RESUMEN
Replicon systems are important tools for investigating viral RNA synthesis. We have developed an ambisense minigenome system for Rift Valley fever virus (RVFV) with the aim to analyse the effects of L gene mutations on viral transcription versus replication. The overall activity of the replication complex was assessed by expression of a luciferase reporter gene. Northern blot analysis enabled differentiation between synthesis of viral mRNA and replication intermediates. The functionality of the system was demonstrated by probing residues predictably involved in the cap-snatching endonuclease active site in the L protein. Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other bunyaviruses. The analysis of further L gene mutants revealed an essential role of a C-terminal region in the RVFV L protein in viral transcription. In summary, the established minigenome system is suitable for functional testing of the relevance of residues for viral transcription and replication.
Asunto(s)
Genoma Viral , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Proteínas Virales/metabolismo , Replicación Viral , Regulación Viral de la Expresión Génica , Mutación , Virus de la Fiebre del Valle del Rift/fisiología , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.
Asunto(s)
Virus Lassa/fisiología , ARN Mensajero/genética , ARN Viral/genética , Proteínas Virales/metabolismo , Línea Celular , Humanos , Virus Lassa/genética , Proteínas Virales/genética , Replicación ViralRESUMEN
OBJECTIVES: In this study, we investigated the causes of measles-like illnesses (MLI) in the Uganda national surveillance program in order to inform diagnostic assay selection and vaccination strategies. METHODS: We used metagenomic next-generation sequencing (M-NGS) on the Illumina platform to identify viruses associated with MLI (defined as fever and rash in the presence of either cough, coryza or conjunctivitis) in patient samples that had tested IgM negative for measles between 2010 and 2019. RESULTS: Viral genomes were identified in 87/271 (32%) of samples, of which 44/271 (16%) contained 12 known viral pathogens. Expected viruses included rubella, human parvovirus B19, Epstein Barr virus, human herpesvirus 6B, human cytomegalovirus, varicella zoster virus and measles virus (detected within the seronegative window-period of infection) and the blood-borne hepatitis B virus. We also detected Saffold virus, human parvovirus type 4, the human adenovirus C2 and vaccine-associated poliovirus type 1. CONCLUSIONS: The study highlights the presence of undiagnosed viruses causing MLI in Uganda, including vaccine-preventable illnesses. NGS can be used to monitor common viral infections at a population level, especially in regions where such infections are prevalent, including low and middle income countries to guide vaccination policy and optimize diagnostic assays.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Sarampión , Humanos , Uganda/epidemiología , Preescolar , Sarampión/epidemiología , Sarampión/virología , Lactante , Niño , Masculino , Femenino , Adolescente , Virus/aislamiento & purificación , Virus/genética , Virus/clasificación , Genoma Viral , Adulto , Adulto Joven , Virosis/epidemiología , Virosis/virología , Metagenómica , Virus del Sarampión/genética , Virus del Sarampión/aislamiento & purificación , Virus del Sarampión/clasificaciónRESUMEN
Le Dantec virus (LDV), assigned to the species Ledantevirus ledantec, genus Ledantevirus, family Rhabdoviridae has been associated with human disease but has gone undetected since the 1970s. We describe the detection of LDV in a human case of undifferentiated fever in Uganda by metagenomic sequencing and demonstrate a serological response using ELISA and pseudotype neutralisation. By screening 997 individuals sampled in 2016, we show frequent exposure to ledanteviruses with 76% of individuals seropositive in Western Uganda, but lower seroprevalence in other areas. Serological cross-reactivity as measured by pseudotype-based neutralisation was confined to ledanteviruses, indicating population seropositivity may represent either exposure to LDV or related ledanteviruses. We also describe the discovery of a closely related ledantevirus in blood from the synanthropic rodent Mastomys erythroleucus. Ledantevirus infection is common in Uganda but is geographically heterogenous. Further surveys of patients presenting with acute fever are required to determine the contribution of these emerging viruses to febrile illness in Uganda.
