in MS, as suggested originally by Dr. Pierre Marie over 100 years ago, may explain the difficulty in identifying a single agent responsible for this highly variable and chronic disease. Moreover, extreme caution is encouraged in attempts to readily associate viruses in a chronic, progressive neurologic disorder such as MS. This chapter outlines the difficulty to determine cause from effect particularly when a ubiquitous agent is suggested to play a role in disease pathogenesis. Uniformity in assay design, viral isolation techniques, molecular probes, etc. must be employed by different research groups on a large number of MS cohorts to confirm these virus associations.

Multiple sclerosis (MS) is a putative T helper 1 (Th-1) mediated inflammatory disorder of the central nervous system, and levels of pro-inflammatory and anti-inflammatory cytokines have been found to correlate with changes in MS relapses. However, it is unclear if cytokine profiles differ between relapsing-remitting (RRMS) versus secondary progressive (SPMS) disease stages. Cytokine production (IL-2, IL-4, IL-5, IL-10, IL-12p40, TNF-alpha, and IFN-gamma) was assessed by cytometric bead array (CBA) and intracellular cytokine staining from alphaCD3 antibody and mitogen stimulated peripheral blood mononuclear cells (PBMCs) from female RRMS and SPMS patients, and healthy controls. Significantly increased production of IL-12p40 and decreased production of IL-10 were observed in SPMS patients. Differences in immune responsiveness in RRMS and SPMS are important in the understanding of the evolution of the immunopathogenesis of the disease and for the development of disease type specific treatments.

The protective effect of pregnancy on putative Th1-mediated autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, is associated with a Th1 to Th2 immune shift during pregnancy. The hormone estriol increases during pregnancy and has been shown to ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis. In addition, estrogens induce cytokine changes consistent with a Th1 to Th2 shift when administered in vitro to human immune cells and in vivo to mice. In a pilot trial, oral estriol treatment of relapsing remitting multiple sclerosis patients caused significant decreases in enhancing lesions on brain magnetic resonance imaging. Here, the immunomodulatory effects of oral estriol therapy were assessed. PBMCs collected longitudinally during the trial were stimulated with mitogens, recall Ags, and glatiramer acetate. Cytokine profiles of stimulated PBMCs were determined by intracellular cytokine staining (IL-5, IL-10, IL-12 p40, TNF-α, and IFN-γ) and cytometric bead array (IL-2, IL-4, IL-5, IL-10, TNF-α, and IFN-γ). Significantly increased levels of IL-5 and IL-10 and decreased TNF-α were observed in stimulated PBMC isolated during estriol treatment. These changes in cytokines correlated with reductions of enhancing lesions on magnetic resonance imaging in relapsing remitting multiple sclerosis. The increase in IL-5 was primarily due to an increase in CD4+ and CD8+ T cells, the increase in IL-10 was primarily due to an increase in CD64+ monocytes/macrophages with some effect in T cells, while the decrease in TNF-α was primarily due to a decrease in CD8+ T cells. Further study of oral estriol therapy is warranted in Th1-mediated autoimmune diseases with known improvement during pregnancy.