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.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing.

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host's immune response in different virus-host combinations.

A novel lncRNA linc-AhRA negatively regulates innate antiviral response in murine microglia upon neurotropic herpesvirus infection.

Microglia are the primary cellular source of type I interferons (I-IFNs) in the brain upon neurotropic virus infection. Although the I-IFN-based antiviral innate immune response is crucial for eliminating viruses, overproduction led to immune disorders. Therefore, the relatively long-lasting I-IFNs must be precisely controlled, but the regulatory mechanism for the innate antiviral response in microglia remains largely unknown. Long non-coding RNAs (lncRNAs) are being recognized as crucial factors in numerous diseases, but their regulatory roles in the innate antiviral response in microglia are undefined. Methods: The high-throughput RNA sequencing was performed to obtain differentially expressed lncRNAs (DELs) in primary microglia infected with or without the neurotropic herpes simplex virus type 1 (HSV-1). We selected four DELs ranked in the top 15 in basic level and their fold change induced by HSV-1, i.e., FPKMHSV-1/FPKMCells.We subsequently found a key lncRNA affecting the innate antiviral response of microglia significantly. We next used dual-luciferase reporter assays, bioinformatical tools, and truncation mutants of both lncRNA and targeted proteins to elucidate the downstream and upstream mechanism of action of lncRNA. Further, we established microglia-specific knock-in (KI) mice to investigate the role of lncRNA in vivo. Results: We identified a long intergenic non-coding RNA, linc-AhRA, involved in regulating the innate antiviral response in murine microglia. linc-AhRA is activated by aryl hydrocarbon receptor (AhR) and restricts I-IFN production in microglia upon neurotropic herpesvirus infection and innate immune stimulation. Mechanistically, linc-AhRA binds to both tripartite motif-containing 27 (TRIM27) and TANK-binding kinase 1 (TBK1) through its conserved 117nt fragment as a molecular scaffold to enhance TRIM27-TBK1 interaction. This interaction facilitates the TRIM27-mediated ubiquitination of TBK1 and results in ubiquitin-proteasome-dependent degradation of TBK1. Consequently, linc-AhRA suppresses I-IFN production through facilitating TBK1 degradation and limits the microglial innate immune response against neurotropic herpesvirus infection. Microglia-specific KI of linc-AhRA mice shows a weakened antiviral immune response upon neurotropic herpesvirus challenge due to a reduction of TBK1 in microglia. Conclusion: Our findings indicate that linc-AhRA is a negative regulator of I-IFN production in microglia to avoid excessive autoimmune responses. These findings uncover a previously unappreciated role for lncRNA conserved fragments in the innate antiviral response, providing a strong foundation for developing nucleotide drugs based on conserved functional fragments within lncRNAs.

Viral proteins recognized by different TLRs.

Virus invasion activates the host's innate immune response, inducing the production of numerous cytokines and interferons to eliminate pathogens. Except for viral DNA/RNA, viral proteins are also targets of pattern recognition receptors. Membrane-bound receptors such as Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, and TLR10 relate to the recognition of viral proteins. Distinct TLRs perform both protective and detrimental roles for a specific virus. Here, we review viral proteins serving as pathogen-associated molecular patterns and their corresponding TLRs. These viruses are all enveloped, including respiratory syncytial virus, hepatitis C virus, measles virus, herpesvirus human immunodeficiency virus, and coronavirus, and can encode proteins to activate innate immunity in a TLR-dependent way. The TLR-viral protein relationship plays an important role in innate immunity activation. A detailed understanding of their pathways contributes to a novel direction for vaccine development.

Viral and Prion Infections Associated with Central Nervous System Syndromes in Brazil.

Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.

Herpesvirus Regulation of Selective Autophagy.

Selective autophagy has emerged as a key mechanism of quality and quantity control responsible for the autophagic degradation of specific subcellular organelles and materials. In addition, a specific type of selective autophagy (xenophagy) is also activated as a line of defense against invading intracellular pathogens, such as viruses. However, viruses have evolved strategies to counteract the host's antiviral defense and even to activate some proviral types of selective autophagy, such as mitophagy, for their successful infection and replication. This review discusses the current knowledge on the regulation of selective autophagy by human herpesviruses.

Berberine in Human Oncogenic Herpesvirus Infections and Their Linked Cancers.

Human herpesviruses are known to induce a broad spectrum of diseases, ranging from common cold sores to cancer, and infections with some types of these viruses, known as human oncogenic herpesviruses (HOHVs), can cause cancer. Challenges with viral latency, recurrent infections, and drug resistance have generated the need for finding new drugs with the ability to overcome these barriers. Berberine (BBR), a naturally occurring alkaloid, is known for its multiple biological activities, including antiviral and anticancer effects. This paper comprehensively compiles all studies that have featured anti-HOHV properties of BBR along with promising preventive effects against the associated cancers. The mechanisms and pathways induced by BBR via targeting the herpesvirus life cycle and the pathogenesis of the linked malignancies are reviewed. Approaches to enhance the therapeutic efficacy of BBR and its use in clinical practice as an anti-herpesvirus drug are also discussed.

Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics.

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species.

Clinical Manifestations and Epigenetic Regulation of Oral Herpesvirus Infections.

The oral cavity is often the first site where viruses interact with the human body. The oral epithelium is a major site of viral entry, replication and spread to other cell types, where chronic infection can be established. In addition, saliva has been shown as a primary route of person-to-person transmission for many viruses. From a clinical perspective, viral infection can lead to several oral manifestations, ranging from common intraoral lesions to tumors. Despite the clinical and biological relevance of initial oral infection, little is known about the mechanism of regulation of the viral life cycle in the oral cavity. Several viruses utilize host epigenetic machinery to promote their own life cycle. Importantly, viral hijacking of host chromatin-modifying enzymes can also lead to the dysregulation of host factors and in the case of oncogenic viruses may ultimately play a role in promoting tumorigenesis. Given the known roles of epigenetic regulation of viral infection, epigenetic-targeted antiviral therapy has been recently explored as a therapeutic option for chronic viral infection. In this review, we highlight three herpesviruses with known roles in oral infection, including herpes simplex virus type 1, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. We focus on the respective oral clinical manifestations of these viruses and their epigenetic regulation, with a specific emphasis on the viral life cycle in the oral epithelium.

Anguillid herpesvirus 1 (AngHV) ORF95 encodes a late, structural envelope protein.

Anguillid herpesvirus 1 (AngHV) is one of the vital pathogenic agents found in the wild and cultured eel populations, which has brought significant losses to eel culture industry in China. In this study, AngHV ORF95 was characterized. Bioinformatics analysis showed that ORF95 putatively encodes a structural protein that is homologous to hemagglutinin-esterase (HE) protein of infectious salmon anemia virus (ISAV). Temporal transcription and expression analysis indicated that ORF95 is a viral late gene. Subcellular localization analysis revealed that ORF95 was predominantly localized in the cytoplasm. Further, western blot analysis indicated that ORF95 is a structural protein of virion envelope. These results provide a novel basis to make further efforts to clarify the function of ORF95 in the process of AngHV infection and the possibility to use ORF95 as antigen to develop AngHV subunit vaccine.

[The role of herpesviruses in development of diseases of the urogenital tract and infertility in women].

This review presents the data on the spreading of all known human herpesviruses (НHVs) in female urogenital tract. According to the WHO almost 500 million people worldwide suffer from genital infection caused by НHVs. НHVs were detected in various inflammatory diseases of female upper and lower genital tract (vaginitis and cervicitis), in extrauterine pregnancy (in fallopian tubes), in infertility (cervical channel, endometrium and ovaries). Herpes simplex virus 1 (HSV­1) was identified for the first time in oocytes after failed in vitro fertilization (IVF). НHVs produce negative effect on the entire reproductive process from conception to childbirth. It was established that HSV, cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) markedly increase the risk of spontaneous abortion, preterm birth and stillbirth. Intrauterine НHV infection is a major cause of congenital malformations. Data on humoral and cell immunity in genital herpesvirus infections (НHVI) are also reviewed. Intravaginal HSV­2 infection changes cell composition of vaginal mucosa, i.e., together with cells mobilized from the blood, protective role is performed by resident memory T­cells (TRM), natural killer cells (NK­cells) and regulatory T­cells (Treg) whose function consists in maintaining the balance of the activities of lymphocytes. Constant НHVI spreading is largely explained by transition of primary infection to potentially reactivating latent form, since latent virus is unavailable to immune recognition and medicines. The genome editing system CRISPR/Cas9 can recognize and modify not only active but also latent viruses. The promising pilot results with the use of this system offer the possibility of developing innovative technologies for НHV elimination and НHVI eradication.

Virome-wide serological profiling reveals association of herpesviruses with obesity.

The relationship between viral infection and obesity has been known for several decades but epidemiological data is limited to only a few viral pathogens. The association between obesity and a wide range of viruses was assessed using VirScan, a pan-viral serological profiling tool. Serum specimens from 457 Qatari adults (lean = 184; obese = 273) and 231 Qatari children (lean = 111; obese = 120) were analyzed by VirScan. Associations with obesity were determined by odds ratio (OR) and Fisher's test (p values), and by multivariate regression analysis to adjust for age and gender. Although there was no association of viral infections with obesity in the pediatric population, a nominal association of obesity with seropositivity to members of the Herpesviridae family is observed for the adult population (OR = 1.5-3.3; p < 0.05). After adjusting p values for multiple comparisons (Bonferroni correction) the odds of being obese is significantly higher in herpes simplex virus 1 (HSV-1) seropositive Qatari adults (OR = 3.3; 95% CI 2.15-4.99; p = 2.787E - 08). By VirScan, the sero-prevalence of HSV1 is 81.3% and 57.1% among Qatari obese and lean adult populations, respectively. Higher prevalence of antibodies against several peptide epitopes of HSV-1/2 is positively associated with obesity (OR = 2.35-3.82; p ≤ 3.981E - 05). By multivariate regression analysis, HSV-1 was independently associated with obesity irrespective of age and gender. Our results suggest that obesity among Qataris may be associated with a higher prevalence of herpesvirus infections, in particular HSV-1. Furthermore, the high prevalence of antibodies against peptide antigens specific to HSV-1 and -2 in the obese population suggests that these viral peptides may play a role in adipogenesis. Further studies with these candidate peptides in cell culture or animal models may confirm their adipogenic roles.

Susceptibility of Pimephales promelas and Carassius auratus to a strain of koi herpesvirus isolated from wild Cyprinus carpio in North America.

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

The effects of human immunodeficiency virus, human papillomavirus, herpes simplex virus-1 and -2, human herpesvirus-6 and -8, cytomegalovirus, and hepatitis B and C virus on female fertility and pregnancy.

Female infertility may be defined as a woman of reproductive age being unable to become pregnant after a year of regular unprotected sexual intercourse. Social, genetic, endocrine, physiological, and psychological factors as well as lifestyle habits (i.e., smoking and alcohol consumption), either alone or in combination with male factors, are major causes. However, approximately 15-30% of cases of female infertility remain unexplained. Numerous investigations have also indicated that microbiomes play an important role in human reproduction. All parts of the female reproductive system may be influenced by infectious and pathological agents, especially viruses, and these may interfere with reproductive function and so are risk factors for infertility, although in many cases an exact role is unclear. We present an overview of the impact of common viral infections on female reproduction, searching Medline, PubMed, Scopus, and Google scholar databases for potentially relevant studies of viruses known to have a potential effect. Human immunodeficiency virus (HIV), herpes simplex virus (HSV) and human herpesvirus (HHV) increase infertility rates whilst human papillomavirus (HPV), cytomegalovirus (CMV), and hepatitis B and C virus (HBV, HCV) infections mostly lead to higher abortion and miscarriage rates. Moreover, HPV infection is linked to increased tubal infertility, endometriosis, and pelvic inflammatory disease. HPV was the most frequently observed infection and with lower pregnancy rate and foetal death in women undergoing IVF treatments. Assisted reproductive treatment could be a safe and effective approach for HIV and HBV infected women.

Inhibitors of APE1 redox function effectively inhibit γ-herpesvirus replication in vitro and in vivo.

APE1 is a multi-functional protein with a redox function in its N-terminal domain and an apurinic/apyrimidinic endonuclease activity in the C-terminal domain. APE1 redox function plays an important role in regulating cell proliferation and survival through activating specific transcriptional activators. APE1 redox function is also found to be associated with some cancer occurrence. In this study, we demonstrated that APE1 redox function is essential for Epstein-Barr virus (EBV) lytic replication as the silencing of APE1 expression or treatment with APE1 redox inhibitors C10 and E3330 can inhibit EBV lytic replication and virion production. Furthermore, C10 and E3330 also inhibit MHV-68 replication in vitro and in vivo. C10 and E3330 were able to significantly reduce the loss of pulmonary alveoli and thickening of alveolar septa in mice caused by MHV-68 infection. Altogether, (i) APE1 redox function is validated as a new antiviral target; (ii) APE1 redox inhibitors, especially C10, have potentials to be used for the treatment of γ-herpesvirus infection and associated diseases; (iii) MHV-68 is validated to be a surrogate for the study of the pathogenesis and therapy of EBV and KSHV infection in vivo.

Antiviral Activities of Quercetin and Isoquercitrin Against Human Herpesviruses.

We previously reported that the ethyl acetate (EtOAc) fraction of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) inhibits varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) replication in vitro. PGG (1,2,3,4,6-penta-O-galloyl-ß-D-glucose) is a major chemical constituent of the EtOAc fraction of ESE that inhibits VZV but not HCMV replication. In this study, we comprehensively screened the chemical compounds identified in the EtOAc fraction of ESE for potential antiviral properties. Among the examined compounds, quercetin and isoquercitrin displayed potent antiviral activities against both VZV and HCMV with no significant cytotoxic effects. Both compounds strongly suppressed the expression of VZV and HCMV immediate-early (IE) genes. Our collective results indicated that, in addition to PGG, quercetin and isoquercitrin are bioactive compounds in the EtOAc fraction of ESE that effectively inhibit human herpesvirus replication.

Herpesviruses in Head and Neck Cancers.

Head and neck cancers arise in the mucosa lining the oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract, and nasopharynx. The etiology of head and neck cancers is complex and involves many factors, including the presence of oncogenic viruses. These types of cancers are among the most common cancers in the world. Thorough knowledge of the pathogenesis of viral infections is needed to fully understand their impact on cancer development.

Koi herpesvirus (KHV) and KHV disease (KHVD) - a recently updated overview.

Over the last years, there has been an enormous increase in the knowledge on koi herpesvirus (KHV), koi herpesvirus disease (KHVD), pathogenesis and virus variants. Different KHV lineages have clearly been identified, possible genomic changes during replication in different cell cultures at different temperatures but also in several hosts have been identified, a persistent stage of infection has been specified and it has been shown that infection with KHV is not host specific at all, but KHVD is. Additionally, it has been shown that it is possible to combat KHVD by immunization with inactivated and attenuated live vaccines using different delivery systems but also to benefit from alternative treatments with e.g. exopolysaccharids obtained from Arthrospira platensis.

Lethal Hemorrhagic Disease and Clinical Illness Associated with Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, both in captivity and in the wild. Most deaths associated with the virus are caused by two chimeric variants of EEHV1 (EEHV1A and EEHV1B), while two other EEHVs endemic within Asian elephants (EEHV4 and EEHV5) have been recognized but cause death less often. Whether lethal EEHV infections are due to primary infection or reactivation of latent virus remains unknown, and knowledge of the anti-EEHV antibody levels in young elephants is limited. To close these gaps, we sought to develop a serologic assay capable of distinguishing among infections with different EEHVs using a luciferase immunoprecipitation system (LIPS) for antibody profiling and a panel of conserved EEHV recombinant proteins and proteins unique to EEHV1. The results showed that elephants dying from EEHV1 hemorrhagic disease or ill from EEHV infection were seronegative for the EEHV species that caused the disease or illness, indicating that the events were associated with primary infection rather than reactivation of latent virus. We also demonstrated that waning of EEHV1-specific antibodies can occur in the first 2 years of life, when a threshold protective level of antibody may be needed to prevent severe EEHV1-related disease. Use of the LIPS assay to identify putative "diagnostic" proteins would be a valuable asset in determining the EEHV immune status of young elephants and responses to candidate EEHV vaccines in the future.IMPORTANCE Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a longstanding question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of the viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHVs and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.