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1.
Nat Commun ; 11(1): 4812, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968075

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Benchmarking , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Primers do DNA/genética , Temperatura Alta , Humanos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade , Suécia/epidemiologia , Ensaio de Placa Viral/métodos
2.
Nat Commun ; 11(1): 4059, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792628

RESUMO

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.


Assuntos
Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Vacinas Virais/imunologia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Testes Sorológicos/métodos , Células Vero , Ensaio de Placa Viral
3.
Virology ; 548: 39-48, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838945

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.


Assuntos
Betacoronavirus/fisiologia , Cultura de Vírus/métodos , Inativação de Vírus , Animais , Antígenos Virais/análise , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/imunologia , Linhagem Celular , Chlorocebus aethiops , Contenção de Riscos Biológicos , Meios de Cultura , Citometria de Fluxo , RNA Viral/análise , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Ensaio de Placa Viral , Replicação Viral
4.
Methods Mol Biol ; 2203: 135-143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833210

RESUMO

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).


Assuntos
Coronavirus/patogenicidade , Ensaio de Placa Viral/métodos , Animais , Células Cultivadas , Coronavirus/crescimento & desenvolvimento , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Vírus da Bronquite Infecciosa/patogenicidade , Traqueia/citologia
5.
Antiviral Res ; 181: 104882, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32738255

RESUMO

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Pneumonia Viral/virologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Betacoronavirus/isolamento & purificação , Chlorocebus aethiops , Infecções por Coronavirus/diagnóstico , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Síndrome Respiratória Aguda Grave/diagnóstico , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral
6.
CMAJ ; 192(31): E871-E874, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32646870

RESUMO

BACKGROUND: Provision of pasteurized donor human milk, as a bridge to mother's own milk, is the standard of care for very low-birth-weight infants in hospital. The aim of this research was to confirm that Holder pasteurization (62.5°C for 30 min) would be sufficient to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in donated human milk samples. METHODS: We spiked frozen milk samples from 10 donors to the Rogers Hixon Ontario Human Milk Bank with SARS-CoV-2 to achieve a final concentration of 1 × 107 TCID50/mL (50% of the tissue culture infectivity dose per mL). We pasteurized samples using the Holder method or held them at room temperature for 30 minutes and plated serial dilutions on Vero E6 cells for 5 days. We included comparative controls in the study using milk samples from the same donors without addition of virus (pasteurized and unpasteurized) as well as replicates of Vero E6 cells directly inoculated with SARS-CoV-2. We reported cytopathic effects as TCID50/mL. RESULTS: We detected no cytopathic activity in any of the SARS-CoV-2-spiked milk samples that had been pasteurized using the Holder method. In the SARS-CoV-2-spiked milk samples that were not pasteurized but were kept at room temperature for 30 minutes, we observed a reduction in infectious viral titre of about 1 log. INTERPRETATION: Pasteurization of human milk by the Holder method (62.5°C for 30 min) inactivates SARS-CoV-2. Thus, in the event that donated human milk contains SARS-CoV-2 by transmission through the mammary gland or by contamination, this method of pasteurization renders milk safe for consumption and handling by care providers.


Assuntos
Betacoronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/prevenção & controle , Bancos de Leite , Leite Humano/virologia , Pandemias/prevenção & controle , Pasteurização/métodos , Pneumonia Viral/prevenção & controle , Inativação de Vírus , Temperatura Alta , Humanos , Leite Humano/química , Ontário , Fatores de Tempo , Ensaio de Placa Viral
7.
Curr Protoc Cytom ; 93(1): e77, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-532562

RESUMO

SARS-CoV-2 is a novel coronavirus that causes the acute respiratory disease-Coronavirus disease 2019 (COVID-19)-which has led to a global health crisis. Currently, no prophylactics or therapies exist to control virus spread or mitigate the disease. Thus, the risk of infection for physicians and scientists is high, requiring work to be conducted in Biosafety Level-3 (BSL-3) facilities if virus will be isolated or propagated. However, inactivation of the virus can enable safe handling at a reduced biosafety level, making samples accessible to a diverse array of institutions and investigators. Institutions of all types have an immediate need for guidelines that outline safe collection, handling, and inactivation of samples suspected to contain active virus. Here we provide a practical guide for physicians and researchers wishing to work with materials from patients who are COVID-19 positive or suspected positive. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Practical guidelines for the safe collection and handling of specimens collected from COVID-19 and suspected COVID-19 patients Basic Protocol 2: Inactivating SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Guias como Assunto , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Manejo de Espécimes , Inativação de Vírus , Humanos , Pandemias , Ensaio de Placa Viral
8.
Curr Protoc Microbiol ; 57(1): ecpmc105, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-437154

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.


Assuntos
Betacoronavirus/isolamento & purificação , Protocolos Clínicos , Ensaio de Placa Viral/métodos , Animais , Chlorocebus aethiops , Humanos , Coloração e Rotulagem , Células Vero
9.
Mem Inst Oswaldo Cruz ; 115: e200012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520074

RESUMO

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.


Assuntos
Aedes/virologia , Culex/virologia , Flavivirus/fisiologia , Insetos Vetores/virologia , Superinfecção/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Argentina , Linhagem Celular , Ensaio de Placa Viral
10.
Curr Protoc Microbiol ; 57(1): ecpmc105, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32475066

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.


Assuntos
Betacoronavirus/isolamento & purificação , Protocolos Clínicos , Ensaio de Placa Viral/métodos , Animais , Chlorocebus aethiops , Humanos , Coloração e Rotulagem , Células Vero
11.
Viruses ; 12(6)2020 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-32517266

RESUMO

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ensaio de Placa Viral/métodos , Inativação de Vírus , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/patogenicidade , Celulose , Chlorocebus aethiops , Meios de Cultura/química , Formaldeído , Guanidinas/farmacologia , Células HEK293 , Humanos , Pandemias , Fenóis/farmacologia , Propiolactona/farmacologia , Sefarose , Células Vero
12.
Viruses ; 12(6)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32532085

RESUMO

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) signals an urgent need for an expansion in treatment options. In this study, we investigated the anti-SARS-CoV-2 activities of 22 antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. They were first evaluated in our primary screening in VeroE6 cells and then the most potent anti-SARS-CoV-2 antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. In addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types I and II recombinant interferons, 25-hydroxycholesterol, and AM580 as the most potent anti-SARS-CoV-2 agents among the 22 antiviral agents. Betaferon (interferon-ß1b) exhibited the most potent anti-SARS-CoV-2 activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. The lipogenesis modulators 25-hydroxycholesterol and AM580 exhibited EC50 at low micromolar levels and selectivity indices of >10.0. Combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the SARS-CoV-2 replication cycle should be evaluated in animal models and/or clinical trials.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Animais , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Humanos , Interferons/metabolismo , Lipogênese/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Transdução de Sinais/efeitos dos fármacos , Células Vero , Carga Viral/efeitos dos fármacos , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
13.
Curr Protoc Cytom ; 93(1): e77, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502333

RESUMO

SARS-CoV-2 is a novel coronavirus that causes the acute respiratory disease-Coronavirus disease 2019 (COVID-19)-which has led to a global health crisis. Currently, no prophylactics or therapies exist to control virus spread or mitigate the disease. Thus, the risk of infection for physicians and scientists is high, requiring work to be conducted in Biosafety Level-3 (BSL-3) facilities if virus will be isolated or propagated. However, inactivation of the virus can enable safe handling at a reduced biosafety level, making samples accessible to a diverse array of institutions and investigators. Institutions of all types have an immediate need for guidelines that outline safe collection, handling, and inactivation of samples suspected to contain active virus. Here we provide a practical guide for physicians and researchers wishing to work with materials from patients who are COVID-19 positive or suspected positive. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Practical guidelines for the safe collection and handling of specimens collected from COVID-19 and suspected COVID-19 patients Basic Protocol 2: Inactivating SARS-CoV-2.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Guias como Assunto , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Manejo de Espécimes , Inativação de Vírus , Humanos , Pandemias , Ensaio de Placa Viral
14.
PLoS One ; 15(4): e0231039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267861

RESUMO

Influenza B virus (IBV) belongs to the Orthomyxoviridae family and generally causes sporadic epidemics but is occasionally deadly to individuals. The current research mainly focuses on clinical and pathological characteristics of IBV. However, to better prevent or treat the disease, one must determine the strategies developed by IBV to invade and disrupt cellular proteins and approach to replicate itself, to suppress antiviral innate immunity, and understand how the host responds to IBV infection. The B/Shanghai/PD114/2018 virus was able to infect alveolar epithelial cells (A549) cells, with good potential for replication. To identify host cellular responses against IBV infection, differentially expressed genes (DEGs) were obtained using RNA sequencing. The GO and KEGG pathway term enrichment analyses with the DEGs were performed, and we found that the DEGs were primary involved in metabolic processes and cellular function, which may be related to the host response, including the innate immune response against the virus. Our transcriptome analysis results demonstrated robust induction of interferon and interferon-stimulated gene expression by IBV in human cells during the early stages of infection, providing a foundation for further studies focused on antiviral drug development and interactions between the virus and host.


Assuntos
Vírus da Influenza B/metabolismo , Influenza Humana/metabolismo , Interferons/metabolismo , Células A549/metabolismo , Células A549/virologia , Western Blotting , Imunofluorescência , Regulação Viral da Expressão Gênica , Humanos , Vírus da Influenza B/genética , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real , Ensaio de Placa Viral , Replicação Viral
15.
Braz J Infect Dis ; 24(1): 13-24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31843340

RESUMO

Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Sorogrupo , Replicação Viral/genética , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Colômbia , Citometria de Fluxo , Formazans , Humanos , Fígado/citologia , RNA Viral/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Fatores de Tempo , Ensaio de Placa Viral
16.
PLoS One ; 14(12): e0225895, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31830142

RESUMO

BACKGROUND: Arboviruses and protozoans can cause neurologic disorders in horses. In Brazilian Amazon, several horses presenting signs compatible with disorders caused by these infectious agents have been observed. OBJECTIVE: To contribute to the knowledge of this epidemiological picture, we sought to construct a serological diagnostic panel for neurotrophic infectious agents in local horses. MATERIAL AND METHODS: A total of 213 blood samples from horses were collected from 29 farms in three municipalities. Samples were evaluated and considered positive when they met the following criteria: titers ≥ 1:80 with the indirect fluorescent antibody test (IFAT) for apicomplexan protozoans; positive recombinant enzyme-linked immunosorbent assay (ELISA) with subsequent titers ≥ 1:10 by the PRNt for viruses; and detection under direct microscopic examination for Trypanosoma evansi. RESULTS: No horses were found to be infected by T. evansi, and only two were infected Toxoplasma gondii and/or Neospora spp. The highest protozoan infection rate was observed for Sarcocystis neurona (40.3%; n = 86/213). Among the positive ELISA samples tested by the plaque reduction neutralization test (PRNT90), 92% (n = 76/83) were positive for St Louis Encephalitis virus, 43% (n = 6/14) were positive for West Nile virus and 33% (n = 16/48) were positive for Mayaro virus. Eighteen percent (n = 39/213) of horses were co-infected by S. neurona and at least one arbovirus, particularly SLEV and/or MAYV. CONCLUSION: Samples positive for SLEV associated with S. neurona, including samples from horses that had recovered from neurological signs were frequent, and must be considered when investigating the possible causes of neurological diseases in South Roraima horses.


Assuntos
Infecções por Arbovirus/veterinária , Arbovirus , Coccídios , Coccidiose/veterinária , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/parasitologia , Doenças dos Cavalos/virologia , Animais , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Geografia , Cavalos , Testes de Neutralização , Estudos Soroepidemiológicos , Ensaio de Placa Viral
17.
Viruses ; 11(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861087

RESUMO

Listeria phage LP-018 is the only phage from a diverse collection of 120 phages able to form plaques on a phage-resistant Listeria monocytogenes strain lacking rhamnose in its cell wall teichoic acids. The aim of this study was to characterize phage LP-018 and to identify what types of mutations can confer resistance to LP-018. Whole genome sequencing and transmission electron microscopy revealed LP-018 to be a member of the Homburgvirus genus. One-step-growth curve analysis of LP-018 revealed an eclipse period of ~60-90 min and a burst size of ~2 PFU per infected cell. Despite slow growth and small burst size, LP-018 can inhibit the growth of Listeria monocytogenes at a high multiplicity of infection. Ten distinct LP-018-resistant mutants were isolated from infected Listeria monocytogenes 10403S and characterized by whole genome sequencing. In each mutant, a single mutation was identified in either the LMRG_00278 or LMRG_01613 encoding genes. Interesting, LP-018 was able to bind to a representative phage-resistant mutant with a mutation in each gene, suggesting these mutations confer resistance through a mechanism independent of adsorption inhibition. Despite forming plaques on the rhamnose deficient 10403S mutant, LP-018 showed reduced binding efficiency, and we did not observe inhibition of the strain under the conditions tested. Two mutants of LP-018 were also isolated and characterized, one with a single SNP in a gene encoding a BppU domain protein that likely alters its host range. LP-018 is shown to be a unique Listeria phage that, with additional evaluation, may be useful in biocontrol applications that aim to reduce the emergence of phage resistance.


Assuntos
Bacteriófagos/fisiologia , Interações Hospedeiro-Patógeno , Listeria monocytogenes/virologia , Bacteriólise , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Genoma Viral , Genômica/métodos , Especificidade de Hospedeiro , Mutação , Fases de Leitura Aberta , Especificidade da Espécie , Ensaio de Placa Viral
18.
Virol J ; 16(1): 122, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660997

RESUMO

BACKGROUND: Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. RESULTS: A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1-5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. CONCLUSIONS: The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.


Assuntos
Técnicas Microbiológicas/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Poliomielite/virologia , Vacina Antipólio Oral/isolamento & purificação , Linhagem Celular Tumoral , Fezes/virologia , Ensaios de Triagem em Larga Escala , Humanos , Técnicas Microbiológicas/normas , Poliovirus/genética , Poliovirus/isolamento & purificação , Vacina Antipólio Oral/genética , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sorogrupo , Ensaio de Placa Viral , Eliminação de Partículas Virais
19.
Intervirology ; 62(3-4): 134-144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31533107

RESUMO

OBJECTIVES: Differences have been observed in the susceptibility of macrophage cell lines to respiratory syncytial virus (RSV) infection. In this study, we evaluated whether the type of macrophage cell line and RSV strain used have an influence on the infectivity and production of progeny virus. METHODS: Both human and murine macrophage-like cell lines were infected with different RSV strains, both lab strains as well as clinical isolates. The infection was evaluated after 24 and 72 h by immunofluorescence staining and microscopic analysis, and the production of new virus particles was determined by plaque assay. RESULTS: Susceptibility of macrophages to RSV was influenced by the RSV strain used but was mostly dependent on the macrophage cell line. Numbers of infected cells and virus production were generally very low or absent in murine cell lines. In human cell lines, clear infection was observed associated with production of new virus particles. CONCLUSION: Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain.


Assuntos
Especificidade de Hospedeiro , Macrófagos/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Replicação Viral , Animais , Linhagem Celular , Humanos , Camundongos , Carga Viral , Ensaio de Placa Viral
20.
Invest Ophthalmol Vis Sci ; 60(12): 3952-3962, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560369

RESUMO

Purpose: γδ T cells offer an important early immune defense against many different pathogens, both bacterial and viral. Herein, we examined the capacity of γδ T cell subsets to provide protection in the cornea against herpes simplex virus-1 (HSV-1). Methods: C57Bl/6 (wild-type [WT]), γδ T-cell deficient (TCRδ-/-) and CCR6-deficient (CCR6-/-) mice were infected intracorneally with HSV-1. At multiple time points following infection, corneas were excised, and cells were immunostained for surface markers, intracellular cytokines, and analyzed using flow cytometry. WT and CCR6-/- γδ T cells were adoptively transferred into TCRδ-/- mice and corneal scores and survival were measured. Results: Intracorneal infection of mice lacking γδ T cells exhibited increased corneal opacity scores, elevated viral titers, and higher mortality compared with WT mice. Both CCR6+ and CCR6neg γδ T cell subsets were observed in corneas after virus infection. CCR6+ γδ T cells produced IL-17A and were predominantly CD44+CD62L+, consistent with natural IL-17+ γδ T cells. In contrast IL-17A production by CCR6neg γδ T cells was infrequent, and this subset was largely single positive for CD62L or CD44. The CCR6+ subset appeared to provide protection against HSV-1 as follows: (1) CCR6-/- mice had more severe corneal opacity compared with WT mice; and (2) adoptive transfer of γδ T cells from WT mice restored protection in TCRδ-/- mice whereas transfer of γδ T cells from CCR6-/- mice did not. Conclusions: γδ T cells in the cornea can be divided into CCR6+ and CCR6neg subsets with the former conferring protection early after intracorneal HSV-1 infection.


Assuntos
Opacidade da Córnea/prevenção & controle , Herpesvirus Humano 1/fisiologia , Linfócitos Intraepiteliais/imunologia , Ceratite Herpética/prevenção & controle , Receptores CCR6/imunologia , Transferência Adotiva , Animais , Córnea/virologia , Opacidade da Córnea/imunologia , Opacidade da Córnea/virologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-17/metabolismo , Ceratite Herpética/imunologia , Ceratite Herpética/virologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gânglio Trigeminal/virologia , Ensaio de Placa Viral
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