RESUMO
In a pattern repeated across a range of ecological niches, arenaviruses have evolved a compact four-gene genome to orchestrate a complex life cycle in a narrow range of susceptible hosts. A number of mammalian arenaviruses cross-infect humans, often causing a life-threatening viral hemorrhagic fever. Among this group of geographically bound zoonoses, Lassa virus has evolved a unique niche that leads to significant and sustained human morbidity and mortality. As a biosafety level 4 pathogen, direct study of the pathogenesis of Lassa virus is limited by the sparse availability, high operating costs, and technical restrictions of the high-level biocontainment laboratories required for safe experimentation. In this chapter, we introduce the relationship between genome structure and the life cycle of Lassa virus and outline reverse genetic approaches used to probe and describe functional elements of the Lassa virus genome. We then review the tools used to obtain viral genomic sequences used for phylogeny and molecular diagnostics, before shifting to a population perspective to assess the contributions of phylogenetic analysis in understanding the evolution and ecology of Lassa virus in West Africa. We finally consider the future outlook and clinical applications for genetic study of Lassa virus.
Assuntos
Febre Lassa , Vírus Lassa , Animais , Humanos , Vírus Lassa/genética , Febre Lassa/epidemiologia , Febre Lassa/genética , Filogenia , África Ocidental/epidemiologia , Zoonoses , MamíferosRESUMO
During 2018, an unusual increase in Lassa fever cases occurred in Nigeria, raising concern among national and international public health agencies. We analyzed 220 Lassa virus genomes from infected patients, including 129 from the 2017-2018 transmission season, to understand the viral populations underpinning the increase. A total of 14 initial genomes from 2018 samples were generated at Redeemer's University in Nigeria, and the findings were shared with the Nigerian Center for Disease Control in real time. We found that the increase in cases was not attributable to a particular Lassa virus strain or sustained by human-to-human transmission. Instead, the data were consistent with ongoing cross-species transmission from local rodent populations. Phylogenetic analysis also revealed extensive viral diversity that was structured according to geography, with major rivers appearing to act as barriers to migration of the rodent reservoir.
Assuntos
Genoma Viral , Febre Lassa/virologia , Vírus Lassa/genética , RNA Viral/análise , Adolescente , Adulto , Animais , Teorema de Bayes , Reservatórios de Doenças , Feminino , Variação Genética , Humanos , Febre Lassa/epidemiologia , Febre Lassa/transmissão , Masculino , Cadeias de Markov , Pessoa de Meia-Idade , Nigéria/epidemiologia , Filogenia , Filogeografia , Roedores , Análise de Sequência de RNA , Zoonoses/transmissãoAssuntos
Febre Lassa/epidemiologia , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/virologia , Adolescente , Adulto , Gerenciamento Clínico , Feminino , Idade Gestacional , Humanos , Febre Lassa/diagnóstico , Febre Lassa/tratamento farmacológico , Vírus Lassa , Nigéria/epidemiologia , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Resultado da Gravidez , Vigilância em Saúde Pública , Estudos Retrospectivos , Índice de Gravidade de Doença , Avaliação de Sintomas , Adulto JovemRESUMO
Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.
Assuntos
Febre Lassa , Humanos , Febre Lassa/genética , Febre Lassa/diagnóstico , Febre Lassa/epidemiologia , Estudo de Associação Genômica Ampla , Estudos Soroepidemiológicos , Vírus Lassa/genética , Febre , Genética HumanaRESUMO
Effective infectious disease surveillance in high-risk regions is critical for clinical care and pandemic preemption; however, few clinical diagnostics are available for the wide range of potential human pathogens. Here, we conduct unbiased metagenomic sequencing of 593 samples from febrile Nigerian patients collected in three settings: i) population-level surveillance of individuals presenting with symptoms consistent with Lassa Fever (LF); ii) real-time investigations of outbreaks with suspected infectious etiologies; and iii) undiagnosed clinically challenging cases. We identify 13 distinct viruses, including the second and third documented cases of human blood-associated dicistrovirus, and a highly divergent, unclassified dicistrovirus that we name human blood-associated dicistrovirus 2. We show that pegivirus C is a common co-infection in individuals with LF and is associated with lower Lassa viral loads and favorable outcomes. We help uncover the causes of three outbreaks as yellow fever virus, monkeypox virus, and a noninfectious cause, the latter ultimately determined to be pesticide poisoning. We demonstrate that a local, Nigerian-driven metagenomics response to complex public health scenarios generates accurate, real-time differential diagnoses, yielding insights that inform policy.
Assuntos
Febre Lassa , Vírus , Humanos , Nigéria/epidemiologia , Metagenômica , Febre Lassa/diagnóstico , Febre Lassa/epidemiologia , Vírus Lassa/genética , Vírus/genéticaRESUMO
Developing and deploying new diagnostic tests are difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important. During a pandemic, laboratories play a key role in helping healthcare providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, PCR remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular-based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions.
Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , Serviço Hospitalar de Emergência , Laboratórios Hospitalares , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , COVID-19/virologia , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
Haptic perception is an active process that provides an awareness of objects that are encountered as an organism scans its environment. In contrast to the sensation of touch produced by contact with an object, the perception of object location arises from the interpretation of tactile signals in the context of the changing configuration of the body. A discrete sensory representation and a low number of degrees of freedom in the motor plant make the ethologically prominent rat vibrissa system an ideal model for the study of the neuronal computations that underlie this perception. We found that rats with only a single vibrissa can combine touch and movement to distinguish the location of objects that vary in angle along the sweep of vibrissa motion. The patterns of this motion and of the corresponding behavioral responses show that rats can scan potential locations and decide which location contains a stimulus within 150 ms. This interval is consistent with just one to two whisk cycles and provides constraints on the underlying perceptual computation. Our data argue against strategies that do not require the integration of sensory and motor modalities. The ability to judge angular position with a single vibrissa thus connects previously described, motion-sensitive neurophysiological signals to perception in the behaving animal.
Assuntos
Comportamento Animal/fisiologia , Atividade Motora/fisiologia , Percepção Espacial/fisiologia , Vibrissas/fisiologia , Animais , Feminino , Ratos , Ratos Long-Evans , Fatores de TempoRESUMO
Jamestown Canyon virus (JCV) is a neuroinvasive arbovirus that is found throughout North America and increasingly recognized as a public health concern. From 2004 to 2012, an average of 1.7 confirmed cases were reported annually in the United States, whereas from 2013 to 2018 this figure increased over seventeen-fold to 29.2 cases per year. The rising number of reported human infections highlights the need for better understanding of the clinical manifestations and epidemiology of JCV. Here, we describe nine patients diagnosed with neuroinvasive JCV infection in Massachusetts from 2013, the year of the first reported case in the state, to 2017. Because current diagnostic testing relies on serology, which is complicated by cross-reactivity with related orthobunyaviruses and can be negative in immunosuppressed patients, we developed and evaluated an RT-qPCR assay for detection of JCV RNA. We tested this on the available archived serum from two patients, but did not detect viral RNA. JCV is transmitted by multiple mosquito species and its primary vector in Massachusetts is unknown, so we additionally applied the RT-qPCR assay and confirmatory RNA sequencing to assess JCV prevalence in a vector candidate, Ochlerotatus canadensis. We identified JCV in 0.6% of mosquito pools, a similar prevalence to neighboring Connecticut. We assembled the first Massachusetts JCV genome directly from a mosquito sample, finding high identity to JCV isolates collected over a 60-year period. Further studies are needed to reconcile the low vector prevalence and low rate of viral evolutionary change with the increasing number of reported cases.
Assuntos
Culicidae/virologia , Vírus da Encefalite da Califórnia , Encefalite/virologia , Meningite/virologia , Ochlerotatus/virologia , Adulto , Idoso , Animais , Vetores de Doenças , Encefalite/diagnóstico , Vírus da Encefalite da Califórnia/genética , Vírus da Encefalite da Califórnia/imunologia , Vírus da Encefalite da Califórnia/isolamento & purificação , Feminino , Genoma Viral , Humanos , Masculino , Massachusetts/epidemiologia , Meningite/diagnóstico , Pessoa de Meia-Idade , Mosquitos Vetores/virologia , Filogenia , Prevalência , RNA ViralRESUMO
Fifty patients with unexplained fever and poor outcomes presented at Irrua Specialist Teaching Hospital (ISTH) in Edo State, Nigeria, an area endemic for Lassa fever, between September 2018 - January 2019. After ruling out Lassa fever, plasma samples from these epidemiologically-linked cases were sent to the African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer's University, Ede, Osun State, Nigeria, where we carried out metagenomic sequencing which implicated yellow fever virus (YFV) as the etiology of this outbreak. Twenty-nine of the 50 samples were confirmed positive for YFV by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), 14 of which resulted in genome assembly. Maximum likelihood phylogenetic analysis revealed that these YFV sequences formed a tightly clustered clade more closely related to sequences from Senegal than sequences from earlier Nigerian isolates, suggesting that the YFV clade responsible for this outbreak in Edo State does not descend directly from the Nigerian YFV outbreaks of the last century, but instead reflects a broader diversity and dynamics of YFV in West Africa. Here we demonstrate the power of metagenomic sequencing for identifying ongoing outbreaks and their etiologies and informing real-time public health responses, resulting in accurate and prompt disease management and control.
Assuntos
Sistemas Computacionais , Surtos de Doenças , Metagenoma , Doenças não Diagnosticadas/epidemiologia , Doenças não Diagnosticadas/genética , Febre Amarela/epidemiologia , Febre Amarela/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Funções Verossimilhança , Masculino , Pessoa de Meia-Idade , Nigéria/epidemiologia , Filogenia , Doenças não Diagnosticadas/virologia , Febre Amarela/virologia , Adulto JovemRESUMO
Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.
RESUMO
Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.
Assuntos
Anticorpos Antivirais/metabolismo , Testes Diagnósticos de Rotina/métodos , Febre Lassa/diagnóstico , Vírus Lassa/isolamento & purificação , RNA Viral/genética , Adulto , Antígenos Virais/imunologia , Surtos de Doenças , Feminino , Humanos , Vírus Lassa/genética , Vírus Lassa/imunologia , Masculino , Pessoa de Meia-Idade , Nigéria , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Análise de Sequência de RNA , Adulto JovemRESUMO
Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK's use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.
Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/diagnóstico , Febre Lassa/diagnóstico , Vírus Lassa/patogenicidade , Anticorpos Antivirais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ebolavirus/genética , Doença pelo Vírus Ebola/virologia , Febre Lassa/virologia , Vírus Lassa/genéticaRESUMO
How are two prominent environmental features, surface texture and object location, transduced and encoded as rats whisk? Recent papers show that textures may excite intrinsic mechanical vibrations of the vibrissae. Although these vibrations are too rapid to be directly followed by cortical neurons, there is evidence that their speed is encoded by contact-dependent sensory signals. In addition to contact, sensory signals exist that report the angular position of the vibrissae. The combination of contact and reference signals may be used to decode spatial variations in the environment, particularly the location of objects in head-centered coordinates.
Assuntos
Neurônios Aferentes/fisiologia , Vibrissas/inervação , Vias Aferentes/fisiologia , Animais , Discriminação Psicológica , Estimulação Física , Ratos , Propriedades de SuperfícieRESUMO
To explore mechanisms governing the formation, stability, and elimination of synapses during neuronal development, we used FM 1-43 fluorescence imaging to track vesicle turnover at >7000 individually identified developing synapses between embryonic rat hippocampal neurons in culture. The majority of presynaptic boutons were stable in efficacy and position over a period of 1.5 hr. Activity, evoked by burst-patterned field stimulation, decreased presynaptic function across the population of boutons, an effect that required NMDA receptor activation. Decreased FM 1-43 staining correlated with low synapsin-I and synaptophysin immunoreactivities, suggesting that decreased presynaptic function was commensurate with synaptic disassembly. These observations provide new information on the stability of developing presynaptic function and suggest that NMDA receptor activation may regulate the stability of developing synapses.
Assuntos
Hipocampo/ultraestrutura , Plasticidade Neuronal/fisiologia , Sinapses/ultraestrutura , Animais , Células Cultivadas , Estimulação Elétrica , Corantes Fluorescentes , Hipocampo/embriologia , Hipocampo/metabolismo , Imuno-Histoquímica , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismoRESUMO
Chronux is an open-source software package developed for the analysis of neural data. The current version of Chronux includes software for signal processing of neural time-series data including several specialized mini-packages for spike-sorting, local regression, audio segmentation, and other data-analysis tasks typically encountered by a neuroscientist. Chronux is freely available along with user tutorials, sample data, and extensive documentation from http://chronux.org/.
Assuntos
Potenciais de Ação/fisiologia , Neurônios/fisiologia , Software , Estatística como Assunto/métodos , Animais , Humanos , Funções Verossimilhança , Análise de Regressão , Análise EspectralRESUMO
Leaf stomata close in response to high carbon dioxide levels and open at low CO(2). CO(2) concentrations in leaves are altered by daily dark/light cycles, as well as the continuing rise in atmospheric CO(2). Relative to abscisic acid and blue light signaling, little is known about the molecular, cellular, and genetic mechanisms of CO(2) signaling in guard cells. Interestingly, we report that repetitive Ca(2+) transients were observed during the stomatal opening stimulus, low [CO(2)]. Furthermore, low/high [CO(2)] transitions modulated the cytosolic Ca(2+) transient pattern in Arabidopsis guard cells (Landsberg erecta). Inhibition of cytosolic Ca(2+) transients, achieved by loading guard cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and not adding external Ca(2+), attenuated both high CO(2)-induced stomatal closing and low CO(2)-induced stomatal opening, and also revealed a Ca(2+)-independent phase of the CO(2) response. Furthermore, the mutant, growth controlled by abscisic acid (gca2) shows impairment in [CO(2)] modulation of the cytosolic Ca(2+) transient rate and strong impairment in high CO(2)-induced stomatal closing. Our findings provide insights into guard cell CO(2) signaling mechanisms, reveal Ca(2+)-independent events, and demonstrate that calcium elevations can participate in opposed signaling events during stomatal opening and closing. A model is proposed in which CO(2) concentrations prime Ca(2+) sensors, which could mediate specificity in Ca(2+) signaling.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Cálcio/metabolismo , Dióxido de Carbono/metabolismo , Transdução de Sinais , Ácido Abscísico/farmacologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Dióxido de Carbono/farmacologia , Citosol/metabolismo , Mutação/genéticaRESUMO
The discovery of a postsynaptically expressed form of cerebellar parallel fiber-Purkinje cell long-term potentiation (LTP) raises the question whether this is the long-sought resetting mechanism for long-term depression (LTD). Extracellular monitoring of PC spikes enables stable prolonged recordings of parallel fiber-Purkinje cell synaptic efficacy. LTD, saturated by repeated induction protocols, can be reversed by a single round of postsynaptic LTP or nitric oxide (NO), enabling LTD to be reinduced. Conversely, after postsynaptic LTP has been saturated, one round of LTD permits fresh postsynaptic LTP. By contrast, after saturation of LTD, induction of presynaptic LTP or application of forskolin leaves LTD still saturated. Likewise, presynaptic LTP cannot be reversed by LTD. Therefore postsynaptic LTP mediated by NO without postsynaptic Ca2+ elevation, unlike presynaptic LTP mediated by cAMP, is a true counterbalance to LTD mediated by coincidence of NO plus postsynaptic Ca2+