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Abstract The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

(1) Background: Crimean–Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease endemic in Africa, Asia, the Middle East, and the Balkan and Mediterranean regions of Europe. Although no human CCHF cases have been reported, based on vector presence, serological evidence among small vertebrates, and the general human population, Hungary lies within high evidence consensus for potential CCHF introduction and future human infection. Thus, the aim of our pilot serosurvey was to assess CCHF seropositivity among cattle and sheep as indicator animals for virus circulation in the country. (2) Methods: In total, 1905 serum samples taken from free-range cattle and sheep in 2017 were tested for the presence of anti-CCHF virus IgG antibodies using commercial ELISA and commercial and in-house immunofluorescent assays. (3) Results: We found a total of eleven reactive samples (0.58%) from five administrative districts of Hungary comprising 8 cattle and 3 sheep. The most affected regions were the south–central and northwestern parts of the country. (4) Conclusions: Based on these results, more extended surveillance is advised, especially in the affected areas, and there should be greater awareness among clinicians and other high-risk populations of the emerging threat of CCHF in Hungary and Central Europe.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

Long-read sequencing (LRS) approaches shed new light on the complexity of viral (Kakuk et al., 2021 [1]; Boldogkői et al., 2019 [2]; Depledge et a., 2019 [3]), bacterial (Yan et al., 2018 [4]) and eukaryotic (Tilgner et al., 2014 [5]) transcriptomes. Emerging RNA viruses are zoonotic (Woolhouse et al., 2016 [6]) and create public health problems, e.g. influenza pandemic caused by H1N1 virus in (Fraser et al., 2009 [7]), as well as the current SARS-CoV-2 pandemic (Kim et al., 2020 [8]). In this study, we carried out nanopore sequencing for generating transcriptomic data valuable for structural and kinetic profiling of six important human pathogen RNA viruses, the H1N1 subtype of Influenza A virus (IVA), the Zika virus (ZIKV), the West Nile virus (WNV), the Crimean-Congo hemorrhagic fever virus (CCHFV), the Coxsackievirus [group B serotype 5 (CVB5)] and the Vesicular stomatitis Indiana virus (VSIV), and the response of host cells upon viral infection. The raw sequencing data were filtered during basecalling and only high quality reads (Qscore ≥ 7) were mapped to the appropriate viral and host genomes. Length distribution of sequencing reads were assessed and statistics of data were plotted by the ReadStat.4 python script. The datasets can be used to profile the transcriptomic landscape of RNA viruses, provide information for novel gene annotations, can serve as resource for studying the virus-host interactions, and for the analysis of RNA base modifications. These datasets can be used to compare the different sequencing techniques, library preparation approaches, bioinformatics pipelines, and to analyze the RNA profiles of viruses with small RNA genomes.

Abstract Objective In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Results In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the “regulation of signaling receptor activity” Gene Ontology annotation as a result of viral infection.

This review summarizes the status of resolving the problem of false positive serologic results (FPSR) in Brucella serology, compiles our knowledge on the molecular background of the problem, and highlights some prospects for its resolution. The molecular basis of the FPSRs is reviewed through analyzing the components of the cell wall of Gram-negative bacteria, especially the surface lipopolysaccharide (LPS) with details related to brucellae. After evaluating the efforts that have been made to solve target specificity problems of serologic tests, the following conclusions can be drawn: (i) resolving the FPSR problem requires a deeper understanding than we currently possess, both of Brucella immunology and of the current serology tests; (ii) the practical solutions will be as expensive as the related research; and (iii) the root cause of FPSRs is the application of the same type of antigen (S-type LPS) in the currently approved tests. Thus, new approaches are necessary to resolve the problems stemming from FPSR. Such approaches suggested by this paper are: (i) the application of antigens from R-type bacteria; or (ii) the further development of specific brucellin-based skin tests; or (iii) the application of microbial cell-free DNA as analyte, whose approach is detailed in this paper.

We aimed to estimate the proportion of the population infected with SARS-CoV-2 in the first year of the pandemic. The study population consisted of outpatient adults with mild or no COVID-19 symptoms and was divided into subpopulations with different levels of exposure. Among the subpopulation without known previous COVID-19 contacts, 4143 patients were investigated. Of the subpopulation with known COVID-19 contacts, 594 patients were investigated. IgG- and IgA-seroprevalence and RT-PCR positivity were determined in context with COVID-19 symptoms. Our results suggested no significant age-related differences between participants for IgG positivity but indicated that COVID-19 symptoms occurred most frequently in people aged between 20 and 29 years. Depending on the study population, 23.4–74.0% PCR-positive people (who were symptomless SARS-CoV-2 carriers at the time of the investigation) were identified. It was also observed that 72.7% of the patients remained seronegative for 30 days or more after their first PCR-positive results. This study hoped to contribute to the scientific understanding of the significance of asymptomatic and mild infections in the long persistence of the pandemic.

PurposeThe basis of the antitumor immunotherapy, of which the purpose is the stimulation of the immune system. We have used two of the Pathogen Associated Molecular Patterns: unmethylated CpG oligonucleotide, a ligand of Toll-like receptor 9 (TLR9), and lipopolysaccharide (LPS) which is recognized by TLR4, combined with an agonistic OX40 receptor-specific monoclonal antibody (anti-OX40), which is expressed by activated regulatory T-cells (and by other activated T-cell populations as well). The objective of this study was to prove the effectiveness of the aforementioned compounds in an animal model, on a bladder cancer cell line.MethodsWe have instilled MB49 cells subcutaneously, to the left musculus biceps femoris. We have created three observation groups, each containing ten mice. After eleven days, all treated mice bearing the size of 5-8 mm (in diameter) tumor were administered CpG + anti-OX40 or LPS + anti-OX40 three times with a three-day lap between each treatment. Mice in the control group did not receive any treatment.ResultsAll the specimens from the control and LPS + anti-OX40 groups have died by the sixtieth day of the observation period, however, five mice from the CpG + anti-OX40 group were still alive. The experiment lasted until the last surviving mouse died, which occurred on the 357th day after tumor implantation.DiscussionThe treatment with LPS did not make anti-OX40 more potent and did not increase the survival times. However, CpG + anti-OX40 has shown increased antitumor activity compared to the other two groups.

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94-99 and to amino acids 140-147 on the beta chain and it recognizes the amino acids 23-32 and 93-98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity.

Abstract Background Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease. Findings Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents. Conclusions Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

Data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete Freund’s adjuvant (CFA) that can maintain safe and durable immunologic homeostasis in NOD mice. Systemic coinoculation of prediabetic mice with recombinant vaccinia virus rVV-CTB::GAD and undiluted or 10-fold diluted CFA demonstrated a significant decrease in hyperglycemia and pancreatic islet inflammation in comparison with control animals during 17–61 and 17–105 weeks of age, respectively. Synergy in these beneficial effects was observed during 43–61 and 61–105 wks of age, respectively. Inflammatory cytokine and chemokine levels in GAD-stimulated splenocytes isolated from vaccinated mice were generally lower than those detected in unvaccinated mice. The overall health and humoral immune responses of the vaccinated animals remained normal throughout the duration of the experiments.