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1.
Int J Infect Dis ; 91: 129-136, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31821892

RESUMO

OBJECTIVES: Dengue outbreaks, mainly caused by dengue virus serotype 2 (DENV-2), occurred in 2007 and in 2010 in Gabon, Central Africa. However, information on DENV infections has been insufficient since 2010. The aim of this study was to investigate the current DENV infection scenario and the risk of repeated infections in Gabon. METHODS: During 2015-2017, serum samples were collected from enrolled febrile participants and were tested for DENV infection using RT-qPCR. DENV-positive samples were analyzed for a history of previous DENV infection(s) using ELISA. The complete DENV genome was sequenced to analyze the phylogeny of Gabonese DENV strains. RESULTS: DENV-3 was exclusively detected, with a high rate of anti-DENV IgG seropositivity among DENV-3-positive participants. DENV-3 showed higher infection rates in adults and the infection was seasonal with peaks in the rainy seasons. Phylogenetic analysis revealed that Gabonese DENV-3 originated from West African strains and has been circulating continuously in Gabon since at least 2010, when the first DENV-3 case was reported. CONCLUSIONS: These findings indicate stable DENV-3 circulation and the risk of repeated DENV infections in Gabon, highlighting the need for continuous monitoring to control DENV infections.


Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Feminino , Gabão/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Estudos Soroepidemiológicos , Sorogrupo , Adulto Jovem
2.
J Virol Methods ; 269: 30-37, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30974179

RESUMO

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

3.
PLoS Negl Trop Dis ; 12(11): e0006971, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30500827

RESUMO

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.


Assuntos
Febre Lassa/virologia , Vírus Lassa/genética , Vírus Lassa/isolamento & purificação , Evolução Molecular , Variação Genética , Humanos , Febre Lassa/epidemiologia , Vírus Lassa/classificação , Nigéria/epidemiologia , Filogenia , Proteínas Virais/genética
4.
PLoS Pathog ; 14(7): e1007172, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30028868

RESUMO

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo.


Assuntos
Antígenos CD/imunologia , Coriomeningite Linfocítica/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/metabolismo , Proliferação de Células , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Ativação Linfocitária , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL
5.
Biochem Biophys Res Commun ; 503(2): 631-636, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29906459

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection.


Assuntos
Colesterol/metabolismo , Ácidos Graxos/metabolismo , Febre por Flebótomos/metabolismo , Phlebovirus/fisiologia , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Triglicerídeos/metabolismo , Replicação Viral , Animais , Vias Biossintéticas , Células CHO , Linhagem Celular , Cricetulus , Humanos , Febre por Flebótomos/enzimologia
6.
J Gen Virol ; 99(2): 181-186, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29300152

RESUMO

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann-Pick C1.


Assuntos
Ebolavirus/genética , Glicoproteínas/genética , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno , Modelos Estruturais , Proteína C1 de Niemann-Pick/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Ebolavirus/patogenicidade , Glicoproteínas/metabolismo , Humanos , Mamíferos , Mutação , Proteína C1 de Niemann-Pick/genética , Primatas , Alinhamento de Sequência , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
Sci Rep ; 7(1): 13503, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29044149

RESUMO

The recent outbreak of Zika virus (ZIKV) disease caused an enormous number of infections in Central and South America, and the unusual increase in the number of infants born with microcephaly associated with ZIKV infection aroused global concern. Here, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of ZIKV. The assay specifically detected ZIKV strains of both Asian and African genotypes without cross-reactivity with other arboviruses, including Dengue and Chikungunya viruses. The assay detected viral RNA at 14.5 TCID50/mL in virus-spiked serum or urine samples within 15 min, although it was slightly less sensitive than reference real time RT-PCR assay. We then evaluated the utility of this assay as a molecular diagnostic test using 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. The results of this assay were consistent with those of the reference RT-PCR test. This portable RT-LAMP assay was highly specific for ZIKV, and enable rapid diagnosis of the virus infection. Our results provide new insights into ZIKV molecular diagnostics and may improve preparedness for future outbreaks.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecção por Zika virus/sangue , Animais , Chlorocebus aethiops , Sensibilidade e Especificidade , Células Vero , Zika virus/genética , Infecção por Zika virus/urina
8.
J Virol Methods ; 249: 102-110, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28837842

RESUMO

This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86-98% and 67-77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.


Assuntos
Doenças dos Bovinos/diagnóstico , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real , Animais , Bovinos , Doenças dos Bovinos/virologia , Primers do DNA/genética , Vírus da Febre Aftosa/genética , RNA Viral/genética , Sensibilidade e Especificidade , Temperatura
9.
J Virol Methods ; 246: 8-14, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28356221

RESUMO

Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.


Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Primers do DNA , Vírus da Dengue/genética , Ebolavirus/classificação , Ebolavirus/genética , Humanos , Vírus Lassa/genética , Limite de Detecção , Marburgvirus/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Transcrição Reversa , Sensibilidade e Especificidade , Temperatura
10.
Genes Cells ; 22(2): 148-159, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28084671

RESUMO

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence.


Assuntos
Ebolavirus/genética , Mutação , Proteínas do Envelope Viral/genética , Células A549 , Ebolavirus/metabolismo , Evolução Molecular , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Análise de Sequência de DNA/métodos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
11.
J Virol Methods ; 238: 42-47, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27751948

RESUMO

Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals.


Assuntos
Doenças dos Bovinos/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Parapoxvirus/genética , Animais , Bovinos , Doenças dos Bovinos/virologia , Primers do DNA/genética , DNA Viral/análise , Células Epiteliais/virologia , Limite de Detecção , Parapoxvirus/isolamento & purificação , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/virologia , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Temperatura
12.
J Infect Dis ; 214(suppl 3): S229-S233, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27481863

RESUMO

To strengthen the laboratory diagnostic capacity for Ebola virus disease (EVD) in the remote areas of Guinea, we deployed a mobile field laboratory and implemented reverse transcription loop-mediated isothermal amplification (RT-LAMP) for postmortem testing. We tested 896 oral swab specimens and 21 serum samples, using both RT-LAMP and reverse transcription-polymerase chain reaction (RT-PCR). Neither test yielded a positive result, and the results from RT-LAMP and RT-PCR were consistent. More than 95% of the samples were tested within 2 days of sample collection. These results highlight the usefulness of the RT-LAMP assay as an EVD diagnostic testing method in the field or remote areas.


Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ebolavirus/genética , Guiné/epidemiologia , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Humanos , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade
13.
Sci Rep ; 6: 20213, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26863911

RESUMO

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of (18)F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.


Assuntos
Arbovirus/genética , Infecções por Bunyaviridae/virologia , Genoma Viral , Nairovirus/genética , Filogenia , Carrapatos/virologia , Animais , Arbovirus/classificação , Arbovirus/patogenicidade , Infecções por Bunyaviridae/mortalidade , Infecções por Bunyaviridae/patologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Monitoramento Epidemiológico , Fluordesoxiglucose F18/metabolismo , Trato Gastrointestinal/patologia , Trato Gastrointestinal/virologia , Células HEK293 , Humanos , Japão , Camundongos , Camundongos Knockout , Nairovirus/classificação , Nairovirus/patogenicidade , Neuroglia/patologia , Neuroglia/virologia , Neurônios/patologia , Neurônios/virologia , Testes de Neutralização , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Análise de Sequência de RNA , Análise de Sobrevida , Células Vero
14.
PLoS Negl Trop Dis ; 10(2): e0004472, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26900929

RESUMO

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Primers do DNA/genética , Ebolavirus/classificação , Ebolavirus/isolamento & purificação , Guiné , Doença pelo Vírus Ebola/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Transcrição Reversa , Sensibilidade e Especificidade
15.
BMJ Glob Health ; 1(3): e000180, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28588973

RESUMO

With the incidence and mortality rates of Ebola virus disease (EVD) in Guinea, Liberia and Sierra Leone now at zero and reports of the largest and most complex EVD outbreak in history no longer on the front pages of newspapers worldwide, the urgency of that crisis seems to have subsided. During this lull after the storm and before the next one, the international community needs to engage in a 'lessons-learned' exercise with respect to our collective scientific, clinical and public health preparedness. This engagement must identify pragmatic, innovative mechanisms at multinational, national and community levels that allow research and development of next generation diagnostics and therapeutics, the safe and effective practice of medicine, and the maintenance of public health to keep pace with the rapid epidemiological dynamics of EVD and other deadly infectious diseases.

16.
Microbiol Res ; 166(2): 77-86, 2011 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-20347283

RESUMO

Molecular typing is an important tool in the surveillance and investigation of human Legionella infection outbreaks. In this study, two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), were used to discriminate 23 Legionella pneumophila strains. The usefulness of MALDI-TOF-MS was demonstrated. The MALDI-TOF-MS fingerprinting with filtered small acid-soluble molecules gave different molecular profiles among strains, and the clustal analysis with MALDI-TOF-MS showed a high discrimination of strains the same as that with PFGE. In addition, MALDI-TOF-MS data could be generated within a few hours after the initial culture, although PFGE analyses took several days to complete. Thus, MALDI-TOF-MS offers a simple and rapid discrimination technique that could aid in the tracking of fast-spreading outbreaks of Legionella.


Assuntos
Legionella pneumophila/classificação , Tipagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado/métodos , Humanos , Legionella pneumophila/genética , Legionella pneumophila/isolamento & purificação , Reação em Cadeia da Polimerase
17.
Microbiol Immunol ; 55(1): 44-50, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21175773

RESUMO

In this study, a simple one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Lassa virus (LASV) was established. The two primer sets were designed to detect LASV circulating in Sierra Leone and northeastern Nigeria. The RT-LAMP assay using these primer sets was able to detect 100 copies of the in vitro transcribed artificial LASV RNA within 25 min. The assay was also evaluated using intact viral RNA extracted from cell culture-propagated viruses and confirmed to be highly specific for LASV. The RT-LAMP assay developed in this study is rapid, simple, and highly specific for the detection of LASV, although its sensitivity is slightly lower than that of real-time RT-PCR. In addition, because the RT-LAMP assay does not require the use of sophisticated equipment, it would be advantageous for clinical diagnosis of LASV infection in developing countries. It might also be employed in cases of deliberate release during bioterrorism attacks or in epidemiological surveillance for disease outbreaks.


Assuntos
Vírus Lassa/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Transcrição Reversa , Sequência de Bases , Reações Cruzadas , Vírus Lassa/genética , Dados de Sequência Molecular , Fatores de Tempo
18.
J Clin Microbiol ; 48(7): 2330-6, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20421440

RESUMO

Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.


Assuntos
Marburgvirus , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Virologia/métodos , Animais , Sequência de Bases , Chlorocebus aethiops , Humanos , Doença do Vírus de Marburg/diagnóstico , Marburgvirus/genética , Marburgvirus/isolamento & purificação , Dados de Sequência Molecular , RNA Viral/isolamento & purificação , Transcrição Reversa , Ribonucleoproteínas/genética , Sensibilidade e Especificidade , Alinhamento de Sequência , Especificidade da Espécie , Células Vero , Proteínas Virais/genética
19.
J Virol ; 81(11): 5908-18, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17360758

RESUMO

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). To develop a better animal model for the investigation of HTLV-1 infection, we established a transgenic (Tg) rat carrying the human CRM1 (hCRM1) gene, which encodes a viral RNA transporter that is a species-specific restriction factor. At first we found that CRM1 expression is elaborately regulated through a pathway involving protein kinase C during lymphocyte activation, initially by posttranscriptional and subsequently by transcriptional mechanisms. This fact led us to use an hCRM1-containing bacterial artificial chromosome clone, which would harbor the entire regulatory and coding regions of the CRM1 gene. The Tg rats expressed hCRM1 protein in a manner similar to expression of intrinsic rat CRM1 in various organs. HTLV-1-infected T-cell lines derived from these Tg rats produced 100- to 10,000-fold more HTLV-1 than did T cells from wild-type rats, and the absolute levels of HTLV-1 were similar to those produced by human T cells. We also observed enhancement of the dissemination of HTLV-1 to the thymus in the Tg rats after intraperitoneal inoculation, although the proviral loads were low in both wild-type and Tg rats. These results support the essential role of hCRM1 in proper HTLV-1 replication and suggest the importance of this Tg rat as an animal model for HTLV-1.


Assuntos
Animais Geneticamente Modificados , Regulação Neoplásica da Expressão Gênica/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Carioferinas/genética , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/virologia , Receptores Citoplasmáticos e Nucleares/genética , Subpopulações de Linfócitos T/virologia , Replicação Viral/fisiologia , Animais , Linhagem Celular Transformada , Células Cultivadas , Modelos Animais de Doenças , Humanos , Carioferinas/biossíntese , Ratos , Receptores Citoplasmáticos e Nucleares/biossíntese , Subpopulações de Linfócitos T/metabolismo
20.
J Virol Methods ; 141(1): 78-83, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17194485

RESUMO

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10(-3) FFU of the cell-culture propagated viruses. The reaction time needed to detect 10(4) FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.


Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Chlorocebus aethiops , Ebolavirus/genética , Estudos de Avaliação como Assunto , Doença pelo Vírus Ebola/virologia , Humanos , RNA Viral/análise , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Células Vero
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