RESUMEN
The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Virus de la Influenza A/efectos de los fármacos , Naproxeno/análogos & derivados , Células A549 , Animales , Sitios de Unión , Inhibidores de la Ciclooxigenasa 2/química , Perros , Diseño de Fármacos , Reposicionamiento de Medicamentos , Farmacorresistencia Viral/efectos de los fármacos , Femenino , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Gripe Humana/patología , Células de Riñón Canino Madin Darby , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Naproxeno/farmacología , Proteínas de la Nucleocápside , Oseltamivir/farmacología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Resonancia por Plasmón de Superficie , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismoRESUMEN
The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Receptores de Formil Péptido/antagonistas & inhibidores , Receptores de Lipoxina/antagonistas & inhibidores , Células A549 , Animales , Anexina A1/genética , Anticuerpos Monoclonales/administración & dosificación , Endosomas/efectos de los fármacos , Endosomas/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/virología , Receptores de Formil Péptido/genética , Receptores de Lipoxina/genética , Replicación Viral/efectos de los fármacosRESUMEN
Relapsing fever borreliae, produced by ectoparasite-borne Borrelia species, cause mild to deadly bacteremia and miscarriage. In the perspective of developing inexpensive assays for the rapid detection of relapsing fever borreliae, we produced 12 monoclonal antibodies (MAbs) against Borrelia crocidurae and characterized the two exhibiting the highest titers. P3A10 MAb reacts with the 35.6-kDa flagellin B (flaB) of B. crocidurae while P6D9 MAb recognizes a 35.1-kDa variable-like protein (Vlp) in B. crocidurae and a 35.2-kDa Vlp in Borrelia duttonii. Indirect immunofluorescence assay incorporating relapsing fever and Lyme group borreliae and 11 blood-borne organisms responsible for fever in West Africa confirmed the reactivity of these two MAbs. Combining these two MAbs in indirect immunofluorescence assays detected relapsing fever borreliae including B. crocidurae in ticks and the blood of febrile Senegalese patients. Both antibodies could be incorporated into inexpensive and stable formats suited for the rapid point-of-care diagnosis of relapsing fever. These first-ever MAbs directed against African relapsing fever borreliae are available for the scientific community to promote research in this neglected field.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Infecciones por Borrelia/microbiología , Borrelia/clasificación , Borrelia/inmunología , Garrapatas/microbiología , Animales , Especificidad de Anticuerpos , Infecciones por Borrelia/diagnóstico , Humanos , RatonesRESUMEN
In Africa, relapsing fevers caused by ectoparasite-borne Borrelia species are transmitted by ticks, with the exception of Borrelia recurrentis, which is a louse-borne spirochete. These tropical diseases are responsible for mild to deadly spirochetemia. Cultured Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica circulate alongside at least six species that have not yet been cultured in vectors. Direct diagnosis is hindered by the use of non-specific laboratory tools. Indeed, microscopic observation of Borrelia spirochaeta in smears of peripheral blood taken from febrile patients lacks sensitivity and specificity. Although best visualized using dark-field microscopy, the organisms can also be detected using Wright-Giemsa or acridine orange stains. PCR-based detection of specific sequences in total DNA extracted from a specimen can be used to discriminate different relapsing fever Borreliae. In our laboratory, we developed a multiplex real-time PCR assay for the specific detection of B. duttonii/recurrentis and B. crocidurae: multispacer sequence typing accurately identified cultured relapsing fever borreliae and revealed diversity among them. Other molecular typing techniques, such as multilocus sequence analysis of tick-borne relapsing fever borreliae, showed the potential risk of human infection in Africa. Recent efforts to culture and sequence relapsing fever borreliae have provided new information for reassessment of the diversity of these bacteria. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been reported as a means of identifying cultured borreliae and of identifying both vectors and vectorized pathogens such as detecting relapsing fever borreliae directly in ticks. The lack of a rapid diagnosis test restricts the management of such diseases. We produced monoclonal antibodies against B. crocidurae in order to develop cheap assays for the rapid detection of relapsing fever borreliae. In this paper, we review point-of-care diagnosis and confirmatory methods.
RESUMEN
To improve the knowledge base of Borrelia in north Africa, we tested 257 blood samples collected from febrile patients in Oran, Algeria, between January and December 2012 for Borrelia species using flagellin gene polymerase chain reaction sequencing. A sequence indicative of a new Borrelia sp. named Candidatus Borrelia algerica was detected in one blood sample. Further multispacer sequence typing indicated this Borrelia sp. had 97% similarity with Borrelia crocidurae, Borrelia duttonii, and Borrelia recurrentis. In silico comparison of Candidatus B. algerica spacer sequences with those of Borrelia hispanica and Borrelia garinii revealed 94% and 89% similarity, respectively. Candidatus B. algerica is a new relapsing fever Borrelia sp. detected in Oran. Further studies may help predict its epidemiological importance.
Asunto(s)
Borrelia/aislamiento & purificación , Fiebre Recurrente/epidemiología , Fiebre Recurrente/microbiología , Argelia/epidemiología , Secuencia de Bases , Borrelia/clasificación , Borrelia/genética , Cartilla de ADN/genética , ADN Bacteriano/sangre , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Intergénico/química , Flagelina/genética , Geografía , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The draft genome sequence of Borrelia crocidurae strain 03-02, a blood isolate from a febrile Senegalese patient, comprises a 920,021-bp linear chromosome (27.7% G+C content), 32 tRNAs, 818 open reading frames, and one cluster of regularly interspaced short palindromic repeats. Its genotype differs from that of the Achema reference strain.
RESUMEN
BACKGROUND: In Africa, relapsing fever borreliae are neglected vector-borne pathogens that cause mild to deadly septicemia and miscarriage. Screening vectors for the presence of borreliae currently requires technically demanding, time- and resource-consuming molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has recently emerged as a tool for the rapid identification of vectors and the identification of cultured borreliae. We investigated whether MALDI-TOF-MS could detect relapsing fever borreliae directly in ticks. METHODOLOGY/PRINCIPAL FINDINGS: As a first step, a Borrelia MALDI-TOF-MS database was created to house the newly determined Mean Spectrum Projections for four Lyme disease group and ten relapsing fever group reference borreliae. MALDI-TOF-MS yielded a unique protein profile for each of the 14 tested Borrelia species, with 100% reproducibility over 12 repeats. In a second proof-of-concept step, the Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect Borrelia crocidurae in 20 Ornithodoros sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing. A B. crocidurae-specific pattern consisting of 3405, 5071, 5898, 7041, 8580 and 9757-m/z peaks was found in all B. crocidurae-infected ticks and not found in any of the un-infected ticks. In a final blind validation step, MALDI-TOF-MS exhibited 88.9% sensitivity and 93.75% specificity for the detection of B. crocidurae in 50 O. sonrai ticks, including 18 that tested positive for B. crocidurae by PCR-sequencing. MALDI-TOF-MS took 45 minutes to be completed. CONCLUSIONS/SIGNIFICANCE: After the development of an appropriate database, MALDI-TOF-MS can be used to identify tick species and the presence of relapsing fever borreliae in a single assay. This work paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens.