RESUMEN
Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007. We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.
Asunto(s)
Genoma Viral , Inestabilidad Genómica , Monkeypox virus/genética , Filogenia , Adaptación Biológica/genética , Secuencia de Aminoácidos , Animales , República Democrática del Congo/epidemiología , Monitoreo Epidemiológico , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Mpox/epidemiología , Mpox/virología , Monkeypox virus/clasificación , Análisis de Secuencia de ADN , Índice de Severidad de la EnfermedadRESUMEN
To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.
Asunto(s)
Ebolavirus/genética , Genoma Viral , Animales , Técnicas de Cultivo de Célula , Análisis por Conglomerados , Marcadores Genéticos , Glicoproteínas/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Mutación , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Primates/genética , ARN Viral/genética , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Células Vero , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Nipah virus (NiV) is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB) proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains) has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs) and demonstrated that the matrix (M) protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release. RESULTS: Using rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins. CONCLUSION: These data indicate that the YPLGVG motif within the NiV M protein plays an important role in M budding; however, involvement of any specific components of the cellular MVB sorting pathway in henipavirus budding remains to be demonstrated. Further investigation of henipavirus assembly and budding may yet reveal a novel mechanism(s) of viral assembly and release that could be applicable to other enveloped viruses or have therapeutic implications.
Asunto(s)
Virus Nipah/química , Virus Nipah/fisiología , Proteínas de la Matriz Viral/química , Esparcimiento de Virus , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Mutación , Virus Nipah/genética , Alineación de Secuencia , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.
Asunto(s)
Ebolavirus/fisiología , Nucleoproteínas/fisiología , Proteínas del Núcleo Viral/fisiología , Ensamble de Virus , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dimerización , Ebolavirus/química , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , ViriónRESUMEN
The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.
Asunto(s)
Ebolavirus/crecimiento & desarrollo , Luciferasas de Luciérnaga/análisis , Mediciones Luminiscentes/métodos , Línea Celular , Ebolavirus/metabolismo , Genes Reporteros , Humanos , Luciferasas de Luciérnaga/metabolismo , Nucleoproteínas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Ensamble de VirusRESUMEN
The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minigenome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.
Asunto(s)
Ebolavirus/química , Genoma Viral , Nucleoproteínas/fisiología , Proteínas del Núcleo Viral/fisiología , Ensamble de Virus/fisiología , Animales , Células COS , Chlorocebus aethiops , Ebolavirus/ultraestructura , Proteínas de la Nucleocápside , Células VeroRESUMEN
PURPOSE: We developed a 51-mutation extended cystic fibrosis (CF) panel that incorporates the 25 previously recommended CFTR mutations, plus 26 additional mutations including 3199del6, which was associated with I148T. METHODS: This assay utilizes an integrated matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system. RESULTS: CF testing was performed on over 5,000 individuals, including a 3-year-old Hispanic-American patient with a compound heterozygous G542X/3199del6 genotype. He is negative for I148T, or other mutations assessed by CFTR gene sequencing. CONCLUSION: These results demonstrate the successful implementation of MALDI-TOF mass spectrometry in CF clinical testing, and establish 3199del6 as a disease-causing CF mutation.