Structural insights into RNA-mediated transcription regulation in bacteria.

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation.

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Structural Basis for NusA Stabilized Transcriptional Pausing.

Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.

NREM Slow-Wave Activity in Adolescents Is Differentially Associated With ADHD Levels and Normalized by Pharmacological Treatment.

BACKGROUND: A compelling hypothesis about attention-deficit/hyperactivity disorder (ADHD) etiopathogenesis is that the ADHD phenotype reflects a delay in cortical maturation. Slow-wave activity (SWA) of non-rapid eye movement (NREM) sleep electroencephalogram (EEG) is an electrophysiological index of sleep intensity reflecting cortical maturation. Available data on ADHD and SWA are conflicting, and developmental differences, or the effect of pharmacological treatment, are relatively unknown. METHODS: We examined, in samples (Mage = 16.4, SD = 1.2), of ever-medicated adolescents at risk for ADHD (n = 18; 72% boys), medication-naïve adolescents at risk for ADHD (n = 15, 67% boys), and adolescents not at risk for ADHD (n = 31, 61% boys) matched for chronological age and controlling for non-ADHD pharmacotherapy, whether ADHD pharmacotherapy modulates the association between NREM SWA and ADHD risk in home sleep. RESULTS: Findings indicated medication-naïve adolescents at risk for ADHD exhibited greater first sleep cycle and entire night NREM SWA than both ever-medicated adolescents at risk for ADHD and adolescents not at risk for ADHD and no difference between ever-medicated, at-risk adolescents, and not at-risk adolescents. CONCLUSIONS: Results support atypical cortical maturation in medication-naïve adolescents at risk for ADHD that appears to be normalized by ADHD pharmacotherapy in ever-medicated adolescents at risk for ADHD. Greater NREM SWA may reflect a compensatory mechanism in middle-later adolescents at risk for ADHD that normalizes an earlier occurring developmental delay.

Theta and alpha activity are differentially associated with physiological and rating scale measures of affective processing in adolescents with but not without ADHD.

Although atypical theta and alpha activity may be biomarkers of attention-deficit/hyperactivity disorder (ADHD) outcomes such as atypical affective processing and attention, the exact nature of the relations of these characteristics is unknown. We examined in age- and sex-matched adolescents (N = 132; Mage = 14.944, years, SD = .802) with and without ADHD, whether resting state (RS) theta and alpha power or theta and alpha event-related synchronization (ERS) during affect regulation (1) differ between adolescents with and without ADHD; (2) are differentially associated with event-related potential (ERP) and parent- and self-report measures of affective processing and inattention, given ADHD status and sex, and (3) are differentially lateralized, given ADHD status and sex. Adolescents with ADHD exhibited lower RS frontal-midline alpha power than adolescents without ADHD. In adolescents with ADHD, right parietal theta ERS was positively associated with the ERP measure of elaborate affective/motivational processing and right parietal RS alpha power was negatively associated with self-reported positive affectivity. In adolescents without ADHD, associations were nonsignificant. There was no disassociation of theta and alpha activity with affective processing and inattention. Consistent with clinical impressions, the between-group difference in frontal-midline theta ERS was more marked in boys than girls.

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.

Echovirus 9 genetic diversity detected in whole-capsid genome sequences obtained directly from clinical specimens using next generation sequencing.

Echovirus 9 (E9) has been detected in an increased number of symptomatic patient samples received by the National Enterovirus Reference Laboratory in Hungary during 2018 compared to previously reported years.Formerly identified E9 viruses from different specimen types detected from patients of various ages and showing differing clinical signs were chosen for the detailed analysis of genetic relationships and potential variations within the viral populations. We used next generation sequencing (NGS) analysis of 3,900 nucleotide long amplicons covering the entire capsid coding region of the viral genome without isolation, directly from clinical samples.Compared to the E9 reference strain, the viruses showed about 79% nucleotide and around 93% amino acid sequence similarity. The four new viral genome sequences had 1-20 nucleotide differences between them also resulting in 6 amino acid variances in the coding region, including 3 in the structural VP1 capsid protein. One virus from a patient with hand, foot, and mouth disease had two amino acid changes in the VP1 capsid protein. An amino acid difference was also detected in the non-structural 2C gene of one virus sequenced from a throat swab sample from a patient with meningitis, compared to the faecal specimen taken two days later. Two amino acid changes, one in the capsid protein, were found between faecal samples of meningitis patients of different ages.Sequencing the whole capsid genome revealed several nucleotide and amino acid differences between E9 virus strains detected in Hungary in 2018.

Distribution of enterovirus genotypes detected in clinical samples in Hungary, 2010-2018.

This report provides the findings of a retrospective surveillance study on the emergence and circulation of enteroviruses with their associated clinical symptoms over a nine-year period detected at the National Enterovirus Reference Laboratory in Hungary between 2010-2018.Enterovirus (EV) detection and genotyping were performed directly from clinical samples. From 4,080 clinical specimens 25 EV types were identified with a median age of patients of 5 years and 68% of all cases affected children aged 10 years or younger, although infections occurred in all age-groups. In 130 cases neurological symptoms were recorded, in 123 cases the infection presented in skin related signs including hand, foot, and mouth disease (HFMD), herpangina and rash. In 2010 EV-A71 was found to cause the majority of diagnosed EV infections while in 2011 and from 2014-2018, Coxsackievirus (CV)-A6 was identified most often. Echovirus E6 accounted for the most cases in 2012 and Echovirus 30 dominated in 2013. EV-D68 was identified only in 2010 and 2013.Widespread circulation of several EV-A and EV-B viruses with occasional occurrence of EV-C and EV-D was detected. The ability of EVs to cause severe infections in sporadic cases and regular outbreaks highlight the importance of continued monitoring of circulating EV types.

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.

Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018.

BackgroundDuring the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.AimsWe summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.MethodsEvery patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.ResultsUntil the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.ConclusionCareful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Phylogenetic analysis of a transfusion-transmitted hepatitis A outbreak.

A transfusion-associated hepatitis A outbreak was found in the first time in Hungary. The outbreak involved five cases. Parenteral transmission of hepatitis A is rare, but may occur during viraemia. Direct sequencing of nested PCR products was performed, and all the examined samples were identical in the VP1/2A region of the hepatitis A virus genome. HAV sequences found in recent years were compared and phylogenetic analysis showed that the strain which caused these cases is the same as that had spread in Hungary recently causing several hepatitis A outbreaks throughout the country.

Wild type HBx and truncated HBx: Pleiotropic regulators driving sequential genetic and epigenetic steps of hepatocarcinogenesis and progression of HBV-associated neoplasms.

Hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma. The molecular mechanisms of tumorigenesis are complex. One of the host factors involved is apparently the long-lasting inflammatory reaction which accompanies chronic HBV infection. Although HBV lacks a typical viral oncogene, the HBx gene encoding a pleiotropic regulatory protein emerged as a major player in liver carcinogenesis. Here we review the tumorigenic functions of HBx with an emphasis on wild type and truncated HBx variants, and their role in the transcriptional dysregulation and epigenetic reprogramming of the host cell genome. We suggest that HBx acquired by the HBV genome during evolution acts like a cellular proto-onc gene that is activated by deletion during hepatocarcinogenesis. The resulting viral oncogene (v-onc gene) codes for a truncated HBx protein that facilitates tumor progression. Copyright © 2015 John Wiley & Sons, Ltd.

[Detection of West Nile virus in human samples: follow-up studies during the 2015 seasonal period]. / A nyugat-nílusi vírus kimutatása humán betegmintákból: nyomon követéses vizsgálatok a 2015. évi szezonális idoszakban.

INTRODUCTION: West Nile virus, a mosquito-borne viral zoonosis is responsible for human infections in Hungary. Laboratory diagnosis is based on serological tests, however the application of molecular methods has been appreciated. AIM: The aim of the study was to investigate blood, cerebrospinal-fluid and urine samples of acutely ill patients and to follow-up PCR positive cases to ascertain the length of virus excretion. METHOD: Clinical specimens were examined by indirect-immunofluorescent, haemagglutination-inhibition, two PCR tests and Sanger-sequencing. Virus isolation in case of two patients was successful. RESULTS: A follow-up study could be carried out in case of 5 patients. Viral nucleic acid was detectable in urine even for several weeks after symptom onset and viral RNA was present at higher concentration compared with other samples. CONCLUSIONS: PCR analysis of urine could provide useful epidemiological and diagnostic information. Therefore, it is recommended to collect urine samples in order to supplement the serological diagnosis. Orv Hetil. 2017; 158(20): 791-796.

Detection and Phylogenetic Analysis of Torque Teno Virus in Salivary and Tumor Biopsy Samples from Head and Neck Carcinoma Patients.

OBJECTIVES: Because torque teno virus (TTV) has been implicated in tumorigenesis as a cocarcinogen, we studied TTV prevalence in saliva and biopsy samples from head and neck cancer (HNCC) patients, patients with premalignant lesions of oral cancer, and controls. We also wished to determine the TTV genotypes in HNCC patients. METHODS: A seminested polymerase chain reaction (PCR) amplifying the N22 region of the TTV genome, as well as direct sequencing of PCR fragments, was used. RESULTS: TTV prevalence was higher in HNCC patients (saliva: 27/71, 38%; tumor biopsy: 22/74, 30%) than in controls (saliva: 8/56, 14%; oral mucosa: 1/19, 5%). TTV prevalence was also high in patients with premalignant lesions of oral carcinoma (saliva: 9/18, 50%; biopsy: 5/21, 24%). By phylogenetic analysis, TTV belonging mostly to genotypes 1 and 2 was found in HNCC patients. In most of the cases, identical TTV strains were present in the biopsy and salivary sample of the same HNCC patient. In addition, the same TTV strain was detected in 2 laryngeal carcinoma biopsies obtained from 2 independent patients. CONCLUSIONS: Our data are compatible with the idea that TTV might act as a cocarcinogen in certain cases of HNCC. Alternatively, HNCC may facilitate either TTV replication or TTV entry into the saliva.

Detection and sequencing of West Nile virus RNA from human urine and serum samples during the 2014 seasonal period.

West Nile virus, a widely distributed mosquito-borne flavivirus, is responsible for numerous animal and human infections in Europe, Africa and the Americas. In Hungary, the average number of human infections falls between 10 and 20 cases each year. The severity of clinically manifesting infections varies widely from the milder form of West Nile fever to West Nile neuroinvasive disease (WNND). In routine laboratory diagnosis of human West Nile virus infections, serological methods are mainly applied due to the limited duration of viremia. However, recent studies suggest that detection of West Nile virus RNA in urine samples may be useful as a molecular diagnostic test for these infections. The Hungarian National Reference Laboratory for Viral Zoonoses serologically confirmed eleven acute human infections during the 2014 seasonal period. In three patients with neurological symptoms, viral RNA was detected from both urine and serum specimens, albeit for a longer period and in higher copy numbers with urine. Phylogenetic analysis of the NS3 genomic region of three strains and the complete genome of one selected strain demonstrated that all three patients had lineage-2 West Nile virus infections. Our findings reaffirm the utility of viral RNA detection in urine as a molecular diagnostic procedure for diagnosis of West Nile virus infections.

Infectious Agents Associated with Head and Neck Carcinomas.

In addition to traditional risk factors such as smoking habits and alcohol consumption, certain microbes also play an important role in the generation of head and neck carcinomas. Infection with high-risk human papillomavirus types is strongly associated with the development of oropharyngeal carcinoma, and Epstein-Barr virus appears to be indispensable for the development of non-keratinizing squamous cell carcinoma of the nasopharynx. Other viruses including torque teno virus and hepatitis C virus may act as co-carcinogens, increasing the risk of malignant transformation. A shift in the composition of the oral microbiome was associated with the development of oral squamous cell carcinoma, although the causal or casual role of oral bacteria remains to be clarified. Conversion of ethanol to acetaldehyde, a mutagenic compound, by members of the oral microflora as well as by fungi including Candida albicans and others is a potential mechanism that may increase oral cancer risk. In addition, distinct Candida spp. also produce NBMA (N-nitrosobenzylmethylamine), a potent carcinogen. Inflammatory processes elicited by microbes may also facilitate tumorigenesis in the head and neck region.

An archived serum sample as a clue for identifying the primary source of a nosocomial hepatitis C virus outbreak in a haemodialysis unit.

Due to an unexpected technical error, patients at a dialysis unit who were seronegative for hepatitis C virus (HCV) were temporarily transferred to another dialysis unit next to a ward reserved for HCV-seropositive patients. In the following 7 months, 17 patients were diagnosed as anti-HCV positive. The aim of the study was to reveal the cause of this nosocomial infection. Anti-HCV-positive sera were further tested by molecular methods. Data collection and on-site epidemiologic inspections were carried out. The source of the nosocomial infection proved to be a seropositive patient treated at the unit, who died before the outbreak was recognized. The exact date of the infection was determined.

Hepatitis A virus subtype IB outbreak among MSM in Hungary with a link to a frozen berry source.

Men who have sex with men (MSM) are at high risk of acquiring hepatitis A virus (HAV) and in recent years several HAV outbreaks mostly affecting MSM have been described. These outbreaks were caused by subtype IA strains circulating in this high-risk population. After years of low incidence, an outbreak among MSM in Hungary caused a significant increase in reported HAV infections in 2022. Samples from 224 HAV IgM-positive patients diagnosed in 2022 were tested for HAV RNA and positive samples were genotyped by sequencing. In 171 patients a unique subtype IB virus was detected with 99.8-100% sequence identity in the VP1/P2A junction. It was distinct from previously published strains, but most closely related to an Egyptian isolate. Sequence analysis revealed one dominant and three minor variants based on VP1/P2A. Whole genome sequencing revealed limited variation among these variants, suggesting a recent common origin. Epidemiological data indicated that sexual transmission was driving the outbreak for most of the year, suggested by the high male to female ratio and the large number of coinfections with HIV and other sexually transmitted infections among the patients. The outbreak was also associated with a restaurant cluster, in which one of the variants was detected and frozen berries were implicated as the source of infections. The outbreak strain was also detected in other countries around Europe and remained frequently detectable in Hungary in 2023. This study provides insights into the molecular and epidemiological characteristics of the described HAV outbreak. The results show that sequencing is not only useful in connecting cases to an outbreak, but also helps to clarify the relatedness of detected variants. Prevention strategies focusing on vulnerable communities may reduce the burden of HAV infections in the future.

Lymphocytic Choriomeningitis Virus Infections in Hungary between 2017-2023-Investigation of the First Congenital Infections.

Lymphocytic choriomeningitis virus (LCMV) is a neglected rodent-borne arenavirus, primarily spread by common house mouse species. Acquired human infections range from asymptomatic to mild flu-like symptoms and self-resolving neurological diseases. In contrast, intrauterine LCMV infection is associated with high mortality and morbidity. Infection of the fetus often leads to fetal death, and surviving fetuses may develop vision impairment and central nervous system developmental disorders. LCMV is mainly diagnosed by serological methods using in-house indirect immunofluorescence assays. LCMV nucleic acid is detected by the nested RT-PCR method and confirmed by Sanger sequencing. In Hungary, 23 acquired lymphocytic choriomeningitis cases were diagnosed between 2017 and 2023. Ten out of 23 confirmed patients proved to be positive by the PCR method. Two cases of intrauterine LCMV infections were detected in 2019 and 2021, respectively. The IgG antibody titers measured in the infant's serum samples were much higher than the IgG titers of the maternal serum samples. Both IgM and IgA antibodies were detectable in the infants' sera. As the microbiological diagnosis of LCMV is rather challenging and the symptoms are very similar to the clinical picture of other common teratogenic pathogens such as cytomegalovirus or Toxoplasma gondii, intrauterine LCMV infections might still be underdiagnosed.