Seroprevalence of SARS-CoV-2 antibodies in the county town of Slovakia - a pilot study from the Trencin city.

Slovakia is a country with only 5.45 million inhabitants. However, the past two years of the COVID-19 pandemic have shown huge inter-regional differences. These were represented by different numbers of diagnosed SARS-CoV-2 cases and the vaccination rates in the regions, as well as by the willingness of the inhabitants to comply with anti-pandemic measures or to undergo testing. The occurrence of such regional disparities provided a rational basis for monitoring the epidemic situation within smaller areas, e.g. at city level. Trencin is a medium-sized Slovak county town with about 55 000 inhabitants. The city administration gave its residents the opportunity to assess their current level of antibodies against the SARS-CoV-2 virus, and received an additional benefit in the form of data on the real epidemic situation in the city, which helped in further management of anti-pandemic measures. The primary aim of the study, conducted in January and February 2022, was to determine the levels of antibodies against the SARS-CoV-2 virus in the inhabitants of Trencin. The results showed that 75% of the study participants, representing the adult population of the city, had detectable IgG antibodies against the SARS-CoV-2 spike protein. Noteworthy, at the time of the study, 13% of the Trencin city population who were unaware of overcoming COVID-19 had specific antibodies against the virus. Furthermore, the antibody levels in recovered unvaccinated subjects increased not only with the severity of their COVID-19 symptoms, but also after multiple recoveries from the disease. On the other hand, the severity of side effects after vaccination did not influence the antibody levels. The results of the study are in line with the current view that hybrid immunity (vaccination plus SARS-CoV-2 infection in any order) offers greater protection than immunity elicited by vaccination or COVID-19 separately. Keywords: SARS-CoV-2 coronavirus; COVID-19; ELISA; seroprevalence; antibodies; vaccination.

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B.

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Seroprevalence of SARS-CoV-2 IgG antibodies in the staff of the Slovak Academy of Sciences in response to COVID-19 and/or vaccination: situation in August 2021.

Cross-sectional seroprevalence study of SARS-CoV-2 IgG antibodies was accomplished in the Slovak Academy of Sciences to inform authorities of research institutions about the situation at their workplaces, to assess the risk of next exposure to SARS-CoV-2, and to guide decisions on institutional measures sustaining essential research in evolving epidemic situation. Study participants provided informed consent, anamnestic information, and self-collected dry blood spot samples that were analyzed by ELISA for SARS-CoV-2 S protein-specific IgG antibodies. Relative antibody levels detected in 1928 subjects showed seroprevalence of 84.13% and led to the following main findings consistent with the current knowledge: (1) mRNA-based vaccines induce better humoral response compared to adenovirus vaccines, (2) antibody levels reflect severity of COVID-19 symptoms, (3) post-COVID vaccination results in marked elevation of IgG levels particularly in asymptomatic and mild cases, (4) antibody levels decrease with increasing time elapsed from vaccination or COVID-19. In addition, data sorting to distinct research institutes and their clustering to three principal scientific sections of the Slovak Academy of Sciences revealed marked differences in seroprevalence, and allowed to identify workplaces with relatively high seropositivity and response rate that can potentially provide a safer working environment than those, where seroprevalence was low or unknown due to low participation. Thus, findings of this study can have direct implications on management decisions during the next pandemic development, with the necessity to keep in mind the phenomenon of time-dependent immunity waning and current spread of more contagious Delta variant of SARS-CoV-2. Keywords: SARS-CoV-2 coronavirus; COVID-19; spike protein; seroprevalence; antibodies; vaccination.

Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay.

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Clinical and Pre-Clinical Evidence of Carbonic Anhydrase IX in Pancreatic Cancer and Its High Expression in Pre-Cancerous Lesions.

Hypoxia is a common phenomenon that occurs in most solid tumors. Regardless of tumor origin, the evolution of a hypoxia-adapted phenotype is critical for invasive cancer development. Pancreatic ductal adenocarcinoma is also characterized by hypoxia, desmoplasia, and the presence of necrosis, predicting poor outcome. Carbonic anhydrase IX (CAIX) is one of the most strict hypoxia regulated genes which plays a key role in the adaptation of cancer cells to hypoxia and acidosis. Here, we summarize clinical data showing that CAIX expression is associated with tumor necrosis, vascularization, expression of Frizzled-1, mucins, or proteins involved in glycolysis, and inevitably, poor prognosis of pancreatic cancer patients. We also describe the transcriptional regulation of CAIX in relation to signaling pathways activated in pancreatic cancers. A large part deals with the preclinical evidence supporting the relevance of CAIX in processes leading to the aggressive behavior of pancreatic tumors. Furthermore, we focus on CAIX occurrence in pre-cancerous lesions, and for the first time, we describe CAIX expression within intraductal papillary mucinous neoplasia. Our review concludes with a detailed account of clinical trials implicating that treatment consisting of conventionally used therapies combined with CAIX targeting could result in an improved anti-cancer response in pancreatic cancer patients.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection.

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2-4hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.

Antiviral Effect of Interferon Lambda Against Lymphocytic Choriomeningitis Virus.

Lambda interferons inhibit replication of many viruses, but their role in the inhibition of lymphocytic choriomeningitis virus (LCMV) infection remains unclear. In this study, we examined the antiviral effects of interferon (IFN)-λ2 and IFN-λ3 against LCMV in A549 cells. We found that IFN-λ2 is a more potent inhibitor of LCMV strain MX compared with IFN-λ3, whereas both cytokines have similar antiviral effects against an immunosuppressive variant of LCMV, clone-13. We also demonstrated that the antiviral activity of IFN-λ2 is more effective if it is delivered early rather than after establishment of a long-term infection, suggesting that virus replication is only partially responsive to the cytokine. In agreement with this observation, we showed that LCMV infection significantly reduces IFNLR1 mRNA expression in infected cells. In addition, LCMV infection, to some extent, compromises the signal transduction pathway of IFN-λ2. This implies that IFN receptors as well as their downstream signaling components could be selectively targeted either directly by LCMV proteins or indirectly by cellular factor(s) that are induced or activated by LCMV infection.

Hypoxia induces the gene expression and extracellular transmission of persistent lymphocytic choriomeningitis virus.

The physiological context of virus-infected cells can markedly affect multiplication and spread of the virus progeny. During persistent infection, the virus exploits the host cell without disturbing its vital functions. However, microenvironmental hypoxia can uncouple this intimate relationship and escalate virus pathogenesis. Accumulating evidence suggests that hypoxia-inducible factor (HIF) modulates gene expression of the viruses that pass through a DNA stage, contain hypoxia-responsive promoter elements, and replicate in the nucleus. Here we show that hypoxia can influence the gene expression and transmission of the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV), which is a neglected human pathogen and teratogen. The MX strain of LCMV, which we used as a model, replicates in a persistent mode in human HeLa cells, fails to produce mature envelope glycoproteins, and spreads through cell-cell contacts in the absence of extracellular infectious virions. Both exposure of MX-infected HeLa cells to chronic hypoxia and gene transfer approaches led to increased virus RNA transcription and higher levels of the viral proteins via a HIF-dependent mechanism. Moreover, hypoxia enhanced the formation of infectious virions capable of transmitting LCMV by cell-free medium. This LCMV "reactivation" might have health-compromising consequences in hypoxia-associated situations, such as fetal development and ischemia-related pathologies.