Imported Zika virus infections in Hungary between 2016 and 2018

Zika virus is a mosquito-borne flavivirus with significant public health concern due to its association with neurological symptoms and intrauterine malformations. Although it is endemic in tropical and subtropical areas, sexual transmission raises the possibility of autochthonous spreading elsewhere. We describe the first laboratory diagnosed imported Zika-infections of Hungary, to highlight the challenges of microbiological identification of the pathogen, caused by serological cross-reactivity and short viremia. Serological examination was carried out using indirect immunofluorescent assay and enzyme-linked immunosorbent assay. Plaque-reduction neutralization test was used for verification purposes. A wide range of clinical specimens: serum, whole-blood, urine, saliva, and semen were analyzed by molecular methods, and sequencing was applied in case of PCR positive results to identify the virus strain. Zika-infected patients with previous vaccination against flaviviruses or possible flavivirus infection in the past showed high serological cross-reactivity, and even cross-neutralizing antibodies were observed. Zika virus RNA could be detected in urine specimen in case of two patients, and in EDTA-anticoagulated whole-blood sample of one patient. The detected strains belong to the Asian lineage of the virus. We presume that serological investigation of imported Zika virus could be altered by infections, vaccination of endemic flaviviruses in Hungary and vice versa

The Importance Of Epigenetic Alterations In The Development Of Epstein-Barr Virus-Related Lymphomas

Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with a series of malignant tumors. These include lymphomas (Burkitt’s lymphoma, Hodgkin’s disease, T/NK-cell lymphoma, post-transplant lymphoproliferative disease, AIDS-associated lymphoma, X-linked lymphoproliferative syndrome), carcinomas (nasopharyngeal carcinoma, gastric carcinoma, carcinomas of major salivary glands, thymic carcinoma, mammary carcinoma) and a sarcoma (leiomyosarcoma). The latent EBV genomes persist in the tumor cells as circular episomes, co-replicating with the cellular DNA once per cell cycle. The expression of latent EBV genes is cell type specific due to the strict epigenetic control of their promoters. DNA methylation, histone modifications and binding of key cellular regulatory proteins contribute to the regulation of alternative promoters for transcripts encoding the nuclear antigens EBNA1 to 6 and affect the activity of promoters for transcripts encoding transmembrane proteins (LMP1, LMP2A, LMP2B). In addition to genes transcribed by RNA polymerase II, there are also two RNA polymerase III transcribed genes in the EBV genome (EBER 1 and 2). The 5′ and internal regulatory sequences of EBER 1 and 2 transcription units are invariably unmethylated. The highly abundant EBER 1 and 2 RNAs are not translated to protein. Based on the cell type specific epigenetic marks associated with latent EBV genomes one can distinguish between viral epigenotypes that differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Whereas latent EBV genomes are regularly targeted by epigenetic control mechanisms in different cell types, EBV encoded proteins may, in turn, affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. There are EBNA1 binding sites in the human genome. Because high affinity binding of EBNA1 to its recognition sites is known to specify sites of DNA demethylation, we suggest that binding of EBNA1 to its cellular target sites may elicit local demethylation and contribute thereby to the activation of silent cellular promoters. EBNA2 interacts with histone acetyltransferases, and EBNALP (EBNA5) coactivates transcription by displacing histone deacetylase 4 from EBNA2-bound promoter sites. EBNA3C (EBNA6) seems to be associated both with histone acetylases and deacetylases, although in separate complexes. LMP1, a transmembrane protein involved in malignant transformation, can affect both alternative systems of epigenetic memory, DNA methylation and the Polycomb-trithorax group of protein complexes. In epithelial cells LMP1 can up-regulate DNA methyltransferases and, in Hodgkin lymphoma cells, induce the Polycomb group protein Bmi-1. In addition, LMP1 can also modulate cellular gene expression programs by affecting, via the NF-κB pathway, levels of cellular microRNAs miR-146a and miR-155. These interactions may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms, autoimmune phenomena, immunodeficiency). Thus, Epstein-Barr virus, similarly to other viruses and certain bacteria, may induce pathological changes by epigenetic reprogramming of host cells. Elucidation of the epigenetic consequences of EBV-host interactions (within the framework of the emerging new field of patho-epigenetics) may have important implications for therapy and disease prevention, because epigenetic processes are reversible and continuous silencing of EBV genes contributing to patho-epigenetic changes may prevent disease development

Magyarországra behurcolt trópusi arbovírusfertőzések 2016 és 2020 között = Imported tropical arbovirus infections in Hungary between 2016 and 2020

Introduction: Dengue-, Zika-and Chikungunya infections are among the most frequently imported tropical arbovirus infections. Due to their shared endemic regions, vectors and similar clinical symptoms, differential diagnosis is based on serological and molecular analysis. Objective: The aim of our study was to identify the imported arbovirus infections of travellers between 2016 and 2020. Furthermore, to improve the diagnostic sensitivity, anticoagulated whole blood and urine samples were involved in molecular diagnosis. Method: Virus-specific antibody kinetics was tested in paired sera of patients by ELISA method. In case of reactive results, further serological analysis was performed using immunofluorescence assays and/or ELISA tests to exclude serological cross-reactions caused by other members of the flavi-and alphaviruses. Detection of viral RNA was attempted from serum, anticoagulated whole blood and urine specimens using reverse transcription and real-time polymerase chain reaction. Results: Out of the tested 1037 travellers, reactive serological and/or molecular results were obtained in 133 cases. Anticoagulated whole blood proved to be the most suitable specimen for viral RNA detection of the three viruses. Discussion: Parallel testing of Dengue-, Zika-and Chikungunya infections is recommended, as symptom-based differential diagnosis is challenging. Due to the characteristic serological cross-reactivity of flavi-and alphaviruses, microbiological diagnosis relies on both serological and molecular tests. Conclusion: Involving anticoagulated whole blood and urine samples into molecular analysis and early sample collection improve the sensitivity of microbiological diagnostics. Identification of imported tropical arbovirus infections is of high importance as the presence of vector mosquitos in Europe raises the possibility of autochthon transmission

The 5' regulatory sequences of active miR-146a promoters are hypomethylated and associated with euchromatic histone modification marks in B lymphoid cells

Although the microRNA miR-146a is an important regulator of immunological processes and contributes to the pathogenesis of certain B cell lymphoma types, in B cells the epigenetic regulation of miR-146a expresion has not been studied yet. To elucidate the mechanisms controlling miR-146a expression in B lymphoid cells we analysed epigenetic marks, including CpG methylation and histone modifications, at the miR-146a promoter in well characterized Epstein-Barr virus (EBV) positive and EBV negative B cell lines. In addition, EBV positive epithelial cell lines were also studied as controls. In cells with a silent miR-146a promoter the 5' regulatory sequences comprising a CpG island were devoid of activating histone modifications, independently of the methylation pattern of the regulatory region. The regulatory sequences flanking the inactive miR-146 promoter were hypermethylated at CpG dinucleotides in the EBV positive Burkitt's lymphoma (BL) cell lines of memory B cell phenotype (Rael and Akata), partially methylated in the mammary carcinoma cell lines C2G6 and C4A3, and completely unmethylated in the nasopharyngeal carcinoma cell line C666-1. In contrast, in EBV positive cell lines of activated B cell phenotype, and EBV negative BL cell lines the invariably unmethylated 5' regulatory sequences of active miR-146a promoters were enriched in the euchromatic histone modification marks acetylated histone H3, acetylated histone H4, and histone H3 dimethylated at lysine 4. The euchromatic histone modification marks extended over the immediate vicinity of the transcriptional initiation site to the 3' intron, too. We concluded that similarly to the promoters of protein coding genes, both DNA methylation and histone modifications contribute to the host cell dependent expression of miR-146a. © 2013 Elsevier Inc

West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15 WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018 WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations

A 30 KB region of the Epstein-Barr virus genome is colinear with the rearranged human immunoglobulin gene loci: Implications for a “Ping-Pong Evolution” model for persisting viruses and their hosts

The left part of the Epstein-Barr virus (EBV) genome exhibits a strong colinearity of structural and functional elements with the immunoglobulin (Ig) gene loci which is only partially reflected in nucleotide sequence homologies. We propose that this colinearity may be the result of an inter-dependent co-evolution of the immunoglobulin loci together with EBV. Our observation could help elucidating the mechanisms of somatic hypermutation, explaining the ability of EBV to accidentally cause tumors, and shedding more light on the general mechanisms of viral and organismal evolution. We suggest that persisting viruses served as a complement for the organismal germline like in a ping-pong game and outline The Ping-Pong Evolution Hypothesis

The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells

Acetylated Histone H3 and H4 Mark the Upregulated LMP2A Promoter of Epstein-Barr Virus in Lymphoid Cells▿

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap