En España también hay fiebre hemorrágica: el virus Crimea-Congo

Artículo de divulgación publicado en The Conversation España el día 06/03/2022.Cuando escuchamos hablar de fiebres hemorrágicas virales solemos pensar en África y en virus como el ébola. Pero en España convivimos desde hace tiempo con el virus de la Fiebre Hemorrágica de Crimea-Congo, que produce muerte en el 30% de los casos.N

¿Es el filovirus Lloviu una amenaza para la salud mundial?

Artículo de divulgación publicado en The Conversation España el día 18/05/2022.Muchos medios aseguran que el virus Lloviu, hallado en Asturias y estudiado ahora por investigadores húngaros, es una amenaza para la salud humana, y hasta el potencial causante de una pandemia. ¿Hay razones objetivas para semejantes temores?N

Método de amplificación genómica del virus West Nile

Método de amplificación genómica del virus West Nile. El método se basa en la amplificación de una zona de la región 3' no codificante del virus, preferiblemente mediante PCR en Tiempo Real, gracias al uso de una pareja de cebadores mínimamente degenerados, lo que permite la amplificación de virus West Nile presentes en una muestra de cualquiera de los linajes conocidos. El fragmento amplificado se detecta, preferiblemente, con una sonda específica de la zona, también mínimamente degenerada. La pareja de cebadores degenerados elegidos, así como la sonda preferida, dan lugar a una alta sensibilidad en la detección de todos los linajes y también a una alta especificidad para el virus West Nile.REIVINDICACIONES: 1. Un método para la detección del virus West Nile presente en una muestra que comprende las etapas de: a) amplificar los ácidos nucleicos del virus West Nile presentes en dicha muestra utilizando como cebadores los oligonucleótidos con las secuencias: 5'-CGGAAGTYGRGTAKACGGTGCTG-3' (SEQ ID NO:1) (directo) , y 5'-CGGTWYTGAGGGCTTACRTGG-3' (SEQ ID NO:2) (inverso) ; b) detectar el fragmento de ácido nucleico amplificado, en donde la detección del ácido nucleico amplificado indica la presencia del virus West Nile en dicha muestra. 2. Método según la reivindicación 1, en el que la detección del ácido nucleico amplificado se realiza utilizando como sonda la secuencia representada por SEQ ID NO:3. 3. Método según la reivindicación 1 ó 2, en el que la etapa a) de amplificación de los ácidos nucleicos del virus West Nile presentes en la muestra comprende las siguientes subetapas: i) síntesis del cDNA a partir del RNA del virus West Nile presente en la muestra mediante una transcriptasa inversa; ii) amplificación del cDNA mediante PCR en Tiempo Real utilizando como cebadores los oligonucleótidos de SEQ ID NO:1 y SEQ ID NO:2; y la etapa b) de detección del fragmento de ácido nucleico amplificado se lleva a cabo mediante el uso de una sonda fluorogénica que consiste en un oligonucleótido monocatenario, complementario al fragmento de ácido nucleico amplificado en la reacción de PCR, covalentemente unido a al menos un compuesto fluorocromo. 4. Método según la reivindicación 3, en el que el oligonucleótido utilizado como sonda tiene la secuencia de nucleótidos de SEQ ID NO:3. 5. Método según la reivindicación 4, en el que el oligonucleótido utilizado como sonda está covalentemente unido en 5' a un primer compuesto fluorocromo y en 3' a un segundo compuesto fluorocromo quencher capaz de aceptar la energía emitida por el primer fluorocromo cuando ambos están unidos al oligonucleótido de SEQ ID NO:3 y de disiparla en forma de fluorescencia de mayor longitud de onda que la emitida por el primer fluorocromo. 6. Método según la reivindicación 4, en el que el oligonucleótido utilizado como sonda está covalentemente unido en 5' a un compuesto fluorocromo y en 3' a un compuesto quencher no fluorescente, capaz de aceptar la energía emitida por el compuesto fluorocromo cuando ambos están unidos al oligonucleótido de SEQ ID NO:3 y que contiene un resto MGB capaz de unirse al surco menor del DNA. 7. Método según la reivindicación 6, en el que compuesto fluorocromo unido en 5' es 6-carboxifluoresceína (FAM) . 8. Método según una cualquiera de las reivindicaciones anteriores, en el que la amplificación de los ácidos nucleicos del Virus West Nile se lleva a cabo en presencia de una molécula de DNA útil como control interno que comprende un fragmento que puede ser amplificado mediante PCR con los cebadores de SEQ ID NO:1 y SEQ ID NO:2 por comprender secuencias complementarias a ambos cebadores, situadas de manera que cada cadena de la molécula de DNA contiene la secuencia de uno de dichos cebadores y la secuencia complementaria al otro cebador, estando dichas secuencias correspondientes a los cebadores separadas por un fragmento heterólogo, exógeno tanto con respecto al genoma del virus West Nile como al genoma de la especie de la que se ha extraído la muestra en la que se desea comprobar la presencia del virus West Nile. 9. Método según la reivindicación 8, en el que la molécula de DNA útil como control interno comprende la secuencia representada por SEQ ID NO:6. 10. Método según la reivindicación 9, en el que se detecta la amplificación del DNA útil como control interno mediante el uso de una sonda con la secuencia de nucleótidos de SEQ ID NO:7. 11. Método según la reivindicación 10, en el que la secuencia de nucleótidos de SEQ ID NO:7 lleva covalentemente unido un compuesto fluorocromo distinto de cualquier fluorocromo presente en la sonda de SEQ ID NO:3. 12. Método según la reivindicación 11, en el que el compuesto fluorocromo es NED. 13. Método según una cualquiera de las reivindicaciones 9 a 12, que comprende una etapa previa en la que se lleva a cabo una reacción de amplificación por PCR de los oligonucleótidos de SEQ ID NO:4 y SEQ ID NO:5, que sirven tanto como cebadores como moldes, dando lugar a un producto de amplificación que comprende la secuencia de SEQ ID NO:6. 14. Método según una cualquiera de las reivindicaciones precedentes, en el que los ácidos nucleicos amplificados en la etapa a) del método han sido extraídos de una muestra biológica. 15. Método según la reivindicación 14, en el que la muestra biológica ha sido tomada de un ser humano. 16. Método según la reivindicación 15, en el que la muestra biológica se selecciona de líquido cefalorraquídeo, muestras de órganos, muestras de sangre o suero obtenido de la misma. 17. Método según la reivindicación 14, en el que la muestra biológica procede de un artrópodo o de un ave. 18. Un kit que comprende los oligonucleótidos de SEQ ID NO:1 y SEQ ID NO:2. 19. Kit según la reivindicación 18, que adicionalmente comprende una sonda que contiene la secuencia de nucleótidos de SEQ ID NO:3. 20. Kit según la reivindicación 19, en el que la sonda de SEQ ID NO:3 presenta unido al extremo 5' un compuesto fluorocromo y en 3' un compuesto quencher no fluorescente, capaz de aceptar la energía emitida por el compuesto fluorocromo cuando ambos están unidos al oligonucleótido de SEQ ID NO:3, compuesto quencher no fluorescente que contiene un resto MGB capaz de unirse al surco menor del DNA. 21. Kit según la reivindicación 20, que adicionalmente contiene una sonda con la secuencia de nucleótidos de SEQ ID NO:7. 22. Kit según la reivindicación 21, en el que la sonda con la secuencia de nucleótidos de SEQ ID NO:8 lleva unido covalentemente un compuesto fluorocromo distinto de cualquier fluorocromo presente en la sonda de SEQ ID NO:3. 23. Kit según la reivindicación 21 ó 22, que adicionalmente comprende los oligonucleótidos de SEQ ID NO:4 y SEQ ID NO:5. 24. Kit según la reivindicación 21 ó 22, que adicionalmente comprende una molécula de DNA que comprende la secuencia representada por SEQ ID NO:6. 25. Kit según la reivindicación 24, en el que la molécula de DNA es un plásmido.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos IIISolicitud de patent

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control

Monkeypox; PCR; Quality controlVerola del mico; PCR; Control de qualitatViruela del mono; PCR; Control de calidadTo evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.This research was supported by CIBER (Strategic Action for Monkeypox)—Consorcio Centro de Investigación Biomédica en Red—(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. A. d. S. is supported by ‘Instituto de Salud Carlos III’ (grant number JR22/00055)

Oligonucleotide Sensor Based on Selective Capture of Upconversion Nanoparticles Triggered by Target-Induced DNA Interstrand Ligand Reaction

We present a sensor that exploits the phenomenon of upconversion luminescence to detect the presence of specific sequences of small oligonucleotides such as miRNAs among others. The sensor is based on NaYF4:Yb,Er@SiO2 nanoparticles functionalized with ssDNA that contain azide groups on the 3' ends. In the presence of a target sequence, interstrand ligation is possible via the click-reaction between one azide of the upconversion probe and a DBCO-ssDNA-biotin probe present in the solution. As a result of this specific and selective process, biotin is covalently attached to the surface of the upconversion nanoparticles. The presence of biotin on the surface of the nanoparticles allows their selective capture on a streptavidin-coated support, giving a luminescent signal proportional to the amount of target strands present in the test samples. With the aim of studying the analytical properties of the sensor, total RNA samples were extracted from healthy mosquitoes and were spiked-in with a specific target sequence at different concentrations. The result of these experiments revealed that the sensor was able to detect 10-17 moles per well (100 fM) of the target sequence in mixtures containing 100 ng of total RNA per well. A similar limit of detection was found for spiked human serum samples, demonstrating the suitability of the sensor for detecting specific sequences of small oligonucleotides under real conditions. In contrast, in the presence of noncomplementary sequences or sequences having mismatches, the luminescent signal was negligible or conspicuously reduced.The authors are grateful for the financial support from the Bill & Melinda Gates Foundation, with Grant OPP1128411, Asociación Española Contra el Cáncer, Santander-Universidad Complutense project PR26/16-12B-3, and from the Spanish MINECO for the projects MAT2014-55065-R, SAF2014-56763-R, and FIS2013-41709-P.S

Mutations in Coding and Non-Coding Regions in Varicella-Zoster Virus Causing Fatal Hemorrhagic Fever Without Rash in an Immunocompetent Patient: Case Report

Introduction: We report the case of a fatal hemorrhagic varicella primary infection in an immunocompetent man and whole-genome characterization of the virus for the investigation of biomarkers of virulence. Case: A 38-year-old patient born in Nigeria presented to the emergency department with abdominal pain and subsequently developed fatal hemorrhagic disease without skin rash. Extensive laboratory tests including serology and PCR for arenaviruses, bunyaviruses and ebolaviruses were negative. Varicella-zoster virus (VZV) PCR of sera, liver and spleen tissue samples from autopsy revealed the presence of VZV DNA. Primary infection by varicella-zoster virus with hemorrhagic manifestations was diagnosed after virological testing. The VZV genome was sequenced using a mWGS approach. Bioinformatic analysis showed 53 mutations across the genome, 33 of them producing non-synonymous variants affecting up to 14 genes. Some of them, such as ORF11 and ORF 62, encoded for essential functions related to skin or neurotropism. To our knowledge, the mutations reported here have never been described in a VZV causing such a devastating outcome. Discussion: In immunocompetent patients, viral factors should be considered in patients with uncommon symptoms or severe diseases. Some relevant mutations revealed by using whole genome sequencing (WGS) directly from clinical samples may be involved in this case and deserves further investigation. Conclusion: Differential diagnosis of varicella-zoster virus in immunocompetent adults should be considered among patients with suspected VHF, even if the expected vesicular rash is not present at admission and does not arise thereafter. Whole genome sequencing of strains causing uncommon symptoms and/or mortality is needed for epidemiological surveillance and further characterization of putative markers of virulence. Additionally, this report highlights the recommendation for a VZV vaccination policy in non-immunized migrants from developing countries.This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY1372/12. The journal’s Rapid Service fee was paid by Consorcio Centro de Investigación en Red (CIBER). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations

The taxonomy of the family Filoviridae (marburgviruses and ebolaviruses) has changed several times since the discovery of its members, resulting in a plethora of species and virus names and abbreviations. The current taxonomy has only been partially accepted by most laboratory virologists. Confusion likely arose for several reasons: species names that consist of several words or which (should) contain diacritical marks, the current orthographic identity of species and virus names, and the similar pronunciation of several virus abbreviations in the absence of guidance for the correct use of vernacular names. To rectify this problem, we suggest (1) to retain the current species names Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus, but to replace the name Cote d'Ivoire ebolavirus [sic] with Taï Forest ebolavirus and Lake Victoria marburgvirus with Marburg marburgvirus; (2) to revert the virus names of the type marburgviruses and ebolaviruses to those used for decades in the field (Marburg virus instead of Lake Victoria marburgvirus and Ebola virus instead of Zaire ebolavirus); (3) to introduce names for the remaining viruses reminiscent of jargon used by laboratory virologists but nevertheless different from species names (Reston virus, Sudan virus, Taï Forest virus), and (4) to introduce distinct abbreviations for the individual viruses (RESTV for Reston virus, SUDV for Sudan virus, and TAFV for Taï Forest virus), while retaining that for Marburg virus (MARV) and reintroducing that used over decades for Ebola virus (EBOV). Paying tribute to developments in the field, we propose (a) to create a new ebolavirus species (Bundibugyo ebolavirus) for one member virus (Bundibugyo virus, BDBV); (b) to assign a second virus to the species Marburg marburgvirus (Ravn virus, RAVV) for better reflection of now available high-resolution phylogeny; and (c) to create a new tentative genus (Cuevavirus) with one tentative species (Lloviu cuevavirus) for the recently discovered Lloviu virus (LLOV). Furthermore, we explain the etymological derivation of individual names, their pronunciation, and their correct use, and we elaborate on demarcation criteria for each taxon and virus.S

Monitoring monkeypox virus in saliva and air samples in Spain: a cross-sectional study

Background: The transmission of monkeypox virus occurs through direct contact, but transmission through saliva or exhaled droplets and aerosols has not yet been investigated. We aimed to assess the presence of monkeypox virus DNA and infectious virus in saliva samples and droplets and aerosols exhaled from patients infected with monkeypox virus. Methods: We did a cross-sectional study in patients with monkeypox confirmed by PCR who attended two health centres in Madrid, Spain. For each patient, we collected samples of saliva, exhaled droplets within a mask, and aerosols captured by air filtration through newly developed nanofiber filters. We evaluated the presence of monkeypox virus in the samples by viral DNA detection by quantitative PCR (qPCR) and isolation of infectious viruses in cell cultures. Findings: Between May 18 and July 15, 2022, 44 patients with symptomatic monkeypox attended two health centres in Madrid and were included in the study. All were cisgender men, with a median age of 35·0 years (IQR 11·3). We identified high loads of monkeypox virus DNA by qPCR in 35 (85%) of 41 saliva samples. Infectious monkeypox virus was recovered from 22 (67%) of 33 saliva samples positive for monkeypox virus DNA. We also found a significant association between the number of affected cutaneous areas or general symptoms and the viral load present in saliva samples. Droplets exhaled from patients with monkeypox, detected inside a mask, contained monkeypox virus DNA in 32 (71%) of 45 samples, with two of the 32 positive samples showing the presence of the infectious virus. Monkeypox virus DNA in aerosols, collected from the medical consultation room, were detected in 27 (64%) of 42 samples, despite patients wearing an FFP2 mask during the visit. Infectious virus was not recovered from aerosol samples. High levels of monkeypox virus DNA were identified in aerosols collected from a hospital isolation room housing a patient with monkeypox. Interpretation: The identification of high viable monkeypox virus loads in saliva in most patients with monkeypox and the finding of monkeypox virus DNA in droplets and aerosols warrants further epidemiological studies to evaluate the potential relevance of the respiratory route of infection in the 2022 monkeypox virus outbreak.This study was funded by the EU (Nextgeneration EU), Consejo Superior de Investigaciones Científicas (PTI Salud Global), and Ciberinfec (Acción estratégica MKPXV22). We thank Milagros Guerra and the Electron Microscopy Service at CBMSO for their support. We thank the contribution of Grupo Viruela Simio Madrid ISCIII/HCSC/Sandoval.S

Epidemiologic Features and Control Measures during Monkeypox Outbreak, Spain, June 2022

During June 2022, Spain was one of the countries most affected worldwide by a multicountry monkeypox outbreak with chains of transmission without identified links to disease-endemic countries. We provide epidemiologic features of cases reported in Spain and the coordinated measures taken to respond to this outbreak.S

Real time PCR assay for detection of all known lineages of West Nile virus

West Nile virus (WNV) is one of the most widespread arbovirus and a large variety of WNV strains and lineages have been described. The molecular methods for the diagnosis of WNV target mainly lineages 1 and 2, which have caused outbreaks in humans, equines and birds. But the last few years new and putative WNV lineages of unknown pathogenicity have been described. Here we describe a new sensitive and specific real-time PCR assay for the detection and quantification of all the WNV lineages described until now. Primers and probe were designed in the 3'-untranslated region (3'-UTR) of the WNV genome and were designed to match all sequenced WNV strains perfectly. The sensitivity of the assay ranged from 1,5 to 15 copies per reaction depending on the WNV lineage tested. The method was validated for WNV diagnosis using different viral strains, human samples (cerebrospinal fluid, biopsies, serum and plasma) and mosquito pools. The assay did not amplify any other phylogenetically or symptomatically related viruses. All of the above make it a very suitable tool for the diagnosis of WNV and for surveillance studies