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1.
Klin Lab Diagn ; 64(9): 571-577, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31610111

RESUMO

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector¼ (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Febre Hemorrágica da Crimeia/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Kit de Reagentes para Diagnóstico , Humanos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Sensibilidade e Especificidade
2.
Adv Exp Med Biol ; 1172: 157-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628656

RESUMO

RIG-I-like receptors (RLRs) are an important family of pattern recognition receptors. They activate the immunological responses against viral infections by sensing RNAs in the cytoplasm. Recent studies provide significant insights into the activation and transduction mechanisms of RLRs family (members including RIG-I, MDA5, and LGP2). Here we review our current understanding of the structures of RLRs. Structural characterizations of RLRs family have revealed the mechanism of their actions at molecular level. The activation mechanisms of RIG-I and MDA5 are different, despite both of them have similar domain organizations and bind to dsRNA ligands. RIG-I, but not MDA5, adopts an auto-suppression conformation in the absence of RNA. In addition to ligand triggered receptor oligomerization, the activities of these receptors are also regulated by several posttranslational modifications, especially ubiquitination. Overall, these structural studies play critical roles in promoting the understanding of viral RNA recognition mechanisms by the host innate immune system, which also contribute to the designing of drugs for treatment of viral infection. However, much work remains to be done in studying the signaling pathway of MDA5 and LGP2, particularly by structural biology.


Assuntos
Proteína DEAD-box 58 , RNA Helicases DEAD-box , Imunidade Inata , RNA Viral , Animais , Proteína DEAD-box 58/química , Proteína DEAD-box 58/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon , RNA de Cadeia Dupla/metabolismo , RNA Viral/análise , RNA Viral/metabolismo , Transdução de Sinais , Viroses/imunologia
3.
Pan Afr Med J ; 33: 169, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31565130

RESUMO

Introduction: hepatitis C virus (HCV) has several extra-hepatic manifestations including cryoglubulinemia. Cryoglobulinemia is defined as the abnormal presence in the blood of one or several proteins (cryoglobulins) that can precipitate at low temperatures. Method: We conducted a cross-sectional analytical study in the Laboratory of Biology and in the Unit of Hepatology of the General Hospital in Douala (HGD) over a period of 6 months. All patients agreeing to participate to the study and with anti-hepatitis-C antibodies under treatment or not were enrolled. Cryoglobulins were detected using biuret method and the classification was performed using Brouet immunoelectrophoresis. A multivariate analysis was conducted, confounding factors such as age, sex and the length of time after Hepatitis C Virus screening were adjusted. Results: The study enrolled 116 patients. The average age of patients was 58.47±9.95 years. Male sex accounted for 50.86% of cases. Arthralgia was found in 69.80% of cases. Cryoglobulin was found in 63.80% of patients. After adjustment, female sex (OR =2.18; CI 95% [0,97-4,90]; p= 0.059), asthenia alone (OR =2.45;CI 95% [1,04-5,80]; p= 0.041), asthenia combined with arthralgia (OR =2.84;CI 95% [1,13-7, 10]; p= 0.026) and the presence of HCV RNA (OR =2.84;CI 95% [1,13-7,10]; p= 0.028) were factors independently associated with the presence of cryoglobulin. Conclusion: The prevalence of cryoglobubin is high in patients with anti-hepatitis-C antibodies at the HGD. Simple biological methods are used to detect it. Cryoglobulin test in patients with HCV is essential in resource-limited countries.


Assuntos
Crioglobulinemia/epidemiologia , Crioglobulinas/análise , Anticorpos Anti-Hepatite C/sangue , Hepatite C/complicações , Idoso , Artralgia/epidemiologia , Artralgia/etiologia , Camarões , Estudos Transversais , Crioglobulinemia/virologia , Feminino , Humanos , Imunoeletroforese , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prevalência , RNA Viral/análise
4.
Arch Virol ; 164(11): 2683-2690, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428915

RESUMO

Current antiviral therapies against hepatitis B virus (HBV) infections, such as treatment with nucleos(t)ide analogs (NAs) and interferon alpha, can significantly lower HBV DNA titers, eventually to undetectable levels. However, it is still difficult to completely eliminate the stable template of HBV, the covalently closed circular DNA (cccDNA), and this contributes to viral rebound when treatment is discontinued. HBV pregenomic RNA (pgRNA), which was recently found to be present in the enveloped mature HBV viral particle in blood, is tentatively regarded, with still accumulating clinical evidence, as a novel bona fide virological marker reflecting the amount and status of cccDNA when serum HBV DNA becomes undetectable. HBV pgRNA and DNA share almost identical sequences, and it is therefore difficult to differentiate pgRNA from viral DNA using normal PCR methods. To exclude interference from viral DNA, methods for measuring pgRNA usually require a selective DNA degradation step, which is complicated and time-consuming and also compromises the accuracy of detection. In this study, we developed a simplified quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay with improved accuracy achieved by probing the polyA tail of pgRNA. Using clinical serum samples, we observed that not all patients share the same 3' sequence, suggesting slight differences between HBV strains in the way they end transcription. We then designed and evaluated a universal primer and probe set for distinguishing HBV pgRNA from HBV DNA. Our results demonstrated that a one-step qRT-PCR assay could selectively amplify HBV pgRNA from a mixture of HBV RNA and DNA, which is valuable for clinical applications.


Assuntos
Vírus da Hepatite B/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , DNA Viral/análise , DNA Viral/genética , Hepatite B/virologia , Humanos , RNA Viral/análise , RNA Viral/genética
5.
Parasit Vectors ; 12(1): 384, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366369

RESUMO

BACKGROUND: Cache Valley virus (CVV; Bunyavirales, Peribunyaviridae) is a mosquito-borne arbovirus endemic in North America. Although severe diseases are mainly observed in pregnant ruminants, CVV has also been recognized as a zoonotic pathogen that can cause fatal encephalitis in humans. Human exposures to CVV and its related subtypes occur frequently under different ecological conditions in the New World; however, neurotropic disease is rarely reported. High prevalence rates of neutralizing antibodies have been detected among residents in several Latin American cities. However, zoophilic mosquito species involved in the enzootic transmission are unlikely to be responsible for the transmission leading to human exposures to CVV. Mechanisms that lead to frequent human exposures to CVV remain largely unknown. In this study, competence of two anthropophilic mosquitoes, Aedes albopictus and Ae. aegypti, for CVV was determined using per os infection to determine if these species could play a role in the transmission of CVV in the domestic and peridomestic settings of urban and suburban areas. RESULTS: Aedes albopictus were highly susceptible to CVV whereas infection of Ae. aegypti occurred at a significantly lower frequency. Whilst the dissemination rates of CVV were comparable in the two species, the relatively long period to attain maximal infectious titer in Ae. aegypti demonstrated a significant difference in the replication kinetics of CVV in these species. Detection of viral RNA in saliva suggests that both Ae. albopictus and Ae. aegypti are competent vectors for CVV under laboratory conditions. CONCLUSIONS: Differential susceptibility to CVV was observed in Ae. albopictus and Ae. aegypti, reflecting their relatively different capacities for vectoring CVV in nature. The high susceptibility of Ae. albopictus to CVV observed in this study suggests its potential role as an efficient vector for CVV. Complemented by the reports of multiple CVV isolates derived from Ae. albopictus, our finding provides the basis for how the dispersal of Ae. albopictus across the New World may have a significant impact on the transmission and ecology of CVV.


Assuntos
Aedes/virologia , Vírus Bunyamwera/fisiologia , Infecções por Bunyaviridae/transmissão , Mosquitos Vetores/virologia , Zoonoses/transmissão , Zoonoses/virologia , Aedes/fisiologia , Animais , Infecções por Bunyaviridae/virologia , Cidades , Feminino , Humanos , América do Norte , RNA Viral/análise , Saliva/virologia , Carga Viral , Replicação Viral
6.
BMC Infect Dis ; 19(1): 738, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438880

RESUMO

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.


Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção Terciária
7.
Comp Immunol Microbiol Infect Dis ; 65: 219-225, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31300117

RESUMO

The interaction between a low pathogenic avian influenza virus (A/CK/TUN/145/2012), a H9N2 Tunisian isolate, and a vaccine strain (H120) of avian infectious bronchitis, administered simultaneously or sequentially three days apart to chicks during 20 days, was evaluated using ELISA antibody levels, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses and histopathology examination. First, the in vivo replication interference of avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated using qRT-PCR to detect accurately either AIV or IBV genomes or viral copy numbers during dual infections. Second, we have determined the amount of specific antibodies in sera of chick's infected with AIV alone, IBV alone, mixed AIV + IBV, IBV then AIV or AIV IBV 3 days later using an ELISA test. Finally, histopathological analyses of internal organs from inoculated chicks were realized. Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. According to our results, vaccine application was safe and do not interfere with AIV H9N2 infection, and does not enhance such infection. In conclusion, co-infection of chicks with AIV and IBV, simultaneously or sequentially, affected the clinical signs, the virus replication dynamics as well as the internal organ integrity. The results proposed that infection with heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated.


