RESUMO
The SARS-CoV-2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin-converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS-CoV-2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell-free system as demonstrated by enzyme-linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS-CoV-2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti-SARS-CoV-2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Proteínas Virais/metabolismo , Células HEK293 , Ligação Proteica , Compostos de Sulfidrila/farmacologiaRESUMO
Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.
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Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Coronavirus/genética , Genoma Viral/genética , Pneumonia Viral/diagnóstico , RNA Viral/genética , Síndrome Respiratória Aguda Grave/diagnóstico , Viagem , Sequenciamento Completo do Genoma/métodos , Betacoronavirus/isolamento & purificação , COVID-19 , China , Coronavirus/classificação , Coronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Alemanha , Humanos , Itália , Epidemiologia Molecular , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Mutação Puntual , RNA Viral/isolamento & purificação , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/virologiaRESUMO
Data concerning the transmission of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in paucisymptomatic patients are lacking. We report an Italian paucisymptomatic case of coronavirus disease 2019 with multiple biological samples positive for SARS-CoV-2. This case was detected using the World Health Organization protocol on cases and contact investigation. Current discharge criteria and the impact of extra-pulmonary SARS-CoV-2 samples are discussed.
Assuntos
Infecções Assintomáticas , Infecções por Coronavirus/diagnóstico , Coronavirus/isolamento & purificação , Fezes/virologia , Pulmão/diagnóstico por imagem , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , Viagem , Eliminação de Partículas Virais , Anticorpos Antivirais/imunologia , Betacoronavirus , COVID-19 , Teste para COVID-19 , China , Técnicas de Laboratório Clínico , Busca de Comunicante , Coronavirus/genética , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Humanos , Itália , Pulmão/patologia , Masculino , Pandemias , Pneumonia Viral/terapia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Quarentena , Radiografia Torácica , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Tomografia Computadorizada por Raios X , Organização Mundial da Saúde , Adulto JovemRESUMO
Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2-SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-ß and CRM1 play essential roles in localising the RANBP2-SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.
Assuntos
Carioferinas/metabolismo , Cinetocoros/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , beta Carioferinas/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Carioferinas/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , beta Carioferinas/genética , Proteína Exportina 1RESUMO
A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North-eastern Italy.
Assuntos
Anticorpos Antivirais/sangue , Flavivirus/imunologia , Vírus do Nilo Ocidental/imunologia , Anticorpos Neutralizantes/sangue , Doadores de Sangue , Humanos , Itália/epidemiologia , Testes de Neutralização , Estudos Retrospectivos , Estudos SoroepidemiológicosRESUMO
BACKGROUND: Plasmodium vivax infection is known to be rare in West/Central Africa, the most accepted explanation being the lack of expression of erythroid Duffy antigen in the local human populations. Duffy negativity prevents the parasite to exploit the entry mechanism on the red blood cell surface. However, there are a growing number of reported vivax infections in Duffy-negative individuals. Data on P. vivax circulation in Cameroon are limited. The aim of the study was to evaluate the P. vivax presence, and its association with the Duffy genotype in West Cameroon. RESULTS: Overall, 484 blood samples were collected consecutively from febrile outpatients attending the Dschang's Hospital (West Cameroon) during a 3-months period. Plasmodium vivax infection was detected by PCR in 5.6% (n = 27/484) of the cases, representing 38.6% (n = 27/70) of all Plasmodium infections detected. All P. vivax infected individuals showed a Duffy-negative genotype, and the frequency of Duffy-positive individuals in the whole tested population was 1.7%. CONCLUSIONS: The results of this study confirm the circulation of P. vivax in Cameroon, as well as that the lack of expression of Duffy-antigen does not confer full protection against vivax malaria acquisition.
Assuntos
Sistema do Grupo Sanguíneo Duffy/genética , Genótipo , Malária Vivax/epidemiologia , Adolescente , Adulto , Idoso , Camarões/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
We investigated 400 cases of dengue-like illness in persons hospitalized during an outbreak in Al Hudaydah, Yemen, in 2012. Overall, 116 dengue and 49 chikungunya cases were diagnosed. Dengue virus type 2 was the predominant serotype. The co-circulation of these viruses indicates that mosquitoborne infections represent a public health threat in Yemen.
Assuntos
Febre de Chikungunya/epidemiologia , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Coinfecção , Vírus da Dengue/genética , Dengue/epidemiologia , Dengue/virologia , Viremia , Adolescente , Adulto , Febre de Chikungunya/história , Vírus Chikungunya/classificação , Criança , Pré-Escolar , Dengue/história , Vírus da Dengue/classificação , Surtos de Doenças , Geografia , História do Século XXI , Humanos , Incidência , Lactente , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estudos Soroepidemiológicos , Sorotipagem , Iêmen/epidemiologia , Adulto JovemRESUMO
While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. In vitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development in vitro. Here we show that upon in vitro attachment, human blastoids self-organize a BRA+ population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12 days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon in vitro attachment.
Assuntos
Gástrula , Gastrulação , Humanos , Desenvolvimento Embrionário , Blastocisto , MesodermaRESUMO
Herein, we developed a new waste solution-free paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva, by combining vertical and lateral flow. In detail, the device was constituted of a reservoir containing all reagents for the construction of the immunological chain onto the magnetic beads and a lateral flow holder which contained a polyester-based electrode, a magnet, and an adsorbent pad. The measurement was carried out by adding the saliva sample into the reservoir, followed by the addition of this solution in the hole present in the lateral flow holder. The successive additions of washing buffer and TMB solution in the lateral flow holder allowed the detection of N protein in saliva in the range of 0.06 to 4 µg/mL with a detection limit equal to 30 ng/mL. The analysis of several saliva samples with the sensing tool and the reference method, demonstrated the effectiveness of this device, being able to identify positive patients with high values of CT e.g. 35. This new configuration paves the way for the realization of any magnetic beads-based immunosystem without waste solution production, enlarging the application of paper-based devices.
Assuntos
Técnicas Biossensoriais , COVID-19 , Humanos , Saliva , SARS-CoV-2 , Imunoensaio/métodos , Técnicas Biossensoriais/métodos , Smartphone , COVID-19/diagnóstico , Limite de Detecção , Técnicas Eletroquímicas/métodos , EletrodosRESUMO
AIMS: To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory. METHODS: Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry. RESULTS: None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals. CONCLUSIONS: ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos B , Reações Cruzadas , Vacina Antivariólica , Vaccinia virus , Adulto , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Chlorocebus aethiops , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Memória Imunológica , Ativação Linfocitária , Testes de Neutralização , Orthopoxvirus/imunologia , Varíola/imunologia , Varíola/prevenção & controle , Vacina Antivariólica/imunologia , Linfócitos T/imunologia , Vacinação , Vaccinia virus/imunologia , Células Vero , Monkeypox virus/imunologiaRESUMO
Cancer patients (CPs), being immunosuppressed due to the treatment received or to the disease itself, are more susceptible to infections and their potential complications, showing therefore an increased risk of developing severe COVID-19 compared to the general population. We evaluated the immune responses to anti-SARS-CoV-2 vaccination in patients with solid tumors one year after the administration of the third dose and the effect of cancer treatment on vaccine immunogenicity was assessed. Healthy donors (HDs) were enrolled. Binding and neutralizing antibody (Ab) titers were evaluated using chemiluminescence immunoassay (CLIA) and Plaque Reduction Neutralization Test (PRNT) respectively. T-cell response was analyzed using multiparametric flow cytometry. CPs who were administered three vaccine doses showed lower Ab titers than CPs with four doses and HDs. Overall, a lower cell-mediated response was found in CPs, with a predominance of monofunctional T-cells producing TNFα. Lower Ab titers and a weaker T-cell response were observed in CPs without prior SARS-CoV-2 infection when compared to those with a previous infection. While no differences in the humoral response were found comparing immunotherapy and non-immunotherapy patients, a stronger T-cell response in CPs treated with immunotherapy was observed. Our results emphasize the need of booster doses in cancer patients to achieve a level of protection similar to that observed in healthy donors and underlines the importance of considering the treatment received to reach a proper immune response.
Assuntos
COVID-19 , Neoplasias , Humanos , Vacinas contra COVID-19 , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinação , Neoplasias/terapia , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Introduction: Huntington's disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. Methods: In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Results: Using highly reproducible and quantifiable in vitro micropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD like phenotypes akin to HTT depletion. Discussion: By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type HTT function.
RESUMO
In 1918 many countries, but not Spain, were fighting World War I. Spanish press could report about the diffusion and severity of a new infection without censorship for the first-time, so that this pandemic is commonly defined as "Spanish flu", even though Spain was not its place of origin. "Spanish flu" was one of the deadliest pandemics in history and has been frequently compared with the coronavirus disease (COVID)-19 pandemic. These pandemics share similarities, being both caused by highly variable and transmissible respiratory RNA viruses, and diversity, represented by diagnostics, therapies, and especially vaccines, which were made rapidly available for COVID-19, but not for "Spanish flu". Most comparison studies have been carried out in the first period of COVID-19, when these resources were either not yet available or their use had not long started. Conversely, we wanted to analyze the role that the advanced diagnostics, anti-viral agents, including monoclonal antibodies, and innovative COVID-19 vaccines, may have had in the pandemic containment. Early diagnosis, therapies, and anti-COVID-19 vaccines have markedly reduced the pandemic severity and mortality, thus preventing the collapse of the public health services. However, their influence on the reduction of infections and re-infections, thus on the transition from pandemic to endemic condition, appears to be of minor relevance. The high viral variability of influenza and coronavirus may probably be contained by the development of universal vaccines, which are not easy to be obtained. The only effective weapon still remains the disease prevention, to be achieved with the reduction of promiscuity between the animal reservoirs of these zoonotic diseases and humans.
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Yellow fever disease is a viral zoonosis that may result in a severe hemorrhagic disease. A safe and effective vaccine used in mass immunization campaigns has allowed control and mitigation against explosive outbreaks in endemic areas. Since the 1960's, re-emergent of the yellow fever virus has been observed. The timely implementation of control measures, to avoid or contain an ongoing outbreak requires rapid specific viral detection methods. Here a novel molecular assay, expected to detect all known yellow fever virus strains, is described. The method has demonstrated high sensitivity and specificity in real-time RT-PCR as well as in an endpoint RT-PCR set-up. Sequence alignment and phylogenetic analysis reveal that the amplicon resulting from the novel method covers a genomic region whose mutational profile is completely associated to the yellow fever viral lineages. Therefore, sequencing analysis of this amplicon allows for assignment of the viral lineage.
Assuntos
Vacina contra Febre Amarela , Febre Amarela , Humanos , Vírus da Febre Amarela/genética , Febre Amarela/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , FilogeniaRESUMO
In accordance with the World Health Organization, one dose of yellow fever vaccine may guarantee protection lifelong in healthy adults. However, relatively little information is still available from ad hoc studies. We evaluated the persistence of neutralizing antibodies, which are considered to be an immune correlate of protection, in a large number of military personnel vaccinated up to 47 years before. Overall, 322 individuals were studied. The median time from vaccination to blood collection for neutralizing antibody evaluation was 9 years, ranging from <1 to 47 years. Of the 322 participants, 319 had neutralizing antibodies (99.1 %). The highest median PRNT50 value was observed in those vaccinated ≤1 year before (median PRNT50 = 320). In conclusion, our study confirms on a larger scale that, in healthy adults, neutralizing antibodies may persist as long as 47 years after a single yellow fever vaccines dose.
Assuntos
Vacina contra Febre Amarela , Febre Amarela , Humanos , Adulto , Vírus da Febre Amarela , Anticorpos Neutralizantes , Febre Amarela/prevenção & controle , Anticorpos Antivirais , VacinaçãoRESUMO
SARS-CoV-2 is inactivated in aerosol (its primary mode of transmission) by means of radiated microwaves at frequencies that have been experimentally determined. Such frequencies are best predicted by the mathematical model suggested by Taylor, Margueritat and Saviot. The alignment between such mathematical prediction and the outcomes of our experiments serves to reinforce the efficacy of the radiated microwave technology and its promise in mitigating the transmission of SARS-CoV-2 in its naturally airborne state.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Micro-Ondas , Aerossóis e Gotículas Respiratórios , Modelos TeóricosRESUMO
For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/genética , RNA Helicases/metabolismo , Replicação Viral , Antivirais/farmacologiaRESUMO
Coronaviruses are a family of viruses that cause disease in mammals and birds. In humans, coronaviruses cause infections on the respiratory tract that can be fatal. These viruses can cause both mild illnesses such as the common cold and lethal illnesses such as SARS, MERS, and COVID-19. Air transmission represents the principal mode by which people become infected by SARS-CoV-2. To reduce the risks of air transmission of this powerful pathogen, we devised a method of inactivation based on the propagation of electromagnetic waves in the area to be sanitized. We optimized the conditions in a controlled laboratory environment mimicking a natural airborne virus transmission and consistently achieved a 90% (tenfold) reduction of infectivity after a short treatment using a Radio Frequency (RF) wave emission with a power level that is safe for people according to most regulatory agencies, including those in Europe, USA, and Japan. To the best of our knowledge, this is the first time that SARS-CoV-2 has been shown to be inactivated through RF wave emission under conditions compatible with the presence of human beings and animals. Additional in-depth studies are warranted to extend the results to other viruses and to explore the potential implementation of this technology in different environmental conditions.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Micro-Ondas , Aerossóis e Gotículas Respiratórios , Europa (Continente) , MamíferosRESUMO
One consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries.
Assuntos
COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Anticorpos Antivirais , Europa (Continente) , Imunoglobulina G , Anticorpos NeutralizantesRESUMO
BACKGROUND: West Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals. RESULTS: We produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed -1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals. CONCLUSIONS: N-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.