RESUMO
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
Assuntos
Infecções por Flavivirus/genética , Flavivirus/fisiologia , Proteínas de Membrana/metabolismo , Animais , Povo Asiático/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Sistemas CRISPR-Cas , Linhagem Celular , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/fisiologia , Replicação Viral , Vírus da Febre Amarela/fisiologia , Zika virus/fisiologiaRESUMO
Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Betacoronavirus/enzimologia , Ebolavirus/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Betacoronavirus/química , Linhagem Celular , Tolerância a Medicamentos/genética , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Humanos , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action. The SARS-CoV-2 Delta (B.1.617.2 and AY sublineages) variant rose from 1% to >50% of viral lineages circulating nationally during 8 weeks, from May 1-June 26, 2021. Delta-associated infections remained predominant until being rapidly overtaken by infections associated with the Omicron (B.1.1.529 and BA sublineages) variant in December 2021, when Omicron increased from 1% to >50% of circulating viral lineages during a 2-week period. As of the week ending January 22, 2022, Omicron was estimated to account for 99.2% (95% CI = 99.0%-99.5%) of SARS-CoV-2 infections nationwide, and Delta for 0.7% (95% CI = 0.5%-1.0%). The dynamic landscape of SARS-CoV-2 variants in 2021, including Delta- and Omicron-driven resurgences of SARS-CoV-2 transmission across the United States, underscores the importance of robust genomic surveillance efforts to inform public health planning and practice.
Assuntos
COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/genética , Centers for Disease Control and Prevention, U.S. , Genômica , Humanos , Prevalência , Vigilância em Saúde Pública/métodos , Estados Unidos/epidemiologiaRESUMO
During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.
Assuntos
Surtos de Doenças , Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Serviços de Laboratório Clínico , Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Humanos , Laboratórios , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Serra Leoa/epidemiologiaRESUMO
In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.
Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Centers for Disease Control and Prevention, U.S. , Surtos de Doenças , Epidemias , Humanos , Laboratórios , Serra Leoa/epidemiologia , Estados UnidosRESUMO
Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Koch's postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.
Assuntos
Infecções por Bunyaviridae/virologia , Phlebovirus/classificação , Idoso , Animais , Anticorpos Antivirais/sangue , Medula Óssea/virologia , Febre/etiologia , Genoma Viral , Humanos , Imunoglobulina A/sangue , Leucócitos/virologia , Masculino , Pessoa de Meia-Idade , Missouri , Phlebovirus/genética , Phlebovirus/isolamento & purificação , Filogenia , RNA Viral/análise , Doenças Transmitidas por Carrapatos/virologiaRESUMO
No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2'-C-methylcytidine (2'-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2'-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2'-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2'-CMC, AHFV variants with reduced susceptibility to 2'-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2'-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2'-CMC.
Assuntos
Antivirais/farmacologia , Flavivirus/efeitos dos fármacos , Febres Hemorrágicas Virais/virologia , Doenças Transmitidas por Carrapatos/tratamento farmacológico , Doenças Transmitidas por Carrapatos/virologia , Substituição de Aminoácidos , Linhagem Celular , Citidina/análogos & derivados , Citidina/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Farmacorresistência Viral , Flavivirus/genética , Humanos , Modelos Moleculares , Mutação/genética , Replicação Viral/efeitos dos fármacosRESUMO
Viruses in the Ebolavirus and Marburgvirus genera (family Filoviridae) have been associated with large outbreaks of hemorrhagic fever in human and nonhuman primates. The first documented cases occurred in primates over 45 years ago, but the amount of virus genetic diversity detected within bat populations, which have recently been identified as potential reservoir hosts, suggests that the filoviruses are much older. Here, detailed Bayesian coalescent phylogenetic analyses are performed on 97 whole-genome sequences, 55 of which are newly reported, to comprehensively examine molecular evolutionary rates and estimate dates of common ancestry for viruses within the family Filoviridae. Molecular evolutionary rates for viruses belonging to different species range from 0.46 × 10(-4) nucleotide substitutions/site/year for Sudan ebolavirus to 8.21 × 10(-4) nucleotide substitutions/site/year for Reston ebolavirus. Most recent common ancestry can be traced back only within the last 50 years for Reston ebolavirus and Zaire ebolavirus species and suggests that viruses within these species may have undergone recent genetic bottlenecks. Viruses within Marburg marburgvirus and Sudan ebolavirus species can be traced back further and share most recent common ancestors approximately 700 and 850 years before the present, respectively. Examination of the whole family suggests that members of the Filoviridae, including the recently described Lloviu virus, shared a most recent common ancestor approximately 10,000 years ago. These data will be valuable for understanding the evolution of filoviruses in the context of natural history as new reservoir hosts are identified and, further, for determining mechanisms of emergence, pathogenicity, and the ongoing threat to public health.
Assuntos
Ebolavirus/genética , Genoma Viral , Doença pelo Vírus Ebola/genética , Doença do Vírus de Marburg/genética , Marburgvirus/genética , Substituição de Aminoácidos , Animais , Sequência de Bases , Quirópteros/virologia , Ebolavirus/classificação , Evolução Molecular , Variação Genética , Doença pelo Vírus Ebola/epidemiologia , Humanos , Doença do Vírus de Marburg/epidemiologia , Marburgvirus/classificação , Dados de Sequência Molecular , Filogenia , Primatas/virologia , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
There are no approved vaccines or therapeutics for Lassa virus (LASV) infections. To identify compounds with anti-LASV activity, we conducted a cell-based screening campaign at biosafety level 4 and tested almost 60,000 compounds for activity against an infectious reporter LASV. Hits from this screen included several structurally related macrocycles. The most potent, Mac128, had a sub-micromolar EC50 against the reporter virus, inhibited wild-type clade IV LASV, and reduced viral titers by 4 orders of magnitude. Mechanistic studies suggested that Mac128 inhibited viral replication at the level of the polymerase.
Assuntos
Antivirais , Vírus Lassa , Compostos Macrocíclicos , Replicação Viral , Vírus Lassa/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/química , Replicação Viral/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Compostos Macrocíclicos/química , Humanos , Animais , Chlorocebus aethiops , Células Vero , Febre Lassa/virologia , Febre Lassa/tratamento farmacológico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/genéticaRESUMO
Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6â%. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.
RESUMO
During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.
Assuntos
Viroses/diagnóstico , Vírus/isolamento & purificação , Vírus/ultraestrutura , Arenaviridae/isolamento & purificação , Arenaviridae/ultraestrutura , Bunyaviridae/isolamento & purificação , Bunyaviridae/ultraestrutura , Técnicas de Cultura de Células , Coronaviridae/isolamento & purificação , Coronaviridae/ultraestrutura , Flaviviridae/isolamento & purificação , Flaviviridae/ultraestrutura , Humanos , Microscopia Eletrônica , Paramyxoviridae/isolamento & purificação , Paramyxoviridae/ultraestrutura , Estados Unidos/epidemiologia , Viroses/epidemiologia , Viroses/virologiaRESUMO
Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3' terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.
Assuntos
Arenavirus/genética , RNA Viral/genética , Regiões 3' não Traduzidas , Animais , Arenavirus/fisiologia , Sequência de Bases , Cricetinae , DNA Complementar/metabolismo , Genes Reporter , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleotídeos/genética , Plasmídeos/metabolismo , Vírus de RNA/genética , África do Sul , Fatores de Tempo , Virulência , ZâmbiaRESUMO
Two large outbreaks of Ebola hemorrhagic fever occurred in Uganda in 2000 and 2007. In May 2011, we identified a single case of Sudan Ebola virus disease in Luwero District. The establishment of a permanent in-country laboratory and cooperation between international public health entities facilitated rapid outbreak response and control activities.
Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Surtos de Doenças , Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Criança , Doenças Transmissíveis Emergentes/diagnóstico , Ebolavirus/classificação , Ebolavirus/isolamento & purificação , Monitoramento Epidemiológico , Evolução Fatal , Feminino , Doença pelo Vírus Ebola/diagnóstico , Humanos , Uganda/epidemiologiaRESUMO
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Testes de Neutralização , Pandemias , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Vacinas Sintéticas , Vacinas de mRNARESUMO
Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.
Assuntos
Arenavirus do Velho Mundo/genética , Arenavirus do Velho Mundo/isolamento & purificação , Especiação Genética , África Austral/epidemiologia , Infecções por Arenaviridae/mortalidade , Infecções por Arenaviridae/transmissão , Infecções por Arenaviridae/virologia , Sequência de Bases , Infecção Hospitalar , Genoma Viral , Humanos , Filogenia , RNA Viral/genética , Proteínas ViraisRESUMO
SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/sangue , SARS-CoV-2/isolamento & purificação , Animais , Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , COVID-19/imunologia , Teste Sorológico para COVID-19/economia , Teste Sorológico para COVID-19/métodos , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos , SARS-CoV-2/imunologiaRESUMO
The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core), J6/JFH (2a core), and H77/JFH (1a core), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.
Assuntos
Hepacivirus/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas do Core Viral/biossíntese , Códon de Iniciação , Hepacivirus/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Proteínas do Core Viral/genéticaRESUMO
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.
RESUMO
A nosocomial outbreak of disease involving 5 patients, 4 of whom died, occurred in South Africa during September-October 2008. The first patient had been transferred from Zambia to South Africa for medical management. Three cases involved secondary spread of infection from the first patient, and 1 was a tertiary infection. A novel arenavirus was identified. The source of the first patient's infection remains undetermined.
Assuntos
Infecções por Arenaviridae/epidemiologia , Arenavirus/genética , Infecção Hospitalar/epidemiologia , Febres Hemorrágicas Virais/epidemiologia , Febres Hemorrágicas Virais/virologia , Adulto , Antivirais/uso terapêutico , Arenavirus/classificação , Busca de Comunicante , Surtos de Doenças , Evolução Fatal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ribavirina/uso terapêutico , Zâmbia/epidemiologiaRESUMO
There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, which encodes the α and ß subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.