The Anti-Histamine Azelastine, Identified by Computational Drug Repurposing, Inhibits Infection by Major Variants of SARS-CoV-2 in Cell Cultures and Reconstituted Human Nasal Tissue.

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 EC50 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.

Single-particle detection of native SARS-CoV-2 virions by microfluidic resistive pulse sensing.

Microfluidic resistive pulse sensing (MRPS) was used to determine the size -distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on detecting nearly 30,000 single virions. However, the ultrastructure of SARS-CoV-2 is thoroughly described, but ensemble properties of SARS-CoV-2, e.g., its particle size distribution, are sparsely reported. According to the MRPS results, the size distribution of SARS-CoV-2 follows a log-normal function with a mean value of 85.1 nm, which corresponds to an approximate diameter of the viral envelope. This result also confirms the low number (< 50) of spike proteins on the surface of the virions.

Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity.

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.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary.

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.

Natural Apocarotenoids and Their Synthetic Glycopeptide Conjugates Inhibit SARS-CoV-2 Replication.

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 ß-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.

Effectiveness Regarding Hantavirus Detection in Rodent Tissue Samples and Urine.

The natural hosts of Orthohantaviruses are rodents, soricomorphs and bats, and it is well known that they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces throughout the entirety of their lives. We aim to identify the effectiveness of hantavirus detection in rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 deceased animals. All organs and urine were tested using nested reverse transcription polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) by Western blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV and Tula virus (TULV) were clearly identified. Amid the PCR-positive samples, the nucleic acid of the viruses was detected most effectively in the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only three out of 20 rodent urine samples were positive when tested using nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid in any of the tested organs.

Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

Crimean-Congo hemorrhagic fever virus infection triggers the upregulation of the Wnt signaling pathway inhibitor genes.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic agent. Thus far, vaccines and specific antiviral therapies are not available against the threat of infection. Our knowledge regarding its pathogenesis is indeed limited, and thus, developing effective antiviral therapies is hampered. Several studies have demonstrated that the CCHFV infection has an impact on numerous signal transduction pathways. In parallel, the Wnt signaling pathway components are responsible for different important biological processes including cell fate determination, cell migration and cell polarity. Moreover, its implication among several virus infections has been proven, yet little is known in reference to which components of the Wnt pathway are being activated/inhibited as a response to the infection. Our aim was to elicit the influence of the CCHFV infection on adenocarcinomic human alveolar basal epithelial cells in vitro regarding the Wnt signaling pathway-related genes. Gene-expression changes of 92 Wnt-associated genes were examined 48 h post-infection. Furthermore, ß-catenin levels were compared in the infected and uninfected cells. Significant changes were observed in the case of 13 genes. The majority of the upregulated genes are associated with the inhibition of the Wnt/ß-catenin signaling. Additionally, infected cells expressed less ß-catenin. Our findings suggest that CCHFV blocks the Wnt/ß-catenin pathway. Our study corroborates the link between CCHFV infection and the Wnt signaling pathways. In addition, it broadens our knowledge in the CCHFV pathomechanism.

Temporal Dynamics of Two Pathogenic Hantaviruses Among Rodents in Hungary.

Hantaviruses are worldwide pathogens, which often cause serious or even fatal diseases in humans. Hosts are predominantly in the form of rodents and soricomorphs; however, bats are also described as an important reservoir. In Hungary, representatives of two human pathogenic species of the genus Orthohantavirus are present: the Dobrava-Belgrade orthohantavirus and Puumala orthohantavirus. In Hungarian forests, the dominant rodent species are Apodemus flavicollis, Apodemus agrarius, Apodemus sylvaticus, and Myodes glareolus, all of which are natural reservoirs comprising different hantaviruses. The aim of the study was to survey the prevalence of hantaviruses among rodent populations and examine the potential relationship regarding population densities, years, sex, and seroprevalence. Rodents were trapped at 13 sampling plots in a forest reserve located in the Mecsek Mountain range, Hungary, from March to October between 2011 and 2014. Rodent serum samples were tested for IgG antibodies against Dobrava-Belgrade virus and Puumala virus by enzyme-linked immunosorbent assay (ELISA) using a recombinant nucleocapsid protein. During the 4-year sampling period, 2491 specimens were tested and 254 (10.2%) proved seropositive for orthohantaviruses. In 2011, the seroprevalence among Apodemus spp. and M. glareolus was 17.2% (114/661) and 3.9% (3/77), respectively, although this rate had reversed itself in 2014. Seropositivity was substantiated in 18.4% (12/65) of Myodes voles, while only 3.6% (13/359) of the tested Apodemus rodents were found to be IgG positive. Seroconversion was observed in 58 cases, while seroreversion was only detected in 3 individual cases. A significant difference among the number of infected males and females was identified in the first 2 years of our study. Winter survival with respect to rodents was not negatively affected due to the hantavirus infection. Hantavirus seroprevalence was not directly influenced by host abundance. Consequently, we assume that high rodent density alone does not lead to an increased risk of hantavirus infection among the rodent host population.

Genetic characterization of a novel picornavirus in Algerian bats: co-evolution analysis of bat-related picornaviruses.

Bats are reservoirs of numerous zoonotic viruses. The Picornaviridae family comprises important pathogens which may infect both humans and animals. In this study, a bat-related picornavirus was detected from Algerian Minioptreus schreibersii bats for the first time in the country. Molecular analyses revealed the new virus originates to the Mischivirus genus. In the operational use of the acquired sequence and all available data regarding bat picornaviruses, we performed a co-evolutionary analysis of mischiviruses and their hosts, to authentically reveal evolutionary patterns within this genus. Based on this analysis, we enlarged the dataset, and examined the co-evolutionary history of all bat-related picornaviruses including their hosts, to effectively compile all possible species jumping events during their evolution. Furthermore, we explored the phylogeny association with geographical location, host-genus and host-species in both data sets.

Molecular Identification of a Novel Hantavirus in Malaysian Bronze Tube-Nosed Bats (Murina aenea).

In the past ten years, several novel hantaviruses were discovered in shrews, moles, and bats, suggesting the dispersal of hantaviruses in many animal taxa other than rodents during their evolution. Interestingly, the coevolutionary analyses of most recent studies have raised the possibility that nonrodents may have served as the primordial mammalian host and harboured the ancestors of rodent-borne hantaviruses as well. The aim of our study was to investigate the presence of hantaviruses in bat lung tissue homogenates originally collected for taxonomic purposes in Malaysia in 2015. Hantavirus-specific nested RT-PCR screening of 116 samples targeting the L segment of the virus has revealed the positivity of two lung tissue homogenates originating from two individuals, a female and a male of the Murina aenea bat species collected at the same site and sampling occasion. Nanopore sequencing of hantavirus positive samples resulted in partial genomic data from S, M, and L genome segments. The obtained results indicate molecular evidence for hantaviruses in the M. aenea bat species. Sequence analysis of the PCR amplicon and partial genome segments suggests that the identified virus may represent a novel species in the Mobatvirus genus within the Hantaviridae family. Our results provide additional genomic data to help extend our knowledge about the evolution of these viruses.

Serologic survey of the Crimean-Congo haemorrhagic fever virus infection among wild rodents in Hungary.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne pathogen, which causes an increasing number of severe infections in many parts of Africa, Asia and in Europe. The virus is primarily transmitted by ticks, however, the spectrum of natural hosts regarding CCHFV includes a wide variety of domestic and wild animals. Although the presence of CCHFV was hypothesized in Hungary, data in support of CCHFV prevalence has thus far, proven insufficient. In the present study, rodents belonging to four species, the yellow-necked mouse (Apodemus flavicollis), the striped field mouse (A. agrarius), the wood mouse (A. sylvaticus) and the bank vole (Myodes glareolus), were all systematically trapped in the Mecsek Mountain region (Southwest Hungary), from 2011 through 2013. Rodent sera were collected and screened for CCHFV antibodies with dot-blot pre-screening and immunofluorescence assay. Among the 2085 tested rodents, 20 (0.96%) were positive for IgG antibody against CCHFV. Seroprevalence was the highest (1.25%) in A. flavicollis serum samples. Distinctly, we now provide the first data regarding CCHFV occurrence and seroprevalence among wild rodents in Hungary. This observation represents a need for large-scale surveillance to effectively assess the enzootic background and the potential public health risk of CCHFV in Hungary.

Parallel Survey of Two Widespread Renal Syndrome-Causing Zoonoses: Leptospira spp. and Hantavirus in Urban Environment, Hungary.

Rodents are important reservoir hosts for several zoonotic pathogens that cause significant morbidity and mortality in humans. Among others, leptospirosis is one of the most widespread zoonotic diseases worldwide and has the similar clinical manifestation with hantavirus infection in humans. Despite the fact that both pathogens have great epidemiological significance in Europe, no epizootiological data exist for urbanized areas so far. Therefore, this study was conducted to investigate the occurrence and prevalence of Leptospira spp. and hantaviruses in small wild rodents living in close proximity to humans. Altogether, 338 small rodents representing five different species (Apodemus agrarius, A. flavicollis, A. sylvaticus, Microtus arvalis, and Myodes glareolus) were captured in the city of Pécs (Hungary) and screened for pathogens by different types of PCR methods (TaqMan-based real-time PCR/PCR, RT-PCR/PCR). A total of 18.3% of the rodents were positive for Leptospira kirschneri, L. interrogans, and L. borgpetersenii. Nucleic acid of Tula hantavirus and human pathogen Dobrava-Belgrade orthohantavirus were detected in 8% of tested specimens. Furthermore, dual infections with both Leptospira spp. and hantaviruses were shown in 2.6% of animals, suggesting that the same rodent host can be infected with several pathogens at the same time, therefore, representing a serious threat to public health. Overall, this study provides important surveillance data on the prevalence of Leptospira spp. and hantaviruses from rodents in urbanized environment for the first time in Hungary and emphasizes the importance of further ecoepidemiological investigations.

Serologic survey of orthopoxvirus infection among rodents in hungary.

As a result of discontinuing vaccination against smallpox after the late 1970s, different orthopoxviruses (OPVs), such as cowpox virus (CPXV), have become a re-emerging healthcare threat among zoonotic pathogens. In Hungary, data on OPV prevalence among its rodent host species have been absent. Here, rodents belonging to four species, i.e., striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), wood mouse (A. sylvaticus) and bank vole (Myodes glareolus), were live trapped at 13 sampling plots on a 149-ha area in the Mecsek Mountains, Hungary, from March to September in 2011 and 2012. Rodent sera were collected and screened for OPV-reactive antibodies with an immunfluorescence assay (IFA). Among the 1587 tested rodents, 286 (18.0%) harbored OPV-specific antibodies. Seroprevalence was the highest for the bank vole (71.4%) and the striped field mouse (66.7%). Due to a masting event in the autumn of 2011 across Central Europe, the abundance of bank voles increased drastically in the 2012 season, raising the overall OPV seroprevalence. We provide the first data on OPV occurrence and seroprevalence in rodents in Hungary. The circulation of OPV in rodents in densely populated areas warrants further studies to elucidate the zoonotic potential of OPV in humans.

Serosurvey of pathogenic hantaviruses among forestry workers in Hungary.

