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Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.
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Glicopéptidos , Proteómica , Espectrometría de Masas en Tándem , Glicopéptidos/análisis , Glicopéptidos/química , Proteómica/métodos , Animales , Glicosilación , Electrones , Cromatografía Liquida , Ratones , HumanosRESUMEN
Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.
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Proteínas Bacterianas , Toxinas Bacterianas , Yersinia , Yersinia/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Modelos Moleculares , Cristalografía por Rayos XRESUMEN
The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N-glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N-glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N-glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles.
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COVID-19 , SARS-CoV-2 , Humanos , Glicosilación , Glicoproteína de la Espiga del Coronavirus , Anticuerpos AntiviralesRESUMEN
Bitter taste receptors (T2R) are a subfamily of G protein-coupled receptors that enable humans to detect aversive and toxic substances. The ability to discern bitter compounds varies between individuals and is attributed mainly to naturally occurring T2R polymorphisms. T2Rs are also expressed in numerous non-gustatory tissues, including the heart, indicating potential contributions to cardiovascular physiology. In this study. T2Rs that have previously been identified in human cardiac tissues (T2Rs - 10, 14, 30, 31, 46 and 50) and their naturally occurring polymorphisms were functionally characterised. The ligand-dependent signaling responses of some T2R variants were completely abolished (T2R30 Leu252 and T2R46 Met228), whereas other receptor variants had moderate changes in their maximal response, but not potency, relative to wild type. Using a cAMP fluorescent biosensor, we reveal the productive coupling of T2R14, but not the T2R14 Phe201 variant, to endogenous Gαi. Modeling revealed that these variants resulted in altered interactions that generally affected ligand binding (T2R30 Leu252) or Gα protein interactions (T2R46 Met228 and T2R14 Phe201), rather than receptor structural stability. Interestingly, this study is the first to show a difference in signaling for T2R50 Tyr203 (rs1376251) which has been associated with cardiovascular disease. The observation of naturally occurring functional variation in the T2Rs with the greatest expression in the heart is important, as their discovery should prove useful in deciphering the role of T2Rs within the cardiovascular system.
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Receptores Acoplados a Proteínas G , Gusto , Humanos , Gusto/fisiología , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Acute respiratory infections (ARIs) are amongst the leading causes of death and disability, and the greatest burden of disease impacts children, pregnant women, and the elderly. Respiratory viruses account for the majority of ARIs. The unfolded protein response (UPR) is a host homeostatic defence mechanism primarily activated in response to aberrant endoplasmic reticulum (ER) resident protein accumulation in cell stresses including viral infection. The UPR has been implicated in the pathogenesis of several respiratory diseases, as the respiratory system is particularly vulnerable to chronic and acute activation of the ER stress response pathway. Many respiratory viruses therefore employ strategies to modulate the UPR during infection, with varying effects on the host and the pathogens. Here, we review the specific means by which respiratory viruses affect the host UPR, particularly in association with the high production of viral glycoproteins, and the impact of UPR activation and subversion on viral replication and disease pathogenesis. We further review the activation of UPR in common co-morbidities of ARIs and discuss the therapeutic potential of modulating the UPR in virally induced respiratory diseases.
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In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.
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Infecciones por Henipavirus , Henipavirus , Virus Nipah , Humanos , Glicoproteínas/metabolismo , Proteínas Virales/metabolismo , Virus Nipah/metabolismoRESUMEN
The sugars that coat the outsides of viruses and host cells are key to successful disease transmission, but they remain understudied compared to other molecular features. Understanding the comparative zoology of glycosylation - and harnessing it for predictive science - could help close the molecular gap in zoonotic risk assessment.
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Viroma , Virus , Glicosilación , Virus/genéticaRESUMEN
Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.
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Factor IX , Calidad de Vida , Cricetinae , Animales , Humanos , Factor IX/metabolismo , Cricetulus , Proteínas Recombinantes/metabolismo , Células CHO , Ingeniería CelularRESUMEN
Beer and wine are fermented beverages that contain abundant proteins released from barley or grapes, and secreted from yeast. These proteins are associated with many quality attributes including turbidity, foamability, effervescence, flavour and colour. Many grape proteins and secreted yeast proteins are glycosylated, and barley proteins can be glycated under the high temperatures in the beer making process. The emergence of high-resolution mass spectrometry has allowed proteomic and glycoproteomic analyses of these complex mixtures of proteins towards understanding their role in determining beer and wine attributes. In this review, we summarise recent studies of proteomic and glycoproteomic analyses of beer and wine including their strategies for mass spectrometry (MS)-based identification, quantification and characterisation of the glyco/proteomes of fermented beverages to control product quality.
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Hordeum , Vitis , Vino , Cerveza/análisis , Proteínas Fúngicas/análisis , Proteómica/métodos , Saccharomyces cerevisiae , Vino/análisisRESUMEN
BACKGROUND: A subset of Australians who have been bitten by ticks experience a complex of chronic and debilitating symptoms which cannot be attributed to the known pathogenic species of bacteria present in Australia. As a result, there has been a renewed effort to identify and characterise viruses in Australian terrestrial ticks. Recent transcriptome sequencing of Ixodes and Amblyomma ticks has revealed the presence of multiple virus sequences. However, without virus isolates our ability to understand the host range and pathogenesis of newly identified viruses is limited. We have established a successful method for high-throughput virus discovery and isolation in mosquitoes using antibodies to double-stranded RNA. In this study we sought to characterise five archival tick-borne viruses to adapt our virus discovery protocol for Australian ticks. METHODS: We performed virus characterisation using a combination of bioinformatic sequence analysis and in vitro techniques including replication kinetics, antigenic profiling, virus purification and mass spectrometry. RESULTS: Our sequence analysis of Nugget virus, Catch-me-Cave virus and Finch Creek virus revealed marked genetic stability in isolates collected from the same location approximately 30 years apart. We demonstrate that the Ixodes scapularis-derived ISE6 cell line supports replication of Australian members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families, including Saumarez Reef virus (SREV), a flavivirus isolated from the soft tick Ornithodoros capensis. While antibodies against double-stranded RNA could be used to detect replication of a tick-borne reovirus and mosquito-borne flavivirus, the tick-borne flaviviruses Gadgets Gully virus and SREV could not be detected using this method. Finally, four novel virus-like sequences were identified in transcriptome sequencing of the Australian native tick Ixodes holocyclus. CONCLUSIONS: Genetic and antigenic characterisations of archival viruses in this study confirm that three viruses described in 2002 represent contemporary isolates of virus species first identified 30 years prior. Our findings with antibodies to double-stranded RNA highlight an unusual characteristic shared by two Australian tick-borne flaviviruses. Finally, comparative growth kinetics analyses of Australian tick-borne members of the Flaviviridae, Nairoviridae, Phenuiviridae and Reoviridae families in ISE6 and BSR cells will provide a useful resource for isolation of Australian tick-borne viruses using existing cell lines.
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Flavivirus , Ixodes , Virus ARN , Animales , Australia , Virus ADN , Humanos , Ixodes/genéticaRESUMEN
Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.
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Glicopéptidos/sangre , Glicoproteínas/sangre , Informática/métodos , Proteoma/análisis , Proteómica/métodos , Investigadores/estadística & datos numéricos , Programas Informáticos , Glicosilación , Humanos , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.
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Cerveza/análisis , Australia , Grano Comestible/química , Proteínas Fúngicas/análisis , Glicosilación , Hordeum/química , Procesamiento Proteico-Postraduccional , Proteolisis , Proteoma/análisis , Proteómica/métodos , Almidón/análisisRESUMEN
Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.
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Secuencia Conservada , Glicoproteínas/química , Glicoproteínas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Glicosilación , Oryza/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/química , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa/aislamiento & purificación , Superóxido Dismutasa/metabolismoRESUMEN
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
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Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Coagulantes/aislamiento & purificación , Medios de Cultivo/metabolismo , Factor IX/aislamiento & purificación , Células Cultivadas , Cromatografía de Fase Inversa , Humanos , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteómica , Control de Calidad , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Carga de TrabajoRESUMEN
Influenza virus has a high mutation rate, such that within one host different viral variants can emerge. Evidence suggests that influenza virus variants are more prevalent in pregnant and/or obese individuals due to their impaired interferon response. We have recently shown that the non-allergic, paucigranulocytic subtype of asthma is associated with impaired type I interferon production. Here, we seek to address if this is associated with an increased emergence of influenza virus variants. Compared to controls, mice with paucigranulocytic asthma had increased disease severity and an increased emergence of influenza virus variants. Specifically, PB1 mutations exclusively detected in asthmatic mice were associated with increased polymerase activity. Furthermore, asthmatic host-derived virus led to increased disease severity in wild-type mice. Taken together, these data suggest that at least a subset of patients with asthma may be more susceptible to severe influenza and may be a possible source of new influenza virus variants.
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Asma/virología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/virología , Animales , Femenino , Interacciones Huésped-Patógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor para Productos Finales de Glicación Avanzada/deficienciaRESUMEN
OBJECTIVE: To explore the contribution of environmental factors in the unusual pattern of influenza activity observed in Brisbane, Australia during the summer of 2018-2019. METHODS: Distributed lag nonlinear models (DLNMs) were used to estimate the effect of environmental factors on weekly influenza incidence in Brisbane. Next generation sequencing was then employed to analyze minor and majority variants in influenza strains isolated from Brisbane children during this period. RESULTS: There were limited marked differences in the environmental factors observed in Brisbane between the 2018-2019 summer period and the same period of the proceeding years, with the exception of significant reduction in rainfall. DLNM showed that reduced rainfall in Brisbane (at levels consistent with the 2018-2019 period) correlated with a dramatic increase in the relative risk of influenza. Sulfur dioxide (SO2) levels were also increased in the 2018-2019 period, although these levels did not correlate with an increased risk of influenza. Sequencing of a limited number of pediatric influenza virus strains isolated during the 2018-2019 showed numerous mutations within the viral HA. CONCLUSIONS: Taken together, these data suggest a limited role for key environmental factors in the influenza activity observed in Brisbane, Australia during the summer of 2018-2019. One alternative explanation may that viral factors, in addition to other factors not studied herein, contributed to the unusual influenza season. Our findings provide fundamental information that may be beneficial to a better understanding of the seasonal trends of influenza virus.
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Gripe Humana , Australia/epidemiología , Niño , Humanos , Incidencia , Gripe Humana/epidemiología , Estaciones del Año , Dióxido de AzufreRESUMEN
Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types.
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Proteínas Fúngicas/análisis , Glicopéptidos/análisis , Glicoproteínas/análisis , Proteínas de Plantas/análisis , Saccharomyces cerevisiae , Vitis/química , Vino/análisis , Fermentación , Proteínas Fúngicas/metabolismo , Glicopéptidos/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismo , Proteómica , Saccharomyces cerevisiae/metabolismoRESUMEN
Mass spectrometry glycoproteomics is rapidly maturing, allowing unprecedented insights into the diversity and functions of protein glycosylation. However, quantitative glycoproteomics remains challenging. We developed GlypNirO, an automated software pipeline which integrates the complementary outputs of Byonic and Proteome Discoverer to allow high-throughput automated quantitative glycoproteomic data analysis. The output of GlypNirO is clearly structured, allowing manual interrogation, and is also appropriate for input into diverse statistical workflows. We used GlypNirO to analyse a published plasma glycoproteome dataset and identified changes in site-specific N- and O-glycosylation occupancy and structure associated with hepatocellular carcinoma as putative biomarkers of disease.
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The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.