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1.
PLoS Pathog ; 15(11): e1008061, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31697791

RESUMO

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.

2.
Adv Virus Res ; 104: 185-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31439149

RESUMO

Hantaviruses are important zoonotic pathogens of public health importance that are found on all continents except Antarctica and are associated with hemorrhagic fever with renal syndrome (HFRS) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Despite the significant disease burden they cause, no FDA-approved specific therapeutics or vaccines exist against these lethal viruses. The lack of available interventions is largely due to an incomplete understanding of hantavirus pathogenesis and molecular mechanisms of virus replication, including cellular entry. Hantavirus Gn/Gc glycoproteins are the only viral proteins exposed on the surface of virions and are necessary and sufficient to orchestrate virus attachment and entry. In vitro studies have implicated integrins (ß1-3), DAF/CD55, and gC1qR as candidate receptors that mediate viral attachment for both Old World and New World hantaviruses. Recently, protocadherin-1 (PCDH1) was demonstrated as a requirement for cellular attachment and entry of New World hantaviruses in vitro and lethal HPS in vivo, making it the first clade-specific host factor to be identified. Attachment of hantavirus particles to cellular receptors induces their internalization by clathrin-mediated, dynamin-independent, or macropinocytosis-like mechanisms, followed by particle trafficking to an endosomal compartment where the fusion of viral and endosomal membranes can occur. Following membrane fusion, which requires cholesterol and acid pH, viral nucleocapsids escape into the cytoplasm and launch genome replication. In this review, we discuss the current mechanistic understanding of hantavirus entry, highlight gaps in our existing knowledge, and suggest areas for future inquiry.

3.
MBio ; 10(4)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289183

RESUMO

Ebola virus (EBOV) causes highly lethal disease outbreaks against which no FDA-approved countermeasures are available. Although many host factors exploited by EBOV for cell entry have been identified, including host cell surface phosphatidylserine receptors, endosomal cysteine proteases, and the lysosomal cholesterol trafficking protein NPC1, key questions remain. Specifically, late entry steps culminating in viral membrane fusion remain enigmatic. Here, we investigated a set of glycoprotein (GP) mutants previously hypothesized to be entry defective and identified one mutation, R64A, that abolished infection with no apparent impact on GP expression, folding, or viral incorporation. R64A profoundly thermostabilized EBOV GP and rendered it highly resistant to proteolysis in vitro Forward-genetics and cell entry studies strongly suggested that R64A's effects on GP thermostability and proteolysis arrest viral entry at least at two distinct steps: the first upstream of NPC1 binding and the second at a late entry step downstream of fusion activation. Concordantly, toremifene, a small-molecule entry inhibitor previously shown to bind and destabilize GP, may selectively enhance the infectivity of viral particles bearing GP(R64A) at subinhibitory concentrations. R64A provides a valuable tool to further define the interplay between GP stability, proteolysis, and viral membrane fusion; to explore the rational design of stability-modulating antivirals; and to spur the development of next-generation Ebola virus vaccines with improved stability.IMPORTANCE Ebola virus is a medically relevant virus responsible for outbreaks of severe disease in western and central Africa, with mortality rates reaching as high as 90%. Despite considerable effort, there are currently no FDA-approved therapeutics or targeted interventions available, highlighting the need of development in this area. Host-cell invasion represents an attractive target for antivirals, and several drug candidates have been identified; however, our limited understanding of the complex viral entry process challenges the development of such entry-targeting drugs. Here, we report on a glycoprotein mutation that abrogates viral entry and provides insights into the final steps of this process. In addition, the hyperstabilized phenotype of this mutant makes it useful as a tool in the discovery and design of stability-modulating antivirals and next-generation vaccines against Ebola virus.

4.
Viruses ; 11(7)2019 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-31337019

RESUMO

Andes virus (ANDV) and Sin Nombre virus (SNV) are the main causative agents responsible for hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is a severe respiratory disease with a high fatality rate for which there are no approved therapeutics or vaccines available. Some vaccine approaches for HCPS have been tested in preclinical models, but none have been tested in infectious models in regard to their ability to protect against multiple species of HCPS-causing viruses. Here, we utilize recombinant vesicular stomatitis virus-based (VSV) vaccines for Andes virus (ANDV) and Sin Nombre virus (SNV) and assess their ability to provide cross-protection in infectious challenge models. We show that, while both rVSVΔG/ANDVGPC and rVSVΔG/SNVGPC display attenuated growth as compared to wild type VSV, each vaccine is able to induce a cross-reactive antibody response. Both vaccines protected against both homologous and heterologous challenge with ANDV and SNV and prevented HCPS in a lethal ANDV challenge model. This study provides evidence that the development of a single vaccine against HCPS-causing hantaviruses could provide protection against multiple agents.

5.
Water Environ Res ; 91(11): 1455-1465, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31074914

RESUMO

Despite the increased research efforts, full-scale implementation of shortcut nitrogen removal strategies has been challenged by the lack of consistent nitrite-oxidizing bacteria out-selection. This paper proposes an alternative path using partial denitrification (PdN) selection coupled with anaerobic ammonium-oxidizing bacteria (AnAOB). A nitrate residual concentration (>2 mg N/L) was identified as the crucial factor for metabolic PdN selection using acetate as a carbon source, unlike the COD/N ratio which was often suggested. Therefore, a novel and simple acetate dosing control strategy based on maintaining a nitrate concentration was tested in the absence and presence of AnAOB, achieving PdN efficiencies above 80%. The metabolic-based PdN selection allowed for flexibility to move between PdN and full denitrification when required to meet effluent nitrate levels. Due to the independence of this strategy on species selection and management of nitrite competition, this novel approach will guarantee nitrite availability for AnAOB under mainstream conditions unlike shortcut nitrogen removal approaches based on NOB out-selection. Overall, a COD addition of only 2.2 g COD/g TIN removed was needed for the PdN-AnAOB concept showing its potential for significant savings in external carbon source needs to meet low TIN effluent concentrations making this concept a competitive alternative. PRACTITIONER POINTS: Nitrate residual is the key control parameter for partial denitrification selection. Metabolic selection allowed for flexibility of moving from partial to full denitrification. 2.2 g COD/g TIN removed was needed for partial denitrification-anammox process.


Assuntos
Compostos de Amônio , Desnitrificação , Reatores Biológicos , Nitratos , Nitrogênio , Oxirredução
7.
J Gen Virol ; 100(6): 911-912, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31021739

RESUMO

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.

8.
Nat Struct Mol Biol ; 26(3): 204-212, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30833785

RESUMO

The structural features that govern broad-spectrum activity of broadly neutralizing anti-ebolavirus antibodies (Abs) outside of the internal fusion loop epitope are currently unknown. Here we describe the structure of a broadly neutralizing human monoclonal Ab (mAb), ADI-15946, which was identified in a human survivor of the 2013-2016 outbreak. The crystal structure of ADI-15946 in complex with cleaved Ebola virus glycoprotein (EBOV GPCL) reveals that binding of the mAb structurally mimics the conserved interaction between the EBOV GP core and its glycan cap ß17-ß18 loop to inhibit infection. Both endosomal proteolysis of EBOV GP and binding of mAb FVM09 displace this loop, thereby increasing exposure of ADI-15946's conserved epitope and enhancing neutralization. Our work also mapped the paratope of ADI-15946, thereby explaining reduced activity against Sudan virus, which enabled rational, structure-guided engineering to enhance binding and neutralization of Sudan virus while retaining the parental activity against EBOV and Bundibugyo virus.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Cristalografia por Raios X , Humanos , Estrutura Terciária de Proteína , Sobreviventes
9.
Sci Total Environ ; 665: 944-958, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30790764

RESUMO

The objective of this study was to evaluate the impact of bioswales on nutrient pollution in an urban combined sewershed. This evaluation was based on two criteria: the ability of bioswales to (1) remove nutrient pollution from stormwater runoff directly and (2) decrease sewer overflow volumes, which indirectly reduces total sewershed nutrient pollution during a storm event. Bioswales' direct nutrient removal was determined by analyzing nitrogen and phosphorus levels in water samples at seven bioswales located in the Bronx, New York City (NYC) over 42 storm events, while a bioswale's indirect nutrient removal through combined sewer overflow reduction was estimated by quantifying water retention at one of the bioswales. The study results indicated that: 1) the bioswale retained about 40% of stormwater conveyed to it from a drainage area 231 times its size, 2) bioswales leach nutrients into the subsurface, and 3) nitrogen leaching from bioswales varied seasonally, while phosphorus leaching decreased steadily over the study period. Although the studied bioswales leached a median 1.3 kg nitrogen per year into the subsurface, they provided an aggregate decrease in watershed nutrient pollution, from 7.7 to 6 kg nitrogen per year, due to their reduction of combined sewer overflow via stormwater retention.


Assuntos
Nitrogênio/análise , Fósforo/análise , Eliminação de Resíduos Líquidos/instrumentação , Poluentes Químicos da Água/análise , Poluição Química da Água/prevenção & controle , Áreas Alagadas , Biodegradação Ambiental , Cidade de Nova Iorque
10.
Bioresour Technol ; 279: 101-107, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30711750

RESUMO

Organic compounds such as methanol are widely used for enhancing denitrification at wastewater treatment plants (WWTPs) to meet effluent water quality permits. On the other hand, methane, which is the main feedstock for industrial methanol production, is also generated during anaerobic digestion in WWTPs, but is often flared to mitigate its greenhouse impacts. The overarching goal herein was to develop a novel continuous process for methanol production from methane using nitrifying activated sludge. The maximum AOB specific methanol production rate using hydroxylamine as electron donor was 1.61 ±â€¯0.15 and 1.27 ±â€¯0.15 mg-COD-CH3OH/(mg-COD-AOB*d), for hydraulic retention times (HRTs) of 7.5 h and 2 h, respectively. The corresponding production rate using ammonia as electron donor was 0.31 ±â€¯0.08 mg-COD-CH3OH/(mg-COD-AOB*d) at a HRT of 2 h. These results show that nitrifier-mediated methanol production in a continuous-flow system can enhance the efficiency of WWTPs through internal production of biomethanol for denitrification, while simultaneously minimizing wasteful biogas flaring.


Assuntos
Metanol/metabolismo , Amônia/metabolismo , Betaproteobacteria/metabolismo , Biocombustíveis , Desnitrificação , Metano/biossíntese , Nitrificação , Oxirredução , Esgotos/microbiologia , Águas Residuárias/química
11.
MBio ; 10(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622188

RESUMO

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (biosafety level 3 [BSL-3]) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn and Gc (Gn/Gc) entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantaviruses, Hantaan virus (HTNV) or Dobrava-Belgrade virus (DOBV), were refractory to rescue. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations: I532K in the cytoplasmic tail of Gn and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Finally, I532K/S1094L mutations in DOBV Gn/Gc permitted the rescue of rVSV-DOBV Gn/Gc, demonstrating that incorporation of cognate mutations into other hantaviral Gn/Gc proteins could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSVs, bearing HTNV and DOBV Gn/Gc, reported herein may also have utility as vaccines.IMPORTANCE Human hantavirus infections cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSVs encoding Hantaan virus and Dobrava-Belgrade virus glycoproteins described in this work provide a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as hantavirus vaccines.


Assuntos
Vetores Genéticos , Hantavirus/genética , Proteínas Mutantes/genética , Mutação Puntual , Proteínas Recombinantes/genética , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Linhagem Celular , Glicoproteínas/genética , Humanos , Genética Reversa , Inoculações Seriadas , Vesiculovirus/fisiologia , Liberação de Vírus , Replicação Viral
12.
Water Environ Res ; 91(3): 185-197, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30699248

RESUMO

In this study, concurrent operation of anammox and partial denitrification within a nonacclimated mixed culture system was proposed. The impact of carbon sources (acetate, glycerol, methanol, and ethanol) and COD/NO3- -N ratio on partial denitrification selection under both short- and long-term operations was investigated. Results from short-term testing showed that all carbon sources supported partial denitrification. However, acetate and glycerol were preferred due to their display of efficient partial denitrification selection, which may be related to their different electron transport pathways in comparison with methanol. Long-term operation confirmed results of batch tests by showing the contribution of partial denitrification to nitrate removal above 90% after acclimation in both acetate and glycerol reactors. In contrast, methanol showed challenges of maintaining efficient partial denitrification. COD/NO3- -N ratio mainly controlled the rate of nitrate reduction and not directly partial denitrification selection; thus, it should be used to balance between denitrification rate and anammox rate. PRACTITIONER POINTS: The authors aimed to investigate the impact of carbon sources and COD/NO3-N ratio on partial denitrification selection. All the carbon sources supported partial denitrification as long as the nitrite sink was available. 90% partial denitrification could be achieved with both acetate and glycerol in long-term operations. COD/NO3-N ratio did not directly control partial denitrification but can be used to balance between denitrification rate and anammox rate.


Assuntos
Compostos de Amônio/metabolismo , Análise da Demanda Biológica de Oxigênio , Reatores Biológicos/microbiologia , Carbono/metabolismo , Desnitrificação , Nitrogênio/metabolismo , Anaerobiose , Técnicas de Cultura , Nitratos/metabolismo , Oxirredução
13.
Cell Host Microbe ; 25(1): 39-48.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629917

RESUMO

Passive administration of monoclonal antibodies (mAbs) is a promising therapeutic approach for Ebola virus disease (EVD). However, all mAbs and mAb cocktails that have entered clinical development are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against outbreak-causing Bundibugyo virus (BDBV) and Sudan virus (SUDV). Here, we advance MBP134, a cocktail of two broadly neutralizing human mAbs, ADI-15878 from an EVD survivor and ADI-23774 from the same survivor but specificity-matured for SUDV GP binding affinity, as a candidate pan-ebolavirus therapeutic. MBP134 potently neutralized all ebolaviruses and demonstrated greater protective efficacy than ADI-15878 alone in EBOV-challenged guinea pigs. A second-generation cocktail, MBP134AF, engineered to effectively harness natural killer (NK) cells afforded additional improvement relative to its precursor in protective efficacy against EBOV and SUDV in guinea pigs. MBP134AF is an optimized mAb cocktail suitable for evaluation as a pan-ebolavirus therapeutic in nonhuman primates.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/uso terapêutico , Antivirais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Epitopos/imunologia , Feminino , Filoviridae/imunologia , Cobaias , Doença pelo Vírus Ebola/virologia , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Recombinantes/imunologia , Resultado do Tratamento
14.
Cell Host Microbe ; 25(1): 49-58.e5, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30629918

RESUMO

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Ebolavirus/patogenicidade , Furões/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Bem-Estar do Animal , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/administração & dosagem , Linhagem Celular , Cercopithecus aethiops , Modelos Animais de Doenças , Feminino , Filoviridae/imunologia , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/prevenção & controle , Infecções por Filoviridae/virologia , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Células Matadoras Naturais , Macaca , Macaca fascicularis , Masculino , Primatas , Análise de Sobrevida , Resultado do Tratamento , Proteínas Virais/imunologia
15.
Nat Chem Biol ; 15(3): 259-268, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30643282

RESUMO

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Vesículas Transportadoras/fisiologia , Células A549 , Animais , Antígenos de Diferenciação/metabolismo , Antivirais , Endossomos/fisiologia , Células HeLa , Humanos , Lisossomos/fisiologia , Imagem Óptica/métodos , Transporte Proteico , Vírion/patogenicidade , Internalização do Vírus
16.
Arch Virol ; 164(4): 1233-1244, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30663023

RESUMO

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Mononegavirais/classificação , Mononegavirais/genética , Mononegavirais/isolamento & purificação , Filogenia , Virologia/organização & administração
17.
Virus Evol ; 4(2): vey034, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30524754

RESUMO

Ebola virus (EBOV) disease is a viral hemorrhagic fever with a high case-fatality rate in humans. This disease is caused by four members of the filoviral genus Ebolavirus, including EBOV. The natural hosts reservoirs of ebolaviruses remain to be identified. Glycoprotein 2 of reptarenaviruses, known to infect only boa constrictors and pythons, is similar in sequence and structure to ebolaviral glycoprotein 2, suggesting that EBOV may be able to infect reptilian cells. Therefore, we serially passaged EBOV and a distantly related filovirus, Marburg virus (MARV), in boa constrictor JK cells and characterized viral infection/replication and mutational frequency by confocal imaging and sequencing. We observed that EBOV efficiently infected and replicated in JK cells, but MARV did not. In contrast to most cell lines, EBOV-infected JK cells did not result in an obvious cytopathic effect. Surprisingly, genomic characterization of serial-passaged EBOV in JK cells revealed that genomic adaptation was not required for infection. Deep sequencing coverage (>10,000×) demonstrated the existence of only a single nonsynonymous variant (EBOV glycoprotein precursor pre-GP T544I) of unknown significance within the viral population that exhibited a shift in frequency of at least 10 per cent over six serial passages. In summary, we present the first reptilian cell line that replicates a filovirus at high titers, and for the first time demonstrate a filovirus genus-specific restriction to MARV in a cell line. Our data suggest the possibility that there may be differences between the natural host spectra of ebolaviruses and marburgviruses.

18.
Nat Microbiol ; 3(12): 1486, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30410089

RESUMO

In the version of this Article originally published, the bat species for 12 individuals were incorrectly identified in Supplementary Table 1 and 2. After resequencing the MT-CytB and MT-CO1 segments and reviewing the data, the authors have corrected the errors for these 12 animals. In the amended version of the Supplementary Information, Supplementary Tables 1 and 2 have been replaced to include the corrected host species information. None of the 12 bats affected were positive for the Bombali virus, and the conclusions of the study are therefore unchanged.

19.
Nature ; 563(7732): 559-563, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30464266

RESUMO

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

20.
Front Microbiol ; 9: 2544, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30416492

RESUMO

Conventional biological nitrogen removal (BNR), comprised of nitrification and denitrification, is traditionally employed in wastewater treatment plants (WWTPs) to prevent eutrophication in receiving water bodies. More recently, the combination of selective ammonia to nitrite oxidation (nitritation) and autotrophic anaerobic ammonia oxidation (anammox), collectively termed deammonification, has also emerged as a possible energy- and cost-effective BNR alternative. Herein, we analyzed microbial diversity and functional potential within 13 BNR processes in the United States, Denmark, and Singapore operated with varying reactor configuration, design, and operational parameters. Using next-generation sequencing and metagenomics, gene-coding regions were aligned against a custom protein database expanded to include all published aerobic ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), anaerobic ammonia oxidizing bacteria (AMX), and complete ammonia oxidizing bacteria (CMX). Overall contributions of these N-cycle bacteria to the total functional potential of each reactor was determined, as well as that of several organisms associated with denitrification and/or structural integrity of microbial aggregates (biofilm or granules). The potential for these engineered processes to foster a broad spectrum of microbial catabolic, anabolic, and carbon assimilation transformations was elucidated. Seeded sidestream DEMON® deammonification systems and single-stage nitritation-anammox moving bed biofilm reactors (MBBRs) and a mainstream Cleargreen reactor designed to enrich in AOB and AMX showed lower enrichment in AMX functionality than an enriched two-stage nitritation-anammox MBBR system treating mainstream wastewater. Conventional BNR systems in Singapore and the United States had distinct metagenomes, especially relating to AOB. A hydrocyclone process designed to recycle biomass granules for mainstream BNR contained almost identical structural and functional characteristics in the overflow, underflow, and inflow of mixed liquor (ALT) rather than the expected selective enrichment of specific nitrifying or AMX organisms. Inoculum used to seed a sidestream deammonification process unexpectedly contained <10% of total coding regions assigned to AMX. These results suggest the operating conditions of engineered bioprocesses shape the resident microbial structure and function far more than the bioprocess configuration itself. We also highlight the advantage of a systems- and metagenomics-based interrogation of both the microbial structure and potential function therein over targeting of individual populations or specific genes.

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