OBJECTIVES: The aim of the study was to survey the prevalence of human hantavirus infections among forestry workers, who are considered a risk population for contracting the disease. Sera collected from volunteers were tested for antibodies against Dobrava-Belgrade (DOBV) and Puumala (PUUV) viruses. MATERIAL AND METHODS: For serological analyses, full capsid proteins of DOBV and PUUV viruses were produced in a bacterial expression system, while Ni-resin was used for protein purification. Samples were screened for anti-hantavirus antibodies by ELISA, results were confirmed by Western blot analysis. RESULTS: A total of 835 samples collected from 750 males and 85 females were tested by indirect ELISA and positive test results were confirmed by Western blot assay. Out of the 45 ELISA-reactive samples, 38 were confirmed by Western blot analysis. The regional distribution of seropositive individuals was as follows: 1.9% (2/107) in the Danube-Tisza Plateau (Great Plains), 3.1% (10/321) in the Southern Transdanubian region, 5.2% (13/248) in the Northern Transdanubian, and 8.2% (13/159) in the North Hungarian Mountains. CONCLUSIONS: Our data show marked geographic differences in seroprevalence of pathogenic hantaviruses within Hungary, indicating elevated exposure to hantavirus infections in some areas.

Molecular detection and phylogenetic analysis of tick-borne encephalitis virus in rodents captured in the transdanubian region of Hungary.

Abstract Tick-borne encephalitis virus (TBEV) infection is a common zoonotic disease affecting humans in Europe and Asia. To determine whether TBEV is present in small mammalian hosts in Hungary, liver samples of wild rodents were tested for TBEV RNA. Over a period of 7 years, a total of 405 rodents were collected at five different geographic locations of the Transdanubian region. TBEV nucleic acid was identified in four rodent species: Apodemus agrarius, A. flavicollis, Microtus arvalis, and Myodes glareolus. Out of the 405 collected rodents, 17 small mammals (4.2%) were positive for TBEV. The present study provides molecular evidence and sequence data of TBEV from rodents in Hungary.

Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury.

Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

Molecular characterization of Dobrava and Kurkino genotypes of Dobrava-Belgrade hantavirus detected in Hungary and Northern Croatia.

Among the Hantavirus genus, Saaremaa virus (SAAV) has been the subject of taxonomical debates. While the International Committee on Taxonomy of Viruses declares SAAV as a distinct species, several European hantavirus experts proposed that SAAV is in fact a genotype of Dobrava-Belgrade virus (DOBV). In the present study we performed S-segment-based phylogenetic analysis of eight DOBV strains identified in rodents in Hungary and Northern Croatia. These new sequences considerably increase the number of complete nucleoprotein gene sequences deposited in the NCBI database. Our phylogenetic analysis clearly support the taxonomical nomenclature recently proposed for DOBV, i.e., genotypes such as Dobrava, Saaremaa, Kurkino, and Sochi should indeed be classified within the DOBV hantavirus species. Moreover, we found that only the Dobrava and Kurkino genotypes of DOBV species are circulating in Hungary while currently there is no evidence for the presence of Saaremaa genotype.

Identification of tick-borne encephalitis virus in ticks collected in southeastern Hungary.

Tick-borne encephalitis virus (TBEV) is an arthropod-borne viral pathogen causing infections in Europe and is responsible for most arbovirus central nervous system infections in Hungary. Assessing the TBEV prevalence in ticks through detection of genomic RNA is a broadly accepted approach to estimate the transmission risk from a tick bite. For this purpose, 2731 ticks were collected from the neighboring area of the town of Dévaványa, located in southeastern Hungary, which is considered a low-risk-transmission area for TBEV. Altogether, 2300 ticks were collected from the vegetation, while 431 were collected from rodents. Samples were pooled and then screened for TBEV with a newly designed semi-nested RT-PCR (RT-snPCR) targeting the NS1 genomic region. PCR results were confirmed by direct sequencing of the second round amplicons. Among the 3 different collected tick species (Ixodes ricinus, Haemaphysalis concinna, Dermacentor marginatus), I. ricinus was the only species that tested positive for TBEV. TBEV-positive ticks were collected from small mammals or from the vegetation. One nymphal pool and 4 larval pools tested positive for TBEV. The only positive nymphal pool was unfed and came from vegetation, while ticks of the 4 positive larval pools were collected from rodents. Minimal TBEV prevalence in ticks was 0.08% for unfed nymphs and 0.78% for feeding larvae. Our results indicate that further long-term investigations on the occurrence of TBEV are needed to better describe the geographic distribution and the prevalence of infected ticks in Hungary.