Multicentric evaluation of sensitivity of eight commercial anti-SARS-CoV-2 antibody assays and their correlation to virus neutralization titers in seropositive subjects.

Diagnosis of SARS-CoV-2 virus is mainly based on direct detection. Determination of specific antibodies has been used mostly for epidemiological reasons. However, select immunoassays showed good correlation to plaque reduction virus neutralization test (PRNT) in smaller patient cohorts, which suggests their potential as predictors of virus neutralization titer. A total of 3,699 samples from Covid-19 patients were included in the multicentric study performed in the Czech Republic. Anti-SARS-CoV-2 antibody levels were evaluated by 8 commercial antibody assays. Simultaneously, PRNT evaluations were performed with the SARS-CoV-2 B.1.258 variant. All immunoassays showed an overall high true positive diagnostic value ranging from 79.17 to 98.04%. Several commercial EIA methods showed highly positive correlation between the assay results and PRNT levels, e.g., Liaison CoV-2 TrimericS IgG DiaSorin (Spearman r = 0.8833; Architect SASRS-CoV-2 IgG Abbott (r = 0.7298); NovaLisa SARS-CoV-2 IgG NovaTec (r = 0.7103) and Anti-SARS-CoV-2 ELISA IgG Euroimmun (r = 0.7094). While this correlation was less positive for other assays, those, conversely, presented higher true positive values. For most immunoassays, the positive percent agreement of the results was ≥ 95% in sera exhibiting PRNT levels of 1:80 and higher. The assays tested have shown variable correlation to PRNT. Those possessing high positive predictive values serve well as qualitative tests, while others can be utilised as quantitative tests highly predictive of neutralization antibody levels.

Transformation of hydroxylated polychlorinated biphenyls by bacterial 2-hydroxybiphenyl 3-monooxygenase.

Monohydroxylated PCBs (OH-PCBs) are an (eco)toxicologically significant group of compounds, as they arise from the oxidation of polychlorinated biphenyls (PCBs) and, at the same time, may exert even more severe toxic effects than their parent PCB molecules. Despite having been widely detected in environmental samples, plants, and animals, information on the fate of OH-PCBs in the environment is scarce, including on the enzymatic machinery behind their degradation. To date, only a few bacterial taxa capable of OH-PCB transformation have been reported. In this study, we aimed to obtain a deeper insight into the transformation of OH-PCBs in soil bacteria and isolated a Pseudomonas sp. strain P1B16 based on its ability to use o-phenylphenol (2-PP) which, when exposed to the Delor 103-derived OH-PCB mixture, depleted a wide spectrum of mono-, di, and trichlorinated OH-PCBs. In the P1B16 genome, a region designated as hbp was identified, which bears a set of putative genes involved in the transformation of OH-PCBs, namely hbpA encoding for a putative flavin-dependent 2-hydroxybiphenyl monooxygenase, hbpC (2,3-dihydroxybiphenyl-1,2-dioxygenase), hbpD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase), and the transcriptional activator-encoding gene hbpR. The hbpA coding sequence was heterologously expressed, purified, and its substrate specificity was investigated towards the Delor 103-derived OH-PCB mixture, individual OH-PCBs, and multiple (chlorinated) phenolics. Apart from 2-PP and 2-chlorophenol, HbpA was also demonstrated to transform a range of OH-PCBs, including a 3-hydroxy-2,2',4',5,5'-pentachlorobiphenyl. Importantly, this is the first direct evidence of HbpA homologs being involved in the degradation of OH-PCBs. Moreover, using a P1B16-based biosensor strain, the specific induction of hbp genes by 2-PP, 3-phenylphenol, 4-phenylphenol, and the OH-PCB mixture was demonstrated. This study provides direct evidence on the specific enzymatic machinery responsible for the transformation of OH-PCBs in bacteria, with many implications in ecotoxicology, environmental restoration, and microbial ecology in habitats burdened with PCB contamination.

Two novel Bartonella (sub)species isolated from edible dormice (Glis glis): hints of cultivation stress-induced genomic changes.

Bartonelloses are neglected emerging infectious diseases caused by facultatively intracellular bacteria transmitted between vertebrate hosts by various arthropod vectors. The highest diversity of Bartonella species has been identified in rodents. Within this study we focused on the edible dormouse (Glis glis), a rodent with unique life-history traits that often enters households and whose possible role in the epidemiology of Bartonella infections had been previously unknown. We identified and cultivated two distinct Bartonella sub(species) significantly diverging from previously described species, which were characterized using growth characteristics, biochemical tests, and various molecular techniques including also proteomics. Two novel (sub)species were described: Bartonella grahamii subsp. shimonis subsp. nov. and Bartonella gliris sp. nov. We sequenced two individual strains per each described (sub)species. During exploratory genomic analyses comparing two genotypes ultimately belonging to the same species, both factually and most importantly even spatiotemporally, we noticed unexpectedly significant structural variation between them. We found that most of the detected structural variants could be explained either by prophage excision or integration. Based on a detailed study of one such event, we argue that prophage deletion represents the most probable explanation of the observed phenomena. Moreover, in one strain of Bartonella grahamii subsp. shimonis subsp. nov. we identified a deletion related to Bartonella Adhesin A, a major pathogenicity factor that modulates bacteria-host interactions. Altogether, our results suggest that even a limited number of passages induced sufficient selective pressure to promote significant changes at the level of the genome.

Retrospective Analysis Revealed an April Occurrence of Monkeypox in the Czech Republic.

Herein, we present our findings of an early appearance of the Monkeypox virus in Prague, Czech Republic. A retrospective analysis of biological samples, carried out on the 28th of April, revealed a previously unrecognized case of Monkeypox virus (MPxV) infection. Subsequent data analysis confirmed that the virus strain belongs to the ongoing outbreak. Combined with clinical and epidemiological investigations, we extended the roots of the current outbreak at least back to 16th of April, 2022.

Exosomes released by imatinib­resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib­sensitive cells in the presence of imatinib.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR­ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR­ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib­resistant K562 (K562IR) cells. The K562IR­derived exosomes were internalized by imatinib­sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance­associated markers using a deep label­free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon­induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

Characterization of Two Historic Smallpox Specimens from a Czech Museum.

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.

Induction and characterization of a replication competent cervid endogenous gammaretrovirus (CrERV) from mule deer cells.

Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection.

Hematooncologic patients often host rare or fastidious pathogens. Using 16S rDNA sequencing and transmission electron microscopy, we have identified 2 lymphoma patients infected with Candidatus Neoehrlichia mikurensis. In both individuals, the clinical presentation suggested ehrlichiosis-like syndrome. We believe that molecular techniques open new vistas in the field of pathogen detection.

Borna disease virus (BDV) circulating immunocomplex positivity in addicted patients in the Czech Republic: a prospective cohort analysis.

BACKGROUND: Borna disease virus (BDV) is an RNA virus belonging to the family Bornaviridae. Borna disease virus is a neurotropic virus that causes changes in mood, behaviour and cognition. BDV causes persistent infection of the central nervous system. Immune changes lead to activation of infection. Alcohol and drug dependence are associated with immune impairment. METHODS: We examined the seropositivity of BDV circulating immunocomplexes (CIC) in patients with alcohol and drug dependence and healthy individuals (blood donors). We examined 41 addicted patients for the presence of BDV CIC in the serum by ELISA at the beginning of detoxification, and after eight weeks of abstinence. This is the first such study performed in patients with alcohol and drug dependence. RESULTS: BDV CIC positivity was detected in 36.59% of addicted patients on day 0 and in 42.86% on day 56. The control group was 37.3% positive. However, we did not detect higher BDV CIC positivity in addicted patients in comparison with blood donors (p = 0.179). The significantly higher level of BDV CIC was associated with lower levels of GGT (gamma glutamyl transferase) (p = 0.027) and approached statistical significance with the lower age of addicted patients (p = 0.064). We did not find any association between BDV CIC positivity and other anamnestic and demographic characteristics. CONCLUSIONS: In our study addicted patients did not have significantly higher levels of BDV CIC than the control group. The highest levels of BDV CIC were detected in patients with lower levels of GGT and a lower age. TRIAL REGISTRATION: This study was approved by the ethical committee of the University Hospital Medical Faculty of Charles University in Pilsen, Czech Republic (registration number 303/2001).

Borna disease virus circulating immunocomplex positivity and psychopathology in psychiatric patients in the Czech Republic.

OBJECTIVES: Borna disease virus (BDV) is an RNA virus belonging to the family Bornaviridae. BDV is a neurotropic virus that causes changes in mood, behaviour and cognition. Patients with psychiatric disorders have a higher incidence of BDV positivity than healthy individuals. METHODS: We examined the seropositivity of BDV circulating immunocomplexes (CIC) in psychiatric patients and healthy individuals (blood donors). We examined 39 psychiatric inpatients for the presence of BDV CIC in the serum by ELISA on day 0, 28 and 56. During the same period psychopathology was measured using psychiatric scales (CGI, CGI-I, MADRS, SDS, PANSS). This is the first such study performed in the Czech Republic. RESULTS: BDV CIC positivity was detected in 66.7% of psychiatric patients (26/39) on day 0, in 53.9% (14/26) on day 28 and in 52.9% on day 56 (9/17). The control group was 22.2% (28/126) positive. The incidence of BDV CIC was significantly higher in psychiatric patients than in healthy individuals (p=0.001). The significantly higher level of BDV CIC was associated with the higher severity of psychopathology in comparison with patients with mild or moderate psychopathology (p=0.03). We did not find any association between BDV CIC positivity and other characteristics (age, diagnosis, family, personal history, the history of infectious diseases, contact with animals). CONCLUSION: In our study psychiatric patients had significantly higher levels of BDV CIC than the control group. The highest levels of BDV CIC were detected in patients with more severe psychopathology.

Sodium valproate facilitates the propagation of granulocytic ehrlichiae (Anaplasma phagocytophilum) in HL-60 cells.

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC50-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1-2 and < or = 0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (< or = 0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness.