Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.367
Filtrar
1.
Front Immunol ; 11: 565278, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013929

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. For better understanding the viral spike (S) protein variations and its potential effects on the interaction with the host immune system and also in vaccine development, the cell epitopes, glycosylation profile and their changes during the global transmission course were characterized and compared with SARS-CoV for their glycosylation profile. We analyzed totally 7,813 sequences screened from 8,897 whole genome sequences on GISAID database up to April 26, and 18 S protein amino acid variations with relatively high frequency (≥10-3) were identified. A total of 228 sequences of variants had multiple variations, of note, most of them harboring the D614G mutation. Among the predicted 69 linear B cell epitopes, 175 discontinuous B cell epitopes and 41 cytotoxic T lymphocyte epitopes in the viral S protein, we found that the protein structure and its potential function of some sites changed, such as the linear epitope length shortened and discontinuous epitope disappeared of G476S. In addition, we detected 9 predicted N-glycosylation sites and 3 O-glycosylation sites unique to SARS-CoV-2, but no evidently observed variation of the glycan sites so far. Our findings provided an important snapshot of temporal and geographical distributions on SARS-CoV-2 S protein cell epitopes and glycosylation sites, which would be an essential basis for the selection of vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Pandemias , Pneumonia Viral/virologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Sequenciamento Completo do Genoma
2.
Curr Biol ; 30(19): R1124-R1130, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33022254

RESUMO

Since the first recognition that infectious microbes serve as the causes of many human diseases, physicians and scientists have sought to understand and control their spread. For the past 150+ years, these 'microbe hunters' have learned to combine epidemiological information with knowledge of the infectious agent(s). In this essay, I reflect on the evolution of microbe hunting, beginning with the history of pre-germ theory epidemiological studies, through the microbiological and molecular eras. Now in the genomic age, modern-day microbe hunters are combining pathogen whole-genome sequencing with epidemiological data to enhance epidemiological investigations, advance our understanding of the natural history of pathogens and drivers of disease, and ultimately reshape our plans and priorities for global disease control and eradication. Indeed, as we have seen during the ongoing Covid-19 pandemic, the role of microbe hunters is now more important than ever. Despite the advances already made by microbial genomic epidemiology, the field is still maturing, with many more exciting developments on the horizon.


Assuntos
Bactérias/genética , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/epidemiologia , Epidemiologia Molecular/métodos , Prevenção Primária/métodos , Bactérias/patogenicidade , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Genoma Bacteriano/genética , Genoma Viral/genética , História do Século XIX , História do Século XX , Humanos , Microbiota/genética , Pandemias , Pneumonia Viral/epidemiologia
3.
JAMA Netw Open ; 3(10): e2024191, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026453

RESUMO

Importance: In late December 2019, an outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Data on the routes of transmission to Los Angeles, California, the US West Coast epicenter for coronavirus disease 2019 (COVID-19), and subsequent community spread are limited. Objective: To determine the transmission routes of SARS-CoV-2 to Southern California and elucidate local community spread within the Los Angeles metropolitan area. Design, Setting, and Participants: This case series included 192 consecutive patients with reverse transcription-polymerase chain reaction (RT-PCR) test results positive for SARS-CoV-2 who were evaluated at Cedars-Sinai Medical Center in Los Angeles, California, from March 22 to April 15, 2020. Data analysis was performed from April to May 2020. Main Outcomes and Measures: SARS-CoV-2 viral genomes were sequenced. Los Angeles isolates were compared with genomes from global subsampling and from New York, New York; Washington state; and China to determine potential sources of viral dissemination. Demographic data and outcomes were collected. Results: The cohort included 192 patients (median [interquartile range] age, 59.5 [43-75] years; 110 [57.3%] men). The genetic characterization of SARS-CoV-2 isolates in the Los Angeles population pinpointed community transmission of 13 patients within a 3.81 km2 radius. Variation landscapes of this case series also revealed a cluster of 10 patients that contained 5 residents at a skilled nursing facility, 1 resident of a nearby skilled nursing facility, 3 health care workers, and a family member of a resident of one of the skilled nursing facilities. Person-to-person transmission was detected in a cluster of 5 patients who shared the same single-nucleotide variation in their SARS-CoV-2 genomes. High viral genomic diversity was identified: 20 Los Angeles isolates (15.0%) resembled SARS-CoV-2 genomes from Asia, while 109 Los Angeles isolates (82.0%) were similar to isolates originating from Europe. Analysis of other common respiratory viral pathogens did not reveal coinfection in the cohort. Conclusions and Relevance: These findings highlight the precision of detecting person-to-person transmission and accurate contact tracing directly through SARS-CoV-2 genome isolation and sequencing. Development and application of phylogenetic analyses from the Los Angeles population established connections between COVID-19 clusters locally and throughout the US.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Pneumonia Viral/transmissão , Adulto , Idoso , Ásia , California/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Europa (Continente) , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Los Angeles/epidemiologia , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Análise de Sequência de RNA , Proteínas não Estruturais Virais/genética , Washington
4.
PLoS Pathog ; 16(9): e1008758, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881980

RESUMO

The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (ß-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of ß-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of ß-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations.


Assuntos
Animais Selvagens/virologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Animais , Quirópteros/virologia , Genoma Viral/genética , Especificidade de Hospedeiro/fisiologia , Humanos , Pandemias
6.
MMWR Morb Mortal Wkly Rep ; 69(37): 1288-1295, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32966272

RESUMO

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), can spread rapidly in high-risk congregate settings such as skilled nursing facilities (SNFs) (1). In Minnesota, SNF-associated cases accounted for 3,950 (8%) of 48,711 COVID-19 cases reported through July 21, 2020; 35% of SNF-associated cases involved health care personnel (HCP*), including six deaths. Facility-wide, serial testing in SNFs has been used to identify residents with asymptomatic and presymptomatic SARS-CoV-2 infection to inform mitigation efforts, including cohorting of residents with positive test results and exclusion of infected HCP from the workplace (2,3). During April-June 2020, the Minnesota Department of Health (MDH), with CDC assistance, conducted weekly serial testing at two SNFs experiencing COVID-19 outbreaks. Among 259 tested residents, and 341 tested HCP, 64% and 33%, respectively, had positive reverse transcription-polymerase chain reaction (RT-PCR) SARS-CoV-2 test results. Continued SARS-CoV-2 transmission was potentially facilitated by lapses in infection prevention and control (IPC) practices, up to 12-day delays in receiving HCP test results (53%) at one facility, and incomplete HCP participation (71%). Genetic sequencing demonstrated that SARS-CoV-2 viral genomes from HCP and resident specimens were clustered by facility, suggesting facility-based transmission. Residents and HCP working in SNFs are at risk for infection with SARS-CoV-2. As part of comprehensive COVID-19 preparation and response, including early identification of cases, SNFs should conduct serial testing of residents and HCP, maximize HCP testing participation, ensure availability of personal protective equipment (PPE), and enhance IPC practices† (4-5).


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Instituições de Cuidados Especializados de Enfermagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Feminino , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Pandemias , Medição de Risco , Sequenciamento Completo do Genoma , Adulto Jovem
7.
mBio ; 11(5)2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887735

RESUMO

In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China, causing severe morbidity and mortality. Since then, the virus has swept across the globe, causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, the Arizona Department of Health Services, and those collected as part of community surveillance projects at Arizona State University and the University of Arizona. Phylogenetic analysis of 84 genomes from across Arizona revealed a minimum of 11 distinct introductions inferred to have occurred during February and March. We show that >80% of our sequences descend from strains that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related case in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020.IMPORTANCE As the COVID-19 pandemic swept across the United States, there was great differential impact on local and regional communities. One of the earliest and hardest hit regions was in New York, while at the same time Arizona (for example) had low incidence. That situation has changed dramatically, with Arizona now having the highest rate of disease increase in the country. Understanding the roots of the pandemic during the initial months is essential as the pandemic continues and reaches new heights. Genomic analysis and phylogenetic modeling of SARS-COV-2 in Arizona can help to reconstruct population composition and predict the earliest undetected introductions. This foundational work represents the basis for future analysis and understanding as the pandemic continues.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Arizona/epidemiologia , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Evolução Molecular , Genoma Viral/genética , Humanos , Incidência , Mutação , Pandemias , Filogenia , Pneumonia Viral/virologia , Proteínas Virais/genética
8.
mSphere ; 5(5)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968009

RESUMO

Bats are the reservoir for a large number of zoonotic viruses, including members of Coronaviridae (severe acute respiratory syndrome coronavirus [SARS-CoV] and SARS-CoV-2), Paramyxoviridae (Hendra and Nipah viruses), Rhabdoviridae (rabies virus), and Filoviridae (Ebola virus) as exemplars. Many retroviruses, such as human immunodeficiency virus, are similarly zoonotic; however, only infectious exogenous gammaretroviruses have recently been identified in bats. Here, viral metagenomic sequencing of samples from bats submitted for rabies virus testing, largely due to human exposure, identified a novel, highly divergent exogenous Deltaretrovirus from a big brown bat (Eptesicus fuscus) in South Dakota. The virus sequence, corresponding to Eptesicus fuscus deltaretrovirus (EfDRV), comprised a nearly complete coding region comprised of canonical 5'-gag-pro-pol-env-3' genes with 37% to 51% identity to human T-lymphotropic virus (HTLV), an infectious retrovirus that causes T-cell lymphoma. A putative tax gene with 27% identity to HTLV was located downstream of the pol gene along with a gene harbored in an alternative reading frame which possessed a conserved domain for an Epstein-Barr virus nuclear antigen involved in gene transactivation, suggesting a regulatory function similar to that of the deltaretrovirus rex gene. A TaqMan reverse transcriptase PCR (RT-PCR) targeting the pol gene identified 4/60 (6.7%) bats as positive for EfDRV, which, combined with a search of the E. fuscus genome that failed to identify sequences with homology to EfDRV, suggests that EfDRV is an infectious exogenous virus. As all known members of Deltaretrovirus can cause malignancies and E. fuscus is widely distributed in the Americas, often with a colonial roosting behavior in human dwellings, further studies are needed to investigate potential zoonosis.IMPORTANCE Bats host a large numbers of viruses, many of which are zoonotic. In the United States, the big brown bat (Eptesicus fuscus) is widely distributed and lives in small colonies that roost in cavities, often in human dwellings, leading to frequent human interaction. Viral metagenomic sequencing of samples from an E. fuscus bat submitted for rabies testing identified the first exogenous bat Deltaretrovirus The E. fuscus deltaretrovirus (EfDRV) genome consists of the typical deltaretrovial 5'-gag-pro-pol-env-3' genes along with genes encoding two putative transcriptional transactivator proteins distantly related to the Tax protein of human T-cell lymphotrophic virus and nuclear antigen 3B of Epstein-Barr virus. Searches of the E. fuscus genome sequence failed to identify endogenous EfDRV. RT-PCR targeting the EfDRV pol gene identified 4/60 (6.7%) bats with positive results. Together, these results suggest that EfDRV is exogenous. As all members of Deltaretrovirus are associated with T- and B-cell malignancies or neurologic disease, further studies on possible zoonosis are warranted.


Assuntos
Quirópteros/virologia , Deltaretrovirus/genética , Deltaretrovirus/isolamento & purificação , Genoma Viral/genética , Animais , Produtos do Gene tax/genética , Humanos , RNA Viral/genética , South Dakota , Estados Unidos , Zoonoses/virologia
9.
Sci Rep ; 10(1): 15643, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973171

RESUMO

As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses, for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/prevenção & controle , Proteínas do Nucleocapsídeo/genética , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/imunologia , Sequência de Bases , Infecções por Coronavirus/imunologia , Genoma Viral/genética , Humanos , Proteínas do Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Produtos Inativados/imunologia , Sequenciamento Completo do Genoma
10.
Artigo em Inglês | MEDLINE | ID: mdl-32974224

RESUMO

SARS CoV appeared in 2003 in China, transmitted from bats to humans via eating infected animals. It affected 8,096 humans with a death rate of 11% affecting 21 countries. The receptor binding domain (RBD) in S protein of this virus gets attached with the ACE2 receptors present on human cells. MERS CoV was first reported in 2012 in Middle East, originated from bat and transmitted to humans through camels. MERS CoV has a fatality rate of 35% and last case reported was in 2017 making a total of 1,879 cases worldwide. DPP4 expressed on human cells is the main attaching site for RBD in S protein of MERS CoV. Folding of RBD plays a crucial role in its pathogenesis. Virus causing COVID-19 was named as SARS CoV-2 due its homology with SARS CoV that emerged in 2003. It has become a pandemic affecting nearly 200 countries in just 3 months' time with a death rate of 2-3% currently. The new virus is fast spreading, but it utilizes the same RBD and ACE2 receptors along with furin present in human cells. The lessons learned from the SARS and MERS epidemics are the best social weapons to face and fight against this novel global threat.


Assuntos
Infecções por Coronavirus/transmissão , Peptidil Dipeptidase A/genética , Pneumonia Viral/transmissão , Receptores Virais/genética , Síndrome Respiratória Aguda Grave/transmissão , Glicoproteína da Espícula de Coronavírus/genética , Animais , Betacoronavirus/genética , Betacoronavirus/metabolismo , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Evolução Molecular , Furina/metabolismo , Genoma Viral/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Domínios Proteicos/genética , Receptores Virais/metabolismo , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/patologia , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
J Med Internet Res ; 22(10): e22299, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32931441

RESUMO

BACKGROUND: COVID-19 became a global pandemic not long after its identification in late 2019. The genomes of SARS-CoV-2 are being rapidly sequenced and shared on public repositories. To keep up with these updates, scientists need to frequently refresh and reclean data sets, which is an ad hoc and labor-intensive process. Further, scientists with limited bioinformatics or programming knowledge may find it difficult to analyze SARS-CoV-2 genomes. OBJECTIVE: To address these challenges, we developed CoV-Seq, an integrated web server that enables simple and rapid analysis of SARS-CoV-2 genomes. METHODS: CoV-Seq is implemented in Python and JavaScript. The web server and source code URLs are provided in this article. RESULTS: Given a new sequence, CoV-Seq automatically predicts gene boundaries and identifies genetic variants, which are displayed in an interactive genome visualizer and are downloadable for further analysis. A command-line interface is available for high-throughput processing. In addition, we aggregated all publicly available SARS-CoV-2 sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID), National Center for Biotechnology Information (NCBI), European Nucleotide Archive (ENA), and China National GeneBank (CNGB), and extracted genetic variants from these sequences for download and downstream analysis. The CoV-Seq database is updated weekly. CONCLUSIONS: We have developed CoV-Seq, an integrated web service for fast and easy analysis of custom SARS-CoV-2 sequences. The web server provides an interactive module for the analysis of custom sequences and a weekly updated database of genetic variants of all publicly accessible SARS-CoV-2 sequences. We believe CoV-Seq will help improve our understanding of the genetic underpinnings of COVID-19.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Visualização de Dados , Bases de Dados Genéticas , Genoma Viral/genética , Pneumonia Viral/virologia , Software , Biologia Computacional , Infecções por Coronavirus/epidemiologia , Humanos , Pandemias , Pneumonia Viral/epidemiologia
12.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
13.
PLoS Comput Biol ; 16(9): e1008269, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941419

RESUMO

We propose an efficient framework for genetic subtyping of SARS-CoV-2, the novel coronavirus that causes the COVID-19 pandemic. Efficient viral subtyping enables visualization and modeling of the geographic distribution and temporal dynamics of disease spread. Subtyping thereby advances the development of effective containment strategies and, potentially, therapeutic and vaccine strategies. However, identifying viral subtypes in real-time is challenging: SARS-CoV-2 is a novel virus, and the pandemic is rapidly expanding. Viral subtypes may be difficult to detect due to rapid evolution; founder effects are more significant than selection pressure; and the clustering threshold for subtyping is not standardized. We propose to identify mutational signatures of available SARS-CoV-2 sequences using a population-based approach: an entropy measure followed by frequency analysis. These signatures, Informative Subtype Markers (ISMs), define a compact set of nucleotide sites that characterize the most variable (and thus most informative) positions in the viral genomes sequenced from different individuals. Through ISM compression, we find that certain distant nucleotide variants covary, including non-coding and ORF1ab sites covarying with the D614G spike protein mutation which has become increasingly prevalent as the pandemic has spread. ISMs are also useful for downstream analyses, such as spatiotemporal visualization of viral dynamics. By analyzing sequence data available in the GISAID database, we validate the utility of ISM-based subtyping by comparing spatiotemporal analyses using ISMs to epidemiological studies of viral transmission in Asia, Europe, and the United States. In addition, we show the relationship of ISMs to phylogenetic reconstructions of SARS-CoV-2 evolution, and therefore, ISMs can play an important complementary role to phylogenetic tree-based analysis, such as is done in the Nextstrain project. The developed pipeline dynamically generates ISMs for newly added SARS-CoV-2 sequences and updates the visualization of pandemic spatiotemporal dynamics, and is available on Github at https://github.com/EESI/ISM (Jupyter notebook), https://github.com/EESI/ncov_ism (command line tool) and via an interactive website at https://covid19-ism.coe.drexel.edu/.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Infecções por Coronavirus , Genômica/métodos , Pandemias , Pneumonia Viral , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Evolução Molecular , Marcadores Genéticos/genética , Genoma Viral/genética , Humanos , Mutação/genética , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Análise Espaço-Temporal
14.
mBio ; 11(5)2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978313

RESUMO

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.


