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Sindbis virus (SINV) is a mosquito-borne alphavirus capable of causing neurological and immunological symptoms in humans, yet its effects on neural/immune systems remain insufficiently characterized. This study aimed to examine SINV replication, UV-C light inactivation, apoptosis induction, and immune gene modulation in human SH-SY5Y neuroblastoma cells. Following viral adaptation and infectious dose determination, SINV replication and inactivation were assessed using RT-qPCR and dsRNA immunofluorescence. Apoptotic markers (caspase-3, Bax, Bcl-2) were analyzed by immunofluorescence and immune genes expression kinetics (TLR3/7, RIGI, MDA5, IL-1β, IL-6, TNFα, IL-10, IFNβ and β-catenin) were measured at defined time points post-infection by RT-qPCR. SH-SY5Y cells supported productive SINV infection, with viral RNA detectable as early as 3 hpi and marked cytopathic effects by 24 hpi. A custom-built UV-C chamber achieved complete viral inactivation following 3 × 30 s exposures. We observed SINV time-course replication and UV-C inactivation with conspicuous morphological alterations in SH-SY5Y cells. Furthermore, SINV triggered caspase-dependent apoptosis and robust transcriptional upregulation of innate immune genes, peaking between 12–16 hpi and declining by 30 hpi. These findings elucidate the temporal dynamics of SINV replication, cell death mechanisms, and immune activation in a neuronal context, contributing to a better understanding of SINV neuropathogenesis.

Since the late 2000s, a growing diversity of distinct hantaviruses has been identified in shrews across Europe. However, knowledge about shrew-borne hantaviruses in Hungary is limited. This study was initiated to investigate the prevalence and host range of these viruses in shrew populations in Hungary. Between 2007 and 2011, 129 shrew specimens, representing six species, were collected from four locations in Southern Transdanubia. Lung tissues were analyzed for the presence of hantaviral RNA by RT-PCR targeting the L genomic segment. Hantavirus RNA was detected in four shrew species. This study provides the first molecular evidence of hantavirus infection in the pygmy shrew (Sorex minutus), Miller’s water shrew (Neomys milleri), and the Eurasian water shrew (Neomys fodiens) in Hungary, while also confirming infection in the Eurasian common shrew (Sorex araneus). The overall prevalence varied among species, with marked detection rates in S. minutus (50%) and N. fodiens (33.3%). Our findings document the co-circulation of hantaviruses in four distinct shrew species within the same ecosystems in Hungary, thereby expanding the known host range for these viruses within the country. This contributes to the understanding of the complex hantavirus landscape in Central Europe and highlights the need for genetic characterization of identified viruses to assess their evolutionary dynamics.

The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and for other medical applications as a small-molecule inhibitor of the protein–protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concern such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1–5 μM). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63–ACE2, hepatitis C virus E–CD81) as well as others (e.g., IL-2–IL-2Rα) with similar potency. This nonspecificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells, both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 μM) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19.

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity—and not inverse similarity—between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.

Background and purpose: The COVID-19 pandemic continues to pose challenges, especially with the emergence of new SARS-CoV-2 variants that are associated with higher infectivity and/or compromised protection afforded by the current vaccines. There is a high demand for additional preventive and therapeutic strategies effective against this changing virus. Repurposing of approved or clinically tested drugs can provide an immediate solution. Experimental Approach: We applied a novel computational approach to search among approved and commercially available drugs. Antiviral activity of a predicted drug, azelastine, was tested in vitro in SARS-CoV-2 infection assays with Vero E6 cells, Vero cells stably overexpressing the human TMPRSS2 and ACE2 proteins as well as on reconstituted human nasal tissue using the predominant variant circulating in Europe in summer 2020, B.1.177 (D614G variant), and its emerging variants of concern; B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants. The effect of azelastine on viral replication was assessed by quantification of viral genomes by droplet digital PCR or qPCR. Key results: The computational approach identified major drug families, such as anti-infective, anti-inflammatory, anti-hypertensive, antihistamine, and neuroactive drugs. Based on its attractive safety profile and availability in nasal formulation, azelastine, a histamine 1 receptor-blocker was selected for experimental testing. Azelastine reduced the virus-induced cytopathic effect and SARS-CoV-2 copy numbers both in preventive and treatment settings upon infection of Vero cells with an EC 50 of 2.2–6.5 µM. Comparable potency was observed with the alpha, beta and delta variants. Furthermore, five-fold dilution (containing 0.02% azelastine) of the commercially available nasal spray formulation was highly potent in inhibiting viral propagation in reconstituted human nasal tissue. Conclusion and Implications: Azelastine, an antihistamine available as nasal sprays developed against allergic rhinitis may be considered as a topical prevention or treatment of nasal colonization by SARS-CoV-2. A Phase 2 efficacy indicator study with azelastine-containing nasal spray that was designed based on the findings reported here has been concluded recently, confirming accelerated viral clearance in SARS-CoV-2 positive subjects.

Background Parallel to the growth of the oral healthcare market, there is a constantly increasing demand for natural products as well. Many customers prefer products that contain fewer toxic agents, therefore providing an environmentally friendly solution with the benefit of smaller risk to the user. Medieval and early modern medicinal knowledge might be useful when looking for natural, herbal-based components to develop modern products. Along with these considerations we created, tested, and compared an entirely natural mouthwash, named Herba Dei. Methods The manufacturing procedure was standardized, and the created tincture was evaluated by GC/MS analysis for active compounds, experimentally tested in cell-based cytotoxicity, salivary protein integrity, cell-free antioxidant activity, anti-bacterial and anti-viral assays, and compared with three market-leading mouthwashes. Results Our tincture did not show significant damage in the cytotoxicity assays to keratinocyte and Vero E6 cells and did not disrupt the low molecular weight salivary proteins. Its radical scavenging capacity surpassed that of two tested, partly natural, and synthetic mouthwashes, while its antibacterial activity was comparable to the tested products, or higher in the bacterial aerobic respiratory assay. The active compounds responsible for the effects include naturally occurring phenylpropanoids, terpenes, and terpenoids. Our mouthwash proved to be effective in vitro in lowering the copy number of SARS-CoV-2 in circumstances mimicking the salivary environment. Conclusions The developed product might be a useful tool to impede the transmission and spread of SARS-CoV-2 in interpersonal contact and aerosol-generating conditions. Our mouthwash can help reduce the oral bacterial flora and has an antioxidant activity that facilitates wound healing and prevents adverse effects of smoke in the oral cavity.

The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and β-apo-8′carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics.

Background/Objectives: The ongoing COVID-19 pandemic has underscored the need for alternative prophylactic measures, particularly for populations for whom vaccines may not be effective or accessible. This study aims to evaluate the efficacy of intranasally administered IgY antibodies derived from hen egg yolks as a protective agent against SARS-CoV-2 infection in Syrian golden hamsters, a well-established animal model for COVID-19. Methods: Hens were immunized with the spike protein of SARS-CoV-2 to generate IgY antibodies. These antibodies were extracted from the egg yolks, purified, and their neutralizing activity was tested in vitro. Syrian golden hamsters were then treated with the IgY antibodies before being challenged with SARS-CoV-2. Viral loads were quantified using droplet digital PCR (ddPCR), and lung pathology was assessed through histopathological analysis. Results: The in vitro assays showed that IgY effectively neutralized SARS-CoV-2. In the in vivo hamster model, IgY treatment led to a significant reduction in viral loads and a marked decrease in lung consolidation and inflammation compared to the positive control group. Histopathological findings further supported the protective role of IgY in reducing lung damage caused by SARS-CoV-2. Conclusions: The results demonstrate that IgY antibodies exhibit strong antiviral activity and can significantly reduce SARS-CoV-2 viral loads and associated lung pathology in hamsters. These findings suggest that IgY could be a viable prophylactic option for preventing SARS-CoV-2 infection, particularly for individuals who cannot receive or respond to vaccines. Further studies are warranted to optimize dosage and explore the long-term efficacy of IgY antibodies.