Assuntos
Coinfecção/veterinária , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Interferência Viral , Replicação Viral , Animais , Anticorpos Antivirais/sangue , Galinhas/imunologia , Galinhas/virologia , Coinfecção/virologia , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , RNA Viral/análise , Vacinas Virais/imunologia
8.
Vet Ital ; 55(2): 173-179, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31274180

RESUMO

An outbreak of Peste des petits ruminants (PPR) occurred in the Antalya Province in Turkey during  October  2015.  The  Antalya  Province  has  suitable  habitats  for  vectors.  There  is  no  information  available  on  the  role  of  Culicoides  spp.  in  the  transmission  of  Peste  des  petits  ruminants virus (PPRV). In this study we investigated the potential role of the Culicoides spp. in  the  transmission  of  PPRV.  Culicoides  were  trapped  throughout  middle  of  October  and  middle of December, 2015. A total of 12 pools of non-engorged females were analysed with real-time RT-PCR targeting the nucleocapsid (N) gene of the PPRV. PPRV RNA was detected in 7 of 12 Culicoides pools. These pools were negative for the bovine/ovine beta-actin mRNA. Culicoides spp. were identified to the species level by sequence analysis of the mitochondrial cytochrome  oxidase  subunit  I  gene.  The  species  of  Culicoides  found  PPRV  positive  was  Culicoides  imicola.  Molecular  characterization  of  field  isolates  from  recent  outbreaks  and  pools of midges that tested positive for PPRV suggests that PPRV replication might occur in Culicoides imicola, and it may have played a role in transmitting PPRV.


Assuntos
Ceratopogonidae/virologia , Peste dos Pequenos Ruminantes/transmissão , Vírus da Peste dos Pequenos Ruminantes/isolamento & purificação , RNA Viral/análise , Doenças dos Ovinos/transmissão , Animais , Feminino , Filogenia , Ovinos , Turquia
9.
Medicine (Baltimore) ; 98(29): e16376, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31335686

RESUMO

The HIV subtype B is the most frequent in Brazil. The HIV subtype B' codes the amino acids glicine-tryptophan-glicine (GWG) instead of glicine-proline-glicine on the tip of gp120 V3 loop. This variant was associated to a slower HIV progression in mono-infected patients; however, there is no information in coinfected patients. This study evaluated the infection progression of HIV variant B' on the hepatitis C virus presence. RNA isolated from plasma of the 601 infected patients were used to human immunodeficiency virus (HIV) subtyping and to classify the virus according their syncytium-inducing ability. The HIV infection progression was evaluated by clinical and laboratorial data. The results showed a significant association between HIV B' variant and CD4 count and time of AIDS in HIV mono-infected patients. Notwithstanding the fact that we did not find a direct association between GWG variant and AIDS and in HIV coinfected patients no mitigating effect due to GWG presence was found. We did observe that the association between GWG variant and CD4 counts is lost in coinfected patients. This is first work showing influence of the HIV GWG variant in coinfected patients. Nevertheless, the presence of the GWG variant can indicate a better prognostic in the mono-infected patients.


Assuntos
Proteína gp120 do Envelope de HIV/genética , Infecções por HIV , HIV-1/genética , Hepatite C , Adulto , Brasil/epidemiologia , Contagem de Linfócito CD4/métodos , Coinfecção/epidemiologia , Coinfecção/virologia , Progressão da Doença , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Hepatite C/virologia , Humanos , Masculino , Prognóstico , RNA Viral/análise
10.
BMC Infect Dis ; 19(1): 592, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286917