Assuntos
Betacoronavirus/genética , Cromossomos Artificiais Bacterianos/genética , Animais , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Cricetinae , DNA Complementar/genética , Genoma Viral/genética , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Genética Reversa , Células Vero , Replicação Viral
15.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
16.
Biomed Res Int ; 2020: 9089768, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32923488

RESUMO

Purpose: We investigated sequences of the feline coronaviruses (FCoV), which include feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), from China and other countries to gain insight into the adaptive evolution of this virus. Methods: Ascites samples from 31 cats with suspected FIP and feces samples from 8 healthy cats were screened for the presence of FCoV. Partial viral genome sequences, including parts of the nsp12-nsp14, S, N, and 7b genes, were obtained and aligned with additional sequences obtained from the GenBank database. Bayesian phylogenetic analysis was conducted, and the possibility of recombination within these sequences was assessed. Analysis of the levels of selection pressure experienced by these sequences was assessed using methods on both the PAML and Datamonkey platforms. Results: Of the 31 cats investigated, two suspected FIP cats and one healthy cat tested positive for FCoV. Phylogenetic analysis showed that all of the sequences from mainland China cluster together with a few sequences from the Netherlands as a distinct clade when analyzed with FCoV sequences from other countries. Fewer than 3 recombination breakpoints were detected in the nsp12-nsp14, S, N, and 7b genes, suggesting that analyses for positive selection could be conducted. A total of 4, 12, 4, and 4 positively selected sites were detected in the nsp12-nsp14, S, N, and 7b genes, respectively, with the previously described site 245 of the S gene, which distinguishes FIPV from FECV, being a positive selection site. Conversely, 106, 168, 25, and 17 negative selection sites in the nsp12-14, S, N, and 7b genes, respectively, were identified. Conclusion: Our study provides evidence that the FCoV genes encoding replicative, entry, and virulence proteins potentially experienced adaptive evolution. A greater number of sites in each gene experienced negative rather than positive selection, which suggests that most of the protein sequence must be conservatively maintained for virus survival. A few of the sites showing evidence of positive selection might be associated with the more severe pathology of FIPV or help these viruses survive other harmful conditions.


Assuntos
Adaptação Biológica/genética , Coronavirus Felino/genética , Peritonite Infecciosa Felina/virologia , Panleucopenia Felina/virologia , Genoma Viral/genética , Animais , Teorema de Bayes , Gatos , China , Coronavirus Felino/fisiologia , Evolução Molecular , Fezes/virologia , Peritonite Infecciosa Felina/patologia , Panleucopenia Felina/patologia , Mutação , Filogenia , Recombinação Genética , Seleção Genética , Proteínas Virais/genética
17.
Genome Res ; 30(10): 1434-1448, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32878977

RESUMO

The human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the major pandemic of the twenty-first century. We analyzed more than 4700 SARS-CoV-2 genomes and associated metadata retrieved from public repositories. SARS-CoV-2 sequences have a high sequence identity (>99.9%), which drops to >96% when compared to bat coronavirus genome. We built a mutation-annotated reference SARS-CoV-2 phylogeny with two main macro-haplogroups, A and B, both of Asian origin, and more than 160 sub-branches representing virus strains of variable geographical origins worldwide, revealing a rather uniform mutation occurrence along branches that could have implications for diagnostics and the design of future vaccines. Identification of the root of SARS-CoV-2 genomes is not without problems, owing to conflicting interpretations derived from either using the bat coronavirus genomes as an outgroup or relying on the sampling chronology of the SARS-CoV-2 genomes and TMRCA estimates; however, the overall scenario favors haplogroup A as the ancestral node. Phylogenetic analysis indicates a TMRCA for SARS-CoV-2 genomes dating to November 12, 2019, thus matching epidemiological records. Sub-haplogroup A2 most likely originated in Europe from an Asian ancestor and gave rise to subclade A2a, which represents the major non-Asian outbreak, especially in Africa and Europe. Multiple founder effect episodes, most likely associated with super-spreader hosts, might explain COVID-19 pandemic to a large extent.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Genoma Viral/genética , Pneumonia Viral/epidemiologia , Animais , Ásia/epidemiologia , Sequência de Bases/genética , Quirópteros/virologia , Mapeamento Cromossômico , Europa (Continente)/epidemiologia , Evolução Molecular , Variação Genética/genética , Humanos , Pandemias , Filogenia , Filogeografia , Homologia de Sequência do Ácido Nucleico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA