RESUMEN
Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were â¼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.
Asunto(s)
Anticuerpos Neutralizantes , Proteínas de la Cápside , Virus de la Hepatitis E , Hepatitis E , Anticuerpos de Dominio Único , Virus de la Hepatitis E/inmunología , Animales , Proteínas de la Cápside/inmunología , Anticuerpos de Dominio Único/inmunología , Humanos , Anticuerpos Neutralizantes/inmunología , Hepatitis E/inmunología , Camelus/inmunología , Epítopos/inmunología , Células Hep G2 , Reacciones Cruzadas/inmunología , Genotipo , Especificidad de Anticuerpos/inmunologíaRESUMEN
Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.
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Infecciones por Circoviridae , Circovirus , Anticuerpos de Dominio Único , Vacunas Virales , Animales , Humanos , Ratones , Proteínas de la Cápside , Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/veterinaria , Epítopos , Porcinos , Vacunas Virales/química , Vacunas Virales/inmunologíaRESUMEN
Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.
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Virus de la Hepatitis E , Hepatitis E , Hepatitis Viral Humana , Humanos , Virus de la Hepatitis E/genética , Factores Inmunológicos , Proteína Disulfuro Isomerasas/genética , Tiorredoxinas/genética , Virión/metabolismoRESUMEN
IMPORTANCE: SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.
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COVID-19 , ARN Polimerasa Dependiente de ARN de Coronavirus , Proteína 2 con Dominio MARVEL , SARS-CoV-2 , Humanos , Inmunidad Adaptativa , COVID-19/virología , Citosol , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Proteína 2 con Dominio MARVEL/genéticaRESUMEN
The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.
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Ebolavirus/genética , Ebolavirus/fisiología , Genoma Viral/genética , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Clima , Brotes de Enfermedades/estadística & datos numéricos , Ebolavirus/aislamiento & purificación , Geografía , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Internacionalidad , Modelos Lineales , Epidemiología Molecular , Filogenia , Viaje/legislación & jurisprudencia , Viaje/estadística & datos numéricosRESUMEN
Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified "early viral" and "late inflammatory" clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.
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COVID-19/inmunología , Regulación de la Expresión Génica/inmunología , Pulmón/inmunología , SARS-CoV-2/inmunología , Bazo/inmunología , Anciano , Anciano de 80 o más Años , Autopsia , Femenino , Humanos , Inflamación/inmunología , Masculino , ProteómicaRESUMEN
BACKGROUND: Waning antibody levels post-vaccination and the emergence of variants of concern (VOCs) capable of evading protective immunity have raised the need for booster vaccinations. However, which combination of coronavirus disease 2019 (COVID-19) vaccines offers the strongest immune response against the Omicron variant is unknown. METHODS: This randomized, participant-blinded, controlled trial assessed the reactogenicity and immunogenicity of different COVID-19 vaccine booster combinations. A total of 100 BNT162b2-vaccinated individuals were enrolled and randomized 1:1 to either homologous (BNT162b2 + BNT162b2 + BNT162b2; "BBB") or heterologous messenger RNA (mRNA) (BNT162b2 + BNT162b2 + mRNA-1273; "BBM") booster vaccine. The primary end point was the level of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and VOCs at day 28. RESULTS: A total of 51 participants were allocated to BBB and 49 to BBM; 50 and 48, respectively, were analyzed for safety and immunogenicity outcomes. At day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBB (22 382â IU/mL; 95% confidence interval [CI], 18 210 to 27 517) vs BBM (29 751â IU/mL; 95% CI, 25 281 to 35 011; P = .034) as was the median level of neutralizing antibodies: BBB 99.0% (interquartile range [IQR], 97.9% to 99.3%) vs BBM 99.3% (IQR, 98.8% to 99.5%; P = .021). On subgroup analysis, significant higher mean spike antibody titer, median surrogate neutralizing antibody level against all VOCs, and live Omicron neutralization titer were observed only in older adults receiving BBM. Both vaccines were well tolerated. CONCLUSIONS: Heterologous mRNA-1273 booster vaccination compared with homologous BNT123b2 induced a stronger neutralizing response against the Omicron variant in older individuals. CLINICAL TRIALS REGISTRATION: NCT05142319.
Asunto(s)
Vacuna BNT162 , COVID-19 , Humanos , Anciano , SARS-CoV-2 , Formación de Anticuerpos , Vacuna nCoV-2019 mRNA-1273 , Vacunación , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.
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Ebolavirus/genética , Evolución Molecular , Ojo/virología , Fiebre Hemorrágica Ebola/fisiopatología , Fiebre Hemorrágica Ebola/virología , Animales , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Ebolavirus/inmunología , Femenino , Hurones/inmunología , Fiebre Hemorrágica Ebola/inmunología , Masculino , ARN Viral/genéticaRESUMEN
The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.
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Antivirales/metabolismo , Quimiocinas/metabolismo , Proteoma/metabolismo , Mucosa Respiratoria/virología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Bronquios/patología , Línea Celular , Niño , Células Epiteliales/patología , Células Epiteliales/virología , Células Caliciformes/metabolismo , Células Caliciformes/virología , Homeostasis , Humanos , Lactante , Cinética , Nasofaringe/virología , Mucosa Respiratoria/metabolismo , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Tropismo , Proteínas Virales/metabolismoRESUMEN
Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.
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COVID-19/inmunología , Inflamación/virología , Pulmón/inmunología , Insuficiencia Multiorgánica/virología , SARS-CoV-2/inmunología , Anciano , Anciano de 80 o más Años , Autopsia , Biopsia , COVID-19/patología , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19 , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inflamación/inmunología , Inflamación/patología , Pulmón/patología , Pulmón/virología , Masculino , Insuficiencia Multiorgánica/inmunología , Insuficiencia Multiorgánica/patología , SARS-CoV-2/patogenicidad , Índice de Severidad de la EnfermedadRESUMEN
SARS-CoV-2 is thought to have originated in the human population from a zoonotic spillover event. Infection in humans results in a variety of outcomes ranging from asymptomatic cases to the disease COVID-19, which can have significant morbidity and mortality, with over two million confirmed deaths worldwide as of January 2021. Over a year into the pandemic, sequencing analysis has shown that variants of SARS-CoV-2 are being selected as the virus continues to circulate widely within the human population. The predominant drivers of genetic variation within SARS-CoV-2 are single nucleotide polymorphisms (SNPs) caused by polymerase error, potential host factor driven RNA modification, and insertion/deletions (indels) resulting from the discontinuous nature of viral RNA synthesis. While many mutations represent neutral 'genetic drift' or have quickly died out, a subset may be affecting viral traits such as transmissibility, pathogenicity, host range, and antigenicity of the virus. In this review, we summarise the current extent of genetic change in SARS-CoV-2, particularly recently emerging variants of concern, and consider the phenotypic consequences of this viral evolution that may impact the future trajectory of the pandemic.
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Adaptación Fisiológica/genética , COVID-19/virología , SARS-CoV-2/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/transmisión , Humanos , Evasión Inmune/genética , Mutación , ARN Viral/biosíntesis , ARN Viral/genética , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Internalización del Virus , Replicación Viral , Zoonosis/transmisión , Zoonosis/virologíaRESUMEN
This article provides a brief overview of drug resistance to antiviral therapy as well as known and emergent variability in key SARS-CoV-2 viral sequences. The purpose is to stimulate deliberation about the need to consider drug resistance prior to widespread roll-out of antivirals for SARS-CoV-2. Many existing candidate agents have mechanisms of action involving drug targets likely to be critical for future drug development. Resistance emerged quickly with monotherapies deployed for other pulmonary viruses such as influenza virus, and in HIV mutations in key drug targets compromised efficacy of multiple drugs within a class. The potential for drug resistance in SARS-CoV-2 has not yet been rigorously debated or assessed, and we call for more academic and industry research on this potentially important future threat prior to widespread roll-out of monotherapies for COVID-19 treatment and prevention.
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Tratamiento Farmacológico de COVID-19 , Infecciones por Coronavirus , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Farmacorresistencia Viral , Humanos , SARS-CoV-2RESUMEN
Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic.
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Ebolavirus/fisiología , Modelos Biológicos , Replicación Viral/fisiología , Animales , Teorema de Bayes , Chlorocebus aethiops , Biología Computacional , Simulación por Computador , Ebolavirus/genética , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/virología , Interacciones Microbiota-Huesped/fisiología , Humanos , Técnicas In Vitro , Cinética , Cadenas de Markov , Método de Montecarlo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero , Carga Viral/fisiologíaRESUMEN
West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.
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Brotes de Enfermedades/estadística & datos numéricos , Ebolavirus/genética , Evolución Molecular , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Filogenia , Análisis Espacio-Temporal , Sustitución de Aminoácidos/genética , Ebolavirus/aislamiento & purificación , Femenino , Guinea/epidemiología , Fiebre Hemorrágica Ebola/transmisión , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Liberia/epidemiología , Masculino , Malí/epidemiología , Datos de Secuencia Molecular , Sierra Leona/epidemiologíaRESUMEN
Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.
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Hepevirus/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Aniones/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Pollos/virología , Hepatitis E/virología , Virus de la Hepatitis E/metabolismo , Hepatitis Viral Animal/virología , Hepevirus/fisiología , Péptidos/metabolismo , Enfermedades de las Aves de Corral/virología , Proteínas Virales/metabolismoRESUMEN
Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.
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Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Epítopos de Linfocito B/inmunología , Anticuerpos Antihepatitis/inmunología , Virus de la Hepatitis E/inmunología , Hepatitis E/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Epítopos de Linfocito B/genética , Células Hep G2 , Hepatitis E/genética , Hepatitis E/virología , Virus de la Hepatitis E/genética , Humanos , Homología de Secuencia , PorcinosRESUMEN
Differences in T-cell phenotype, particularly the expression of markers of T-cell homeostasis, have been observed in fatal and nonfatal Ebola virus disease (EVD). However, the relationship between these markers with T-cell function and virus clearance during EVD is poorly understood. To gain biological insight into the role of T cells during EVD, combined transcriptomics and T-cell receptor sequencing was used to profile blood samples from fatal and nonfatal EVD patients from the recent West African EVD epidemic. Fatal EVD was characterized by strong T-cell activation and increased abundance of T-cell inhibitory molecules. However, the early T-cell response was oligoclonal and did not result in viral clearance. In contrast, survivors mounted highly diverse T-cell responses, maintained low levels of T-cell inhibitors, and cleared Ebola virus. Our findings highlight the importance of T-cell immunity in surviving EVD and strengthen the foundation for further research on targeting of the dendritic cell-T cell interface for postexposure immunotherapy.
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Fiebre Hemorrágica Ebola/inmunología , Homeostasis , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Biomarcadores , Estudios Transversales , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/mortalidad , Humanos , TranscriptomaRESUMEN
Porcine reproductive and respiratory syndrome virus(PRRSV) is an important immunosuppressive virus which can suppresses infected cells proliferation. In this work, we examined PRRSV ability to manipulate cell cycle progression of MARC-145 cells and explored the potential molecular mechanisms. The results showed that PRRSV infection imposed a growth-inhibitory effect on MARC-145 cells by inducing cell cycle arrest at G2/M phase. This arrest was due to the significant decrease of Cdc2-cyclinB1 complex activity in PRRSV-infected cells and the activity reduction was a result of Cdc2 Tyr15 phosphorylation and the accumulation of Cdc2 and cyclinB1 in the nucleus. Not only elevated Wee1 and Myt1 expression and inactivated Cdc25C, but also increase of p21 and 14-3-3σ in a p53-dependent manner caused the inhibitory Tyr15 phosphorylation of Cdc2. PRRSV infection also activated Chk1. Our data suggest PRRSV infection induces G2/M arrest via various molecular regulatory mechanisms. These results provide a new insights for PRRSV pathogenesis.
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Apoptosis , Puntos de Control del Ciclo Celular , Proliferación Celular , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Transducción de Señal , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Fosforilación , Porcinos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BackgroundThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. AimTo investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.MethodsWe performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.ResultsDirect metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. ConclusionsThis work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.
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Virus Chikungunya/genética , Virus del Dengue/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuenciación Completa del Genoma , Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Fiebre Chikungunya/sangre , Fiebre Chikungunya/diagnóstico , Virus Chikungunya/aislamiento & purificación , Dengue/sangre , Dengue/diagnóstico , Virus del Dengue/aislamiento & purificación , Humanos , Metagenómica , Nanoporos , SerogrupoRESUMEN
UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. IMPORTANCE: It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.