RESUMO

BACKGROUND: Norovirus (NoV) is recognized as a leading cause of acute gastroenteritis (AGE) outbreaks in settings globally. Studies have shown that employees played an important role in the transmission mode during some NoV outbreaks. This study aimed to investigate the prevalence of NoV infection and duration of NoV shedding among employees during NoV outbreaks, as well as factors affecting shedding duration. METHODS: Specimens and epidemiological data were collected from employees who were suspected of being involved in the transmission or with AGE symptoms during NoV outbreaks in Xuhui District, Shanghai, from 2015 to 2017. Specimens were detected using real-time RT-PCR to determine whether or not employees had become infected with NoV. Specimens were collected every 3-7 days from NoV-infected employees until specimens became negative for NoV. RESULTS: A total of 421 employees were sampled from 49 NoV outbreaks, and nearly 90% of them (377/421) were asymptomatic. Symptomatic employees showed significantly higher prevalence of NoV infection than asymptomatic ones (70.5% vs. 17.0%, P < 0.01). The average duration of NoV shedding was 6.9 days (95% confidence interval: 6.1-7.7 days) among 88 NoV-infected individuals, and was significantly longer in symptomatic individuals than in asymptomatic ones (9.8 days vs. 5.6 days, P < 0.01). In Cox proportional-hazards model, after adjusting age and gender, symptoms was the only factor associated with duration of NoV shedding. CONCLUSIONS: Compared with asymptomatic employees, symptomatic employees had higher prevalence of NoV infection and longer durations of NoV shedding. Since NoV shedding duration among NoV-infected employees tends to be longer than their isolation time during outbreaks, reinforcement of hygiene practices among these employees is especially necessary to reduce the risk of virus secondary transmissions after their return to work.


Assuntos
Infecções por Caliciviridae , Surtos de Doenças/estatística & dados numéricos , Gastroenterite , Norovirus/genética , Adulto , Canal Anal/virologia , Infecções Assintomáticas/epidemiologia , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , China/epidemiologia , Feminino , Gastroenterite/diagnóstico , Gastroenterite/epidemiologia , Gastroenterite/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
Anticancer Res ; 39(7): 3855-3862, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262913

RESUMO

BACKGROUND: We examined treatment the efficacy and data on long-term outcomes in real-world Japanese patients infected with hepatitis C virus (HCV) genotype 2 treated with 12-week sofosbuvir/ribavirin combination therapy. PATIENTS AND METHODS: In a total of 86 patients who were treated with sofosbuvir/ribavirin, sustained virological response (SVR) rates and long-term-outcomes were retrospectively analyzed. RESULTS: The adherence to this combination therapy was 98.8%. The rates of SVR at week 24 (SVR24) achieved with this treatment according to the 'intention-to-treat' and 'per-protocol' analyses were 89.5% and 96.2%, respectively. Two patients who experienced relapse did not have any previously reported resistance-associated substitutions in the HCV non-structural protein 5B (NS5B) polymerase region. We did not observe any patients who experienced late relapse but did observe that 50% and 1.3% of patients with and without a previous history of hepatocellular carcinoma (HCC), respectively, developed HCC after achieving SVR24 (with a mean follow-up period of 2.7±0.8 years). CONCLUSION: Patients with SVR should be carefully followed-up to screen for the occurrence of HCC, although it is infrequent.


Assuntos
Antivirais/uso terapêutico , Hepatite C/tratamento farmacológico , Ribavirina/uso terapêutico , Sofosbuvir/uso terapêutico , Idoso , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Quimioterapia Combinada , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/complicações , Hepatite C/virologia , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Resultado do Tratamento
12.
Biomed Environ Sci ; 32(5): 357-362, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31217052

RESUMO

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION: A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , Vírus da Encefalite Transmitidos por Carrapatos/genética
13.
Int J Infect Dis ; 85: 167-174, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202908

RESUMO

OBJECTIVE: The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples. METHODS: The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus. RESULTS: High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5µl sample and the diagnosis could be completed within 2h. CONCLUSIONS: This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.


Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , Adulto , Criança , Feminino , Humanos , Limite de Detecção , Masculino , RNA Viral/análise , RNA Viral/sangue , Sensibilidade e Especificidade , Zika virus/genética
14.
Lancet ; 394(10193): 148-158, 2019 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-31174831

RESUMO

BACKGROUND: Use of oral live-attenuated polio vaccines (OPV), and injected inactivated polio vaccines (IPV) has almost achieved global eradication of wild polio viruses. To address the goals of achieving and maintaining global eradication and minimising the risk of outbreaks of vaccine-derived polioviruses, we tested novel monovalent oral type-2 poliovirus (OPV2) vaccine candidates that are genetically more stable than existing OPVs, with a lower risk of reversion to neurovirulence. Our study represents the first in-human testing of these two novel OPV2 candidates. We aimed to evaluate the safety and immunogenicity of these vaccines, the presence and extent of faecal shedding, and the neurovirulence of shed virus. METHODS: In this double-blind, single-centre phase 1 trial, we isolated participants in a purpose-built containment facility at the University of Antwerp Hospital (Antwerp, Belgium), to minimise the risk of environmental release of the novel OPV2 candidates. Participants, who were recruited by local advertising, were adults (aged 18-50 years) in good health who had previously been vaccinated with IPV, and who would not have any contact with immunosuppressed or unvaccinated people for the duration of faecal shedding at the end of the study. The first participant randomly chose an envelope containing the name of a vaccine candidate, and this determined their allocation; the next 14 participants to be enrolled in the study were sequentially allocated to this group and received the same vaccine. The subsequent 15 participants enrolled after this group were allocated to receive the other vaccine. Participants and the study staff were masked to vaccine groups until the end of the study period. Participants each received a single dose of one vaccine candidate (candidate 1, S2/cre5/S15domV/rec1/hifi3; or candidate 2, S2/S15domV/CpG40), and they were monitored for adverse events, immune responses, and faecal shedding of the vaccine virus for 28 days. Shed virus isolates were tested for the genetic stability of attenuation. The primary outcomes were the incidence and type of serious and severe adverse events, the proportion of participants showing viral shedding in their stools, the time to cessation of viral shedding, the cell culture infective dose of shed virus in virus-positive stools, and a combined index of the prevalence, duration, and quantity of viral shedding in all participants. This study is registered with EudraCT, number 2017-000908-21 and ClinicalTrials.gov, number NCT03430349. FINDINGS: Between May 22 and Aug 22, 2017, 48 volunteers were screened, of whom 15 (31%) volunteers were excluded for reasons relating to the inclusion or exclusion criteria, three (6%) volunteers were not treated because of restrictions to the number of participants in each group, and 30 (63%) volunteers were sequentially allocated to groups (15 participants per group). Both novel OPV2 candidates were immunogenic and increased the median blood titre of serum neutralising antibodies; all participants were seroprotected after vaccination. Both candidates had acceptable tolerability, and no serious adverse events occurred during the study. However, severe events were reported in six (40%) participants receiving candidate 1 (eight events) and nine (60%) participants receiving candidate 2 (12 events); most of these events were increased blood creatinine phosphokinase but were not accompanied by clinical signs or symptoms. Vaccine virus was detected in the stools of 15 (100%) participants receiving vaccine candidate 1 and 13 (87%) participants receiving vaccine candidate 2. Vaccine poliovirus shedding stopped at a median of 23 days (IQR 15-36) after candidate 1 administration and 12 days (1-23) after candidate 2 administration. Total shedding, described by the estimated median shedding index (50% cell culture infective dose/g), was observed to be greater with candidate 1 than candidate 2 across all participants (2·8 [95% CI 1·8-3·5] vs 1·0 [0·7-1·6]). Reversion to neurovirulence, assessed as paralysis of transgenic mice, was low in isolates from those vaccinated with both candidates, and sequencing of shed virus indicated that there was no loss of attenuation in domain V of the 5'-untranslated region, the primary site of reversion in Sabin OPV. INTERPRETATION: We found that the novel OPV2 candidates were safe and immunogenic in IPV-immunised adults, and our data support the further development of these vaccines to potentially be used for maintaining global eradication of neurovirulent type-2 polioviruses. FUNDING: Bill & Melinda Gates Foundation.


Assuntos
Imunogenicidade da Vacina , Vacina Antipólio Oral/efeitos adversos , Vacina Antipólio Oral/imunologia , Poliovirus/imunologia , Adulto , Anticorpos Antivirais/sangue , Método Duplo-Cego , Fezes/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Poliomielite/prevenção & controle , Vacina Antipólio Oral/administração & dosagem , RNA Viral/análise , Método Simples-Cego , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Virulência/imunologia , Eliminação de Partículas Virais/imunologia , Adulto Jovem
15.
Arch Virol ; 164(7): 1843-1850, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31053978

RESUMO

Human respiratory syncytial virus (RSV) is a common viral pathogen that causes lower respiratory tract infections in infants and children globally. In this study, we developed a duplex reverse transcription recombinase-aided amplification (duplex-rtRAA) assay containing an internal control in a single closed tube for the detection of human RSV. The internal control in the amplification effectively eliminated false-negative results and ensured the accuracy of the duplex-rtRAA system. We first developed and evaluated a universal singleplex-rtRAA assay for RSV. The sensitivity of this assay for RSV was determined as 4.4 copies per reaction, and the specificity was 100%. Next, a duplex-rtRAA assay with an internal control was established. The sensitivity of the duplex-rtRAA assay approached 5.0 copies per reaction, and no cross-reaction with other common respiratory viruses was observed. The two detection methods (singleplex-rtRAA and duplex-rtRAA) developed in this study were used to test 278 clinical specimens, and the results showed absolute consistency with RSV RT-qPCR analysis, demonstrating 100% diagnostic sensitivity and specificity. These data indicate that the duplex-rtRAA has great potential for the rapid detection of RSV with a high sensitivity.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/diagnóstico , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Recombinases , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/virologia , Transcrição Reversa , Sensibilidade e Especificidade
16.
Clin Exp Med ; 19(3): 401-406, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127433

RESUMO

Residual HCV-RNA can persist in liver tissue and peripheral blood mononuclear cells (PBMCs) long after antiviral therapy of chronic hepatitis C in patients repeatedly negative for viral RNA in serum. This occult infection associates with impaired immune response and the risk of lymphoproliferative disorders or progressive liver disease. There are currently no monitoring strategies for patients after treatment. We investigated if serum inflammation markers and interferon lambda (IFNL) genotype can be predictors of the presence of HCV-RNA and the replicative HCV-RNA (-) strand in patients who reached sustained virological response after interferon-free therapy. Forty-two consecutive patients who remained HCV-RNA negative in serum 24 weeks after the end of treatment (EOT) and during the follow-up were enrolled. Total HCV-RNA and HCV-RNA (-) strand were detected using ultrasensitive RT-PCR in PBMCs collected 12-15 months after EOT. Polymorphisms within IFNL3-IFNL4 region (rs12979860 and ss469415590) were genotyped with allele-specific PCR. Viral RNA was found in PBMCs from 31 (74%) patients, and of those 29 (69%) were also positive for HCV-RNA (-). Neither normalization of alanine aminotransferase nor IFNL genotype predicted the presence of residual HCV-RNA. A significantly higher neutrocyte-to-lymphocyte ratio (NLR) 24 weeks after the start of treatment predicted elimination of replicative HCV-RNA strand (OR 0.23; 95% CI 0.10-0.86; P = 0.019). Patients with no HCV-RNA (-) in PBMCs showed a greater increase in neutrocyte count between EOT and baseline (P = 0.028). Lack of significant elevation of NLR after therapy with direct-acting antivirals could predict the presence of residual replicative HCV-RNA strand in PBMCs.


Assuntos
Antivirais/uso terapêutico , Monitoramento de Medicamentos/métodos , Hepacivirus/crescimento & desenvolvimento , Hepatite C Crônica/tratamento farmacológico , Linfócitos/imunologia , Neutrófilos/imunologia , Resposta Viral Sustentada , Adulto , Idoso , Técnicas de Apoio para a Decisão , Feminino , Seguimentos , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , RNA Viral/análise
17.
Clin Microbiol Infect ; 25(8): 1042.e1-1042.e4, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075547

RESUMO

OBJECTIVES: Zika virus (ZIKV) is mostly mosquito borne but it can also be transmitted via the sexual route and persists in semen for a prolonged time. Moreover, viral RNA has been detected in breast milk, saliva, lacrimal fluids and urine, suggesting other possible transmission routes. The aim of our research is to better define ZIKV tropism. METHODS: We investigated the tropism of Asian and African strains of ZIKV using human-derived neural, vaginal, intestinal and respiratory tissues. RESULTS: Asian and African strains of ZIKV were able to grow in all tissues tested, although with different efficiency (7.3 log RNA copies released apically in vaginal tissues versus 9.8 log RNA copies released in intestinal tissues), without the need for major adaptation. CONCLUSIONS: Our results underline that ZIKV tropism may be broader than expected in humans and stress the need to better explore all possible virus-shedding sites and transmission routes.


Assuntos
Intestinos/virologia , Tecido Nervoso/virologia , Sistema Respiratório/virologia , Vagina/virologia , Tropismo Viral , Zika virus/crescimento & desenvolvimento , África , Ásia , Epidemias , Feminino , Humanos , RNA Viral/análise , Zika virus/isolamento & purificação , Infecção por Zika virus/transmissão
18.
Analyst ; 144(13): 3972-3979, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31140473

RESUMO

Hepatitis C virus (HCV) is a major cause of chronic liver disease, which affects 2-3% of the world population. Until now, the early detection of HCV has been a great challenge, especially for those who live in developing countries. In this study, we developed a novel and ultrasensitive assay for the detection of HCV RNA based on the reduced graphene oxide nanosheets (rGONS) and hybridization chain reaction (HCR) amplification technique. This detection system contains a pair of single fluorophore-labeled hairpin probes that can freely exist in the solution in the absence of target RNA. The introduction of target RNA can robustly trigger a HCR with the two probes and produce long nanowires containing a double-stranded structure. The weak adsorption to rGONS makes the long nanowires emit a strong fluorescence. Using this enzyme-free amplification strategy, we developed a new method for the HCV RNA assay with a detection limit of 10 fM, which is far more sensitive than the common GO-based fluorescence method. Furthermore, the new method exhibits high selectivity for the discrimination of perfectly complementary and mismatched sequences. Finally, the new method was successfully used as a HCV RNA assay in biological samples with a strong anti-interference capability in complicated environments. In summary, these remarkable characteristics of the new method highlight its potential use in a clinical sample primary screening.


Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Grafite/química , Hepacivirus/isolamento & purificação , RNA Viral/análise , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Grafite/síntese química , Células HEK293 , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oxirredução , Estudo de Prova de Conceito , RNA Viral/genética , Espectrometria de Fluorescência/métodos
19.
Parasit Vectors ; 12(1): 258, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122295

RESUMO

BACKGROUND: Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus of veterinary importance which is transmitted by biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to ruminants. Culicoides sonorensis Wirth & Jones, the only confirmed vector of EHDV in the USA, is rare in the southeastern states where transmission persists, suggesting that other Culicoides species transmit EHDV in this region. The present study aimed to determine which Culicoides species transmitted EHDV in Florida and Alabama, two states in the southeastern USA. Viral RNA was detected in field-collected midges using molecular methods. These data are presented alongside data on Culicoides blood meal analysis, white-tailed deer (Odocoileus virginianus) aspiration, and seasonality to demonstrate an interaction between potential vector species and EHDV hosts. RESULTS: Out of 661 pools tested, 20 pools were positive for EHDV viral RNA, including six pools from Culicoides stellifer (Coquillett) and 14 pools from Culicoides venustus Hoffman. The overall infection rate was 0.06% for C. stellifer and 2.18% for C. venustus. No positive pools were identified for a further 17 species. Serotypes identified in Culicoides included EHDV-2, EHDV-6, and coinfections of EHDV-2 and EHDV-6 and were identified in similar proportions to serotypes in deer at 3 of 4 deer farms. Viral detections conducted in Alabama also identified one positive pool of C. venustus. Blood meal analysis revealed that both Culicoides species fed on white-tailed deer (verified through aspiration), fallow deer, and elk, species for which EHDV viremia has been documented. Seasonality data indicated that both species were present throughout the period in which viral transmission occurred to EHDV hosts in 2016 in addition to the 2017 epizootic. CONCLUSIONS: Our finding of EHDV positive pools of field-collected C. stellifer and C. venustus and an interaction between these species and EHDV hosts satisfy two of the four criteria for vector incrimination as set by the World Health Organization. Determining the vectors of EHDV is an important step towards developing sound strategies for the control of vector Culicoides and management of EHDV in the southeastern USA.


Assuntos
Ceratopogonidae/virologia , Vírus da Doença Hemorrágica Epizoótica/isolamento & purificação , Insetos Vetores/virologia , RNA Viral/análise , Alabama , Animais , Cervos/parasitologia , Cervos/virologia , Feminino , Florida , Insetos Vetores/classificação , RNA Viral/genética , Infecções por Reoviridae/transmissão , Ruminantes/parasitologia , Ruminantes/virologia , Sorogrupo
20.
Virol J ; 16(1): 58, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046802

RESUMO

BACKGROUND: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection. METHODS: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome. RESULTS: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants. CONCLUSIONS: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.


Assuntos
Aquaporinas/genética , Hepacivirus/genética , Hepatócitos/virologia , MicroRNAs/genética , RNA Viral/análise , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genoma Viral , Humanos , RNA Viral/genética , Transfecção , Carga Viral
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