Asunto(s)
Anticuerpos Antivirales , Rhabdoviridae , Humanos , Uganda/epidemiología , Adulto , Masculino , Femenino , Adolescente , Adulto Joven , Persona de Mediana Edad , Anticuerpos Antivirales/sangre , Niño , Rhabdoviridae/aislamiento & purificación , Rhabdoviridae/genética , Rhabdoviridae/clasificación , Preescolar , Infecciones por Rhabdoviridae/epidemiología , Infecciones por Rhabdoviridae/virología , Infecciones por Rhabdoviridae/veterinaria , Estudios Seroepidemiológicos , Animales , Reacciones Cruzadas , Lactante , Anciano , Filogenia , Ensayo de Inmunoadsorción Enzimática , MetagenómicaRESUMEN
The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.
Asunto(s)
Bryopsida/metabolismo , Proteínas Recombinantes/biosíntesis , Bryopsida/genética , Cromatografía Liquida , Factor H de Complemento/biosíntesis , Factor H de Complemento/química , Humanos , Espectrometría de Masas , Fenotipo , Plantas Modificadas GenéticamenteRESUMEN
OBJECTIVES: Travel-associated infections are challenging to diagnose because of the broad spectrum of potential aetiologies. As a proof-of-principle study, we used MNGS to identify viral pathogens in clinical samples from returning travellers in a single center to explore its suitability as a diagnostic tool. METHODS: Plasma samples from 40 returning travellers presenting with a fever of ≥38°C were sequenced using MNGS on the Illumina MiSeq platform and compared with standard-of-care diagnostic assays. RESULTS: In total, 11/40 patients were diagnosed with a viral infection. Standard of care diagnostics revealed 5 viral infections using plasma samples; dengue virus 1 (nâ¯=â¯2), hepatitis E (nâ¯=â¯1), Ebola virus (nâ¯=â¯1) and hepatitis A (nâ¯=â¯1), all of which were detected by MNGS. Three additional patients with Chikungunya virus (nâ¯=â¯2) and mumps virus were diagnosed by MNGS only. Respiratory infections detected by nasal/throat swabs only were not detected by MNGS of plasma. One patient had infection with malaria and mumps virus during the same admission. CONCLUSIONS: MNGS analysis of plasma samples improves the sensitivity of diagnosis of viral infections and has potential as an all-in-one diagnostic test. It can be used to identify infections that have not been considered by the treating physician, co-infections and new or emerging pathogens. SUMMARY: Next generation sequencing (NGS) has potential as an all-in-one diagnostic test. In this study we used NGS to diagnose returning travellers with acute febrile illness in the UK, highlighting cases where the diagnosis was missed using standard methods.
Asunto(s)
Fiebre/virología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Enfermedad Relacionada con los Viajes , Virosis/diagnóstico , Adulto , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Humanos , Metagenómica , Enfermedades Parasitarias/diagnóstico , Enfermedades Parasitarias/parasitología , Prueba de Estudio Conceptual , ARN Viral/genética , Infecciones del Sistema Respiratorio/sangre , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Sensibilidad y Especificidad , Viaje/estadística & datos numéricos , Virosis/sangre , Virus/genética , Virus/patogenicidadRESUMEN
Lassa virus (LASV) causes a deadly haemorrhagic fever in humans, killing several thousand people in West Africa annually. For 40 years, the Natal multimammate rat, Mastomys natalensis, has been assumed to be the sole host of LASV. We found evidence that LASV is also hosted by other rodent species: the African wood mouse Hylomyscus pamfi in Nigeria, and the Guinea multimammate mouse Mastomys erythroleucus in both Nigeria and Guinea. Virus strains from these animals were isolated in the BSL-4 laboratory and fully sequenced. Phylogenetic analyses of viral genes coding for glycoprotein, nucleoprotein, polymerase and matrix protein show that Lassa strains detected in M. erythroleucus belong to lineages III and IV. The strain from H. pamfi clusters close to lineage I (for S gene) and between II &III (for L gene). Discovery of new rodent hosts has implications for LASV evolution and its spread into new areas within West Africa.