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BACKGROUND: Unlike adults, children experienced stronger and longer vector replication in plasma and shedding in saliva following rVSVΔG-ZEBOV-GP vaccination. The resulting risks of immunosuppression or immune hyperactivation leading to increased Adverse Events (AEs) and altered antibody responses are concerns that have been addressed in the present manuscript. METHODS: Children aged 1-12 years living in Gabon received either rVSVΔG-ZEBOV-GP (ERVEBO®) vaccine or the varicella-zoster virus (VZV) vaccine (VZV). The concentration of rVSVΔG vector in blood and saliva, the occurrence of AEs up to day 28; the anti-rVSVΔG-ZEBOV-GP and anti-VZV IgG antibody titres, neutralising and avidity functions of anti-rVSVΔG-ZEBOV-GP by day 365; were assessed in serum. (PACTR202005733552021) FINDINGS: In the rVSVΔG-ZEBOV-GP group, 70% and 7% of children had >0 copies/ml of rVSVΔG respectively in plasma by day 3 and in saliva by day 14 after vaccination, with no detection on day 28. Significantly higher but transient AEs occurred in the rVSVΔG-ZEBOV-GP group. Both vaccines induced seroconversion on day 28 and sustainable IgG antibody titres by day 365. Avidity and neutralisation functions of the anti-rVSVΔG-ZEBOV-GP antibodies peaked at day 28 and were maintained by day 365. INTERPRETATION: The replication and shedding do not affect the favourable risk-benefit balance of the rVSVΔG-ZEBOV-GP in children.
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Due to the discontinuation of routine smallpox vaccination after its eradication in 1980, a large part of the human population remains naïve against smallpox and other members of the orthopoxvirus genus. As a part of biosafety personnel protection programs, laboratory workers receive prophylactic vaccinations against diverse infectious agents, including smallpox. Here, we studied the levels of cross-protecting neutralizing antibodies as well as total IgG induced by either first- or third-generation smallpox vaccines against Monkeypox virus, using a clinical isolate from the 2022 outbreak. Serum neutralization tests indicated better overall neutralization capacity after vaccination with first-generation smallpox vaccines, compared to an attenuated third-generation vaccine. Results obtained from total IgG ELISA, however, did not show higher induction of orthopoxvirus-specific IgGs in first-generation vaccine recipients. Taken together, our results indicate a lower level of cross-protecting neutralizing antibodies against Monkeypox virus in recipients of third-generation smallpox vaccine compared to first-generation vaccine recipients, although total IgG levels were comparable.
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West Nile virus (WNV) is one of the most widespread flaviviruses in the world, and in recent years, it has been frequently present in many Mediterranean and Eastern European countries. A combination of different conditions, such as a favourable climate and higher seasonal average temperatures, probably allowed its introduction and spread to new territories. In Switzerland, autochthonous cases of WNV have never been reported, and the virus was not detected in mosquito vectors until 2022, despite an entomological surveillance in place in Canton Ticino, southern Switzerland, since 2010. In 2022, 12 sites were monitored from July to October, using BOX gravid mosquito traps coupled with honey-baited FTA cards. For the first time, we could detect the presence of WNV in FTA cards and mosquitoes in 8 out of the 12 sampling sites monitored, indicating an unexpectedly widespread circulation of the virus throughout the territory. Positive findings were recorded from the beginning of August until mid-October 2022, and whole genome sequencing analysis identified a lineage 2 virus closely related to strains circulating in Northern Italy. The entomological surveillance has proved useful in identifying viral circulation in advance of possible cases of WNV infection in humans or horses.
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When infecting humans, Andes orthohantavirus (ANDV) may cause a severe disease called hantavirus cardiopulmonary syndrome (HCPS). Following non-specific symptoms, the infection may progress to a syndrome of hemorrhagic fever combined with hyper-acute cardiopulmonary failure. The case fatality rate ranges between 25-40%, depending on the outbreak. In this study, we present the follow-up of a male patient who recovered from HCPS six years ago. We demonstrate that the ANDV genome persists within the reproductive tract for at least 71 months. Genome sequence analysis early and late after infection reveals a low number of mutations (two single nucleotide variants and one deletion), suggesting limited replication activity. We can exclude the integration of the viral genome into the host genome, since the treatment of the specimen with RNAse led to a loss of signal. We demonstrate a long-lasting, strong neutralizing antibody response using pseudovirions expressing the ANDV glycoprotein. Taken together, our results show that ANDV has the potential for sexual transmission.
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Infecções por Hantavirus , Orthohantavírus , Humanos , Masculino , Orthohantavírus/genética , Sêmen , Anticorpos Neutralizantes , RNA Viral/genéticaRESUMO
OBJECTIVES: To report 5-year persistence and avidity of antibodies produced by the live-attenuated recombinant vesicular stomatitis virus (rVSV) expressing the Zaire Ebolavirus (ZEBOV) glycoprotein (GP), known as rVSV-ZEBOV (Ervebo®). METHODS: Healthy adults vaccinated with 300,000 or 10-50 million plaque-forming units of rVSV-ZEBOV in the WHO-coordinated trials of 2014-2015 were followed for up to 4 (Lambaréné, Gabon) and 5 (Geneva, Switzerland) years. We report seropositivity rates, geometric mean titres (GMTs), and population distribution of ZEBOV-GP ELISA IgG antibodies, neutralizing antibodies (pseudovirus and live-virus neutralization) and antibody avidity; the primary outcome was ZEBOV-GP ELISA IgG GMTs at 4 or 5 years compared with 1 year (Y1) after immunization. RESULTS: Among the 168 eligible vaccinees (Geneva: 97 and Lambaréné: 71) enrolled 1 year post-immunization, 146 (87%) remained enrolled at 4 years (Geneva: n = 88, Lambaréné: n = 58), and 84 (87%, Geneva) at 5 years post-vaccination. ZEBOV-GP ELISA IgG GMTs plateaued, with no declining trend from 1 year through the last time point assessed (1147.8 [95% CI 874.3-1507.0] at Y1 versus 1548.1 [95% CI 1136.6-2108.5] at Y5 in Geneva volunteers receiving ≥10 million plaque-forming units of rVSV-ZEBOV), their avidity matching that of ZEBOV convalescents. Live-virus neutralizing antibodies were detected for shorter periods and in fewer vaccinees (53/95 [56%] at Y1 versus 35/84 [42%] at Y5 in Geneva volunteers, all dose levels). DISCUSSION: Titres at Y1 emerged as a correlate of antibody persistence at Y5. The findings of persistent ZEBOV-GP ELISA IgG titres yet shorter-lasting, lower titres of live-virus neutralizing antibodies suggest the contribution of antibody-mediated protective mechanisms other than neutralization. Long-term clinical efficacy of rVSV-ZEBOV, however, requires further study.
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Vacinas contra Ebola , Ebolavirus , Doença pelo Vírus Ebola , Estomatite Vesicular , Adulto , Animais , Humanos , Ebolavirus/genética , Formação de Anticorpos , República Democrática do Congo , Anticorpos Antivirais , Vacinação , Anticorpos Neutralizantes , Imunoglobulina G , Anticorpos BloqueadoresRESUMO
Proper disinfection and inactivation of highly pathogenic viruses is an essential component of public health and prevention. Depending on environment, surfaces, and type of contaminant, various methods of disinfection must be both efficient and available. To test both established and novel chemical disinfectants against risk group 4 viruses in our maximum containment facility, we developed a standardized protocol and assessed the chemical inactivation of the two Ebola virus variants Mayinga and Makona suspended in two different biological soil loads. Standard chemical disinfectants ethanol and sodium hypochlorite completely inactivate both Ebola variants after 30 s in suspension at 70% and 0.5% v/v, respectively, concentrations recommended for disinfection by the World Health Organization. Additionally, peracetic acid is also inactivating at 0.2% v/v under the same conditions. Continued vigilance and optimization of current disinfection protocols is extremely important due to the continuous presence of Ebola virus on the African continent and increased zoonotic spillover of novel viral pathogens. Furthermore, to facilitate general pandemic preparedness, the establishment and sharing of standardized protocols is very important as it allows for rapid testing and evaluation of novel pathogens and chemical disinfectants.
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Desinfetantes , Ebolavirus , Doença pelo Vírus Ebola , Humanos , Desinfetantes/farmacologia , Doença pelo Vírus Ebola/prevenção & controle , Desinfecção , SoloRESUMO
Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.
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COVID-19 , SARS-CoV-2 , Humanos , Proteínas de Repetição de Anquirina Projetadas , Mucosa Respiratória , Anticorpos Monoclonais , Antivirais/farmacologiaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new variants and generally short duration of immunity, the development of effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing drugs for accelerated clinical testing and emergency use authorizations. However, drug-repurposing studies using cellular assays often identify hits that later prove ineffective clinically, highlighting the need for more complex screening models. To this end, we evaluated the activity of single compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Furthermore, we also evaluated drug combinations based on a sub-maximal concentration of molnupiravir. We report the antiviral activity of 95 single compounds and 30 combinations. We show that only a few single agents are highly effective in inhibiting SARS-CoV-2 replication while selected drug combinations containing 10 µM molnupiravir boosted antiviral activity compared to single compound treatment. These data indicate that molnupiravir-based combinations are worthy of further consideration as potential treatment strategies against coronavirus disease 2019 (COVID-19).
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Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Antivirais/uso terapêutico , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Mucosa Nasal , SARS-CoV-2RESUMO
The need for tools that facilitate rapid detection and continuous monitoring of SARS-CoV-2 variants of concern (VOCs) is greater than ever, as these variants are more transmissible and therefore increase the pressure of COVID-19 on healthcare systems. To address this demand, we aimed at developing and evaluating a robust and fast diagnostic approach for the identification of SARS-CoV-2 VOC-associated spike gene mutations. Our diagnostic assays detect the E484K and N501Y single-nucleotide polymorphisms (SNPs) as well as a spike gene deletion (HV69/70) and can be run on standard laboratory equipment or on the portable rapid diagnostic technology platform peakPCR. The assays achieved excellent diagnostic performance when tested with RNA extracted from culture-derived SARS-CoV-2 VOC lineages and clinical samples collected in Equatorial Guinea, Central-West Africa. Simplicity of usage and the relatively low cost are advantages that make our approach well suitable for decentralized and rapid testing, especially in resource-limited settings.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Guiné Equatorial/epidemiologia , Deleção de Genes , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/classificaçãoRESUMO
Ecological changes, population movements and increasing urbanization promote the expansion of hantaviruses, placing humans at high risk of virus transmission and consequent diseases. The currently limited therapeutic options make the development of antiviral strategies an urgent need. Ribavirin is the only antiviral used currently to treat hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV), even though severe side effects are associated with this drug. We therefore investigated the antiviral activity of favipiravir, a new antiviral agent against RNA viruses. Both ribavirin and favipiravir demonstrated similar potent antiviral activity on HTNV infection. When combined, the efficacy of ribavirin is enhanced through the addition of low dose favipiravir, highlighting the possibility to provide better treatment than is currently available.
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Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing.
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Antivirais/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Infecções por Hantavirus/tratamento farmacológico , Orthohantavírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: Serological immunoassays that can identify protective immunity against SARS-CoV-2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of antibodies in patient sera. METHODS: Consecutive patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1), and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralization assays with live SARS-CoV-2 were performed. RESULTS: One thousand four hundred and seventy-seven individuals were included comprising 112 SARS-CoV-2 positives (defined as a positive real-time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS-CoV-2 positive individuals, 96.3% showed full neutralization of live SARS-CoV-2 at serum dilutions ≥ 1:16, while none of the 6 SARS-CoV-2-negative sera revealed neutralizing activity. CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS-CoV-2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS-CoV-2.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
The original version of the acknowledgement unfortunately contained a mistake.
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BACKGROUND: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro. METHODS: To evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium. RESULTS: In the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 µg/ml IC50. Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 µg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50µg/ml Echinaforce®. CONCLUSIONS: These results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.
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Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Coronavirus Humano 229E/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Coronavirus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Resfriado Comum/tratamento farmacológico , Resfriado Comum/virologia , Infecções por Coronavirus/virologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Vírus de RNA/efeitos dos fármacos , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Células VeroRESUMO
Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance.
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Metagenômica/métodos , RNA Viral/genética , RNA Viral/metabolismo , Animais , Febre de Chikungunya , Chlorocebus aethiops , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/transmissão , Encefalite Transmitida por Carrapatos/virologia , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Mosquitos Vetores/virologia , Reação em Cadeia da Polimerase em Tempo Real , Carrapatos/virologia , Células Vero , Zika virus/genética , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologiaRESUMO
Hantaviruses are rodent-borne hemorrhagic fever viruses leading to serious diseases. Viral attachment and entry represent the first steps in virus transmission and are promising targets for antiviral therapeutic intervention. Here we investigated receptor use in human airway epithelium of the Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV). Using a biocontained recombinant vesicular stomatitis virus pseudotype platform, we provide first evidence for a role of the cellular phosphatidylserine (PS) receptors of the T-cell immunoglobulin and mucin (TIM) protein family in HTNV and ANDV infection. In line with previous studies, HTNV, but not ANDV, was able to use glycosaminoglycan heparan sulfate and αvß3 integrin as co-receptors. In sum, our studies demonstrate for the first time that hantaviruses make use of apoptotic mimicry for infection of human airway epithelium, which may explain why these viruses can easily break the species barrier.
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Vírus Hantaan/metabolismo , Glicoproteínas de Membrana/metabolismo , Orthohantavírus/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Bacteriocinas/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vírus Hantaan/efeitos dos fármacos , Vírus Hantaan/patogenicidade , Vírus Hantaan/fisiologia , Orthohantavírus/fisiologia , Haplorrinos , Heparitina Sulfato/farmacologia , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Mimetismo Molecular , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Vesiculovirus/metabolismo , Vesiculovirus/fisiologia , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: The threat of mosquito-borne diseases is increasing in continental Europe as demonstrated by several autochthonous chikungunya, dengue and West Nile virus outbreaks. In Switzerland, despite the presence of competent vectors, routine surveillance of arboviruses in mosquitoes is not being carried out, mainly due to the high costs associated with the need of a constant cold chain and laborious processing of thousands of mosquitoes. An alternative approach is using honey-baited nucleic acid preserving cards (FTA cards) to collect mosquito saliva that may be analysed for arboviruses. Here, we evaluate whether FTA cards could be used to detect potentially emerging viruses in an area of low virus prevalence in combination with an effective mosquito trap. METHODS: In a field trial in southern Switzerland we measured side-by-side the efficacy of the BG-Sentinel 2, the BG-GAT and the Box gravid trap to catch Aedes and Culex mosquitoes in combination with honey-baited FTA cards during 80 trapping sessions of 48 hours. We then screened both the mosquitoes and the FTA cards for the presence of arboviruses using reverse-transcription PCR. The efficacy of the compared trap types was evaluated using generalized linear mixed models. RESULTS: The Box gravid trap collected over 11 times more mosquitoes than the BG-GAT and BG-Sentinel 2 trap. On average 75.9% of the specimens fed on the honey-bait with no significant difference in feeding rates between the three trap types. From the total of 1401 collected mosquitoes, we screened 507 Aedes and 500 Culex females for the presence of arboviruses. A pool of six Cx. pipiens/Cx. torrentium mosquitoes and also the FTA card from the same Box gravid trap were positive for Usutu virus. Remarkably, only two of the six Culex mosquitoes fed on the honey-bait, emphasising the high sensitivity of the method. In addition, two Ae. albopictus collections but no FTA cards were positive for mosquito-only flaviviruses. CONCLUSIONS: Based on our results we conclude that honey-baited FTA cards, in combination with the Box gravid trap, are an effective method for arbovirus surveillance in areas of low prevalence, particularly where resources are limited for preservation and screening of individual mosquitoes.
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Aedes/virologia , Arbovírus/isolamento & purificação , Culex/virologia , Entomologia/métodos , Técnicas de Diagnóstico Molecular/métodos , Mosquitos Vetores/virologia , Animais , Arbovírus/genética , Mel , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , SuíçaRESUMO
Hantaviruses are emerging rodent-borne negative-strand RNA viruses associated with severe human diseases. Zoonotic transmission occurs via aerosols of contaminated rodent excreta and cells of the human respiratory epithelium represent likely early targets. Here we investigated cellular factors involved in entry of the pathogenic Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV) into human respiratory epithelial cells. Screening of a kinase inhibitor library using a biocontained recombinant vesicular stomatitis virus pseudotype platform revealed differential requirement for host kinases for HTNV and ANDV entry and provided first hints for an involvement of macropinocytosis. Examination of a selected panel of well-defined inhibitors of endocytosis confirmed that both HTNV and ANDV enter human respiratory epithelial cells via a pathway that critically depends on sodium proton exchangers and actin, hallmarks of macropinocytosis. However, HTNV and ANDV differed in their individual requirements for regulatory factors of macropinocytosis, indicating virus-specific differences.
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Endocitose , Células Epiteliais/virologia , Infecções por Hantavirus/virologia , Orthohantavírus/fisiologia , Mucosa Respiratória/virologia , Internalização do Vírus , Linhagem Celular , Células Epiteliais/enzimologia , Orthohantavírus/genética , Infecções por Hantavirus/enzimologia , Infecções por Hantavirus/genética , Infecções por Hantavirus/fisiopatologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Mucosa Respiratória/metabolismoRESUMO